Characterization of CD8+ tissue-resident memory T cells in the salivary gland and the impact of MCMV infection on T cell recruitment and retention

Universidade do Minho Escola de Medicina Sofia Caldeira Dantas dezembro de 2019 + Characterization of CD8 tissue-resident memory T cells in the salivary gland and the impact of MCMV infection on T cell recruitment and retention Sofia Caldeira Dantas + Characterization of CD8 tissue-resident memory T cells in the salivary gland and the impact of MCMV infection on T cell recruitment and retention UMinho|2019 Sofia Caldeira Dantas dezembro de 2019 + Characterization of CD8 tissue-resident memory T cells in the salivary gland and the impact of MCMV infection on T cell recruitment and retention Trabalho efetuado sob a orientação do Professor Doutor Christopher M. Snyder e da Professora Doutora Margarida Correia-Neves Tese de Doutoramento Doutoramento em Medicina Universidade do Minho Escola de Medicina ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contatar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-Compartilha Igual CC BY-SA https://creativecommons.org/licenses/by-sa/4.0/ iii Acknowledgments Firstly, I would like to acknowledge Professor Christopher Snyder for all of your guidance and support through these years. I truly believe that your mentorship allowed me to thrive and grow as both a critical thinker and as a person. I would also like to acknowledge Professor Margarida Correia-Neves for always pushing me to be a better scientist. To all the people that along the way helped me ignite a passion for science, especially my colleagues at the Snyder’s lab with whom I share amazing memories. Thank you for all the experiments together (both in and outside of the lab). To my MD/PhD friends (PhDears) for all of the enthusiasm, great adventures and lifesaving moments. I would like to acknowledge the Escola de Medicina at University of Minho and its faculty for establishing the MD/PhD program and for the continuous efforts in advancing medical education in Portugal. Finally, to my family, that despite the distance (and my terrible tendency to forget to call every so often), have always believed in and supported me throughout the years. Thank you for all the calls, the visits, the essential items delivered by mail and all the critical remarks tactfully disguised as sarcasm and wrapped in love that drove me and continues to drive me to achieve excellence. You are the best and I could not have done it without you! Financial support was provided by Fundação para a Ciência e Tecnologia (PD/BD/52319/2013) in the context of the University of Minho MD/PhD Program and by National Institutes of Health (grant AI106810 to C.M.S.) iv Statement of integrity I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v RESUMO Caracterização das células T CD8+ residentes nas glândulas salivares e o impacto da infeção por MCMV no seu recrutamento e retenção. Células T CD8+ residentes (TRM) são mediadores cada vez mais importantes na resposta imunitária em diversos tecidos. Estas células têm como função a patrulha de tecidos, tendo sido descritas como parte da primeira linha de defesa em órgãos como a pele, pulmões, trato digestivo e cérebro. Devido à persistência das TRM nos tecidos e à sua resposta efetora, a promoção destas células em barreiras naturais e em locais onde ocorre replicação viral pode ter um impacto significativo na patogénese destas infeções. Assim, as TRM são alvos interessantes de estudo quando consideramos infeções víricas latentes como a causada pelo Citomegalovirus (CMV). Contudo, para otimizar o papel destas células é crucial compreender os mecanismos envolvidos na sua diferenciação e residência. Para além de descrever as alterações que ocorrem após infeção por CMV, este trabalho focou-se no estudo dos mecanismos envolvidos na diferenciação de TRM na glândula salivar, órgão crucial para a replicação e disseminação do CMV. Os resultados demonstram que embora a infeção por CMV promova o recrutamento de células T CD8+ para a glândula, a diferenciação de TRM ocorre de forma similar mesmo na ausência de infeção ou antigénio, o que contrasta com o que está descrito na maioria dos órgãos. Estes dados sugerem que mesmo nas glândulas salivares de ratinhos naïve existem sinais que possibilitam a diferenciação de TRM. Assim, o recrutamento de células T CD8+ para a glândula salivar torna-se um passo essencial na formação de TRM. Segundo o nosso trabalho a integrina a4b1 surge como um mediador no recrutamento de células T CD8+ para glândula salivar independentemente da infeção por CMV. Por outro lado, embora a infeção resulte no aumento de quimocinas reconhecidas pelos receptores CXCR3 e CCR5 na glândula salivar, nenhum destes receptores tem um papel determinante no recrutamento de células T CD8+. Curiosamente, a expressão do receptor CXCR3 promove a acumulação de células T CD8+ na ausência de infeção por CMV. Este trabalho representa uma mais valia não só pela caracterização das alterações que ocorrem na glândula salivar após infeção por CMV, mas também pelo estudo do impacto que a infeção tem no recrutamento de células T CD8+ e na formação de TRM. Palavras-chave: Células T residentes; Citomegalovirus (CMV); Glândulas salivares vi ABSTRACT Characterization of CD8+ Tissue-resident memory T cells in the salivary gland and the impact of MCMV infection on T cell recruitment and retention. Tissue-resident memory CD8+ T cells (TRM) are crucial members of the adaptive immune system in different organs. These cells function as patrollers and first-responders of the immune response in organs such as the skin, lungs, digestive system and the brain. Due to the long-lasting persistence and the prompt effector ability of the TRM cells within the residing tissues, promoting these cells in barrier sites and organs that permit viral replication can significantly impact the pathogenesis of infection and the resulting disease. Consequently, TRM cells are interesting cells to study in the context of a life-long latent infection such as the one caused by Cytomegalovirus (CMV). However, to take advantage of the potential role of these cells it is vital to understand the mechanisms involved in TRM cells differentiation and residency. Besides describing the changes in the salivary gland following CMV infection, this work focused on understanding the mechanisms involved in TRM differentiation in the salivary gland, where CMV replicates and spreads through saliva. The results presented here demonstrate that although CMV infection promotes CD8+ T cell recruitment to the salivary gland, TRM, in contrast to most of the other organs, are able to differentiate in the absence of local infection or cognate antigen. This result suggests that the cues involved in TRM differentiation exist in the salivary gland at a steady state which indicates that CD8+ T cells entry in the salivary gland is a crucial step in TRM differentiation in the salivary gland. According to our results, CD8+ T cell migration to the gland is mediated by a4b1 integrin both in infected and uninfected mice. Interestingly, while CMV infection increases the expression of chemokines recognized by the CXCR3 and CCR5 receptors, neither receptor was needed for T cell recruitment to the salivary gland during CMV infection. Surprisingly however, CXCR3 expression promoted the accumulation of CD8+ T cells in the uninfected salivary glands. The novelty of this work relies not only on the characterization of the changes caused by CMV in the salivary gland, but also on the impact of CMV infection in CD8+ T cell recruitment and TRM differentiation. Moreover, unveiling some of the mechanisms involved in CD8+ T cell recruitment to the salivary glands may contribute to the development of preventive and therapeutic approaches to salivary gland-related diseases such as CMV. Keywords: Tissue-resident memory T cells; Cytomegalovirus (CMV); Salivary glands. vii Index Chapter 1 - Introduction 2 1.1.Tissue-resident memory T cells 3 CD8+ T cells 3 Tissue-resident memory T cells, a subset of CD8+ T cells 4 TRM phenotype 7 TRM differentiation 9 Migration of CD8+ T cells to the tissue 9 Rolling/tethering of CD8+ T cell 10 Activation 11 Adhesion phase of CD8+ T cells transendothelial migration 12 Variables that impact CD8+ T cell migration to the tissues 12 In situ signals involved in TRM differentiation 14 Cytokines and chemokines 14 Survival cues for TRM in the tissue 14 The role of antigen and local inflammation in TRM differentiation 15 1.2.Cytomegalovirus 17 CMV burden and disease 17 Routes of CMV infection 18 Immune response to CMV 20 HCMV vaccine 24 1.3.Aims 29 1.4.References 30 Chapter 2 - Experimental work 61 2.1.Abstract 63 2.2.Introduction 64 2.3.Materials and methods 67 2.4.Results 74 2.4.1.Impact of MCMV infection in TRM differentiation 74 2 Introduction 1 3 1.1 Tissue-resident memory T cells CD8+ T cells Early thymocyte progenitors migrate from the bone marrow to the thymus where T cells mature.1,2 After a complex differentiation process, multiple subsets of T cells such as cytotoxic, helper, regulatory, gd and natural-killer (NK) T cells emerge and are characterized by the expression of the T cell receptor (TCR).3–5 These subsets differ not only in function but also in the expression of surface molecules. Simplistically, cytotoxic T cells express the CD8 co-receptor and can directly kill other cells and modulate other branches of the immune response.6,7 Whereas helper T cells express the CD4 co-receptor and orchestrate the adaptive immune responses mainly by cytokines and chemokines production.8 Both subsets are an essential part of the adaptive immune system and are commonly described as CD8+ T or CD4+ T cells according to their expression of co-receptors. As the TCR, both CD8 and CD4 glycoproteins also interact with the major histocompatibility complex (MHC)-I or MHC-II respectively, thus promoting T cell activation, differentiation and function.9–11 CD8+ T cells are particularly relevant in response to intracellular pathogens and since they are the main focus of this work, these cells will be further characterized. During the differentiation of T cells in the thymus, TCR recombination occurs providing a unique specificity to CD8+ T cells that can comprise either a/b or g/d chains.12,13 As for the CD8+ T cell in this work, most of CD8+ T cells have TCRs formed by a/b chains.14 T cells leave the thymus in their naïve form to circulate through the blood, lymph and secondary lymphoid organs until antigen recognition and T cell activation.7,15 T cell activation usually happens in the secondary lymphoid organs and is a complex process that will only be succinctly described. Activation of T cells depends on their specificity and ability to recognize an antigen bound to a MHC, which leads to TCR aggregation and conformational changes that promote intracellular signals and T cell activation.16 Besides TCR-MHC interaction (signal 1), costimulatory signals such as CD28/CD80; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L (signal 2) are necessary for T cell activation and play a role in peripheral tolerance.17–20 Finally, pro-inflammatory cytokines such as type I Interferon (IFN-I) and IL12 (signal 3), among several others, are also engaged in this process that ultimately leads to a robust interaction between cells, signal transduction and consequently CD8+ T cell activation with several transcriptional and functional changes.7,20 As mentioned before, despite their multiple roles, activated CD8+ T cells mostly patrol and kill target cells. This can be achieved in different ways. CD8+ T cells can produce and release cytokines such as interferon gamma (IFN-g) and tumor necrosis factor alpha (TNF-a). TNF-a signal, among others, leads 4 to a caspase mediated apoptosis, while IFN-g, among other things, promotes MHC-I expression, both of which promote destruction of the target cell.21–23 Additionally, direct killing also occurs either by the FasFas ligand interaction between CD8+ T cell and the target cells, or by the action of the highly cytotoxic granules with perforin and granzyme.24–26 The fate of activated CD8+ T cells is very heterogenic and depends on multiple factors.27,28 Although most of the CD8+ T cells undergo apoptosis following most of the acute viral infections, a smaller fraction of cells survive and constitute the memory pool.29,30 All the studies done with CD8+ T cells allowed us to use several markers to better define CD8+ T cells and its subsets. Differences exist between human and rodent CD8+ T cells and their markers. Therefore, it is important to note that all the experiments performed and the CD8+ T cell markers used in this introduction refer to the mouse model, if not otherwise specified. Classically, CD8+ T cells can be simply divided into naïve, short lived effector and memory T cells.31,32 After a viral infection and CD8+ T cell activation, this population is dominated early on by short lived effector cells that are characterized by the expression of the killer cell lectin-like receptor subfamily G member 1 (KLRG1) and their brief lifespan and robust cytotoxic activity32. In contrast, memory T cells survive longer due to homeostatic proliferation, mostly through IL-15 and IL-7 signals, that lead to a higher expression of anti-apoptotic molecules, such as B-cell lymphoma (Bcl) 2.30,33 For most viral infections, the short-lived effector population contracts after the first week, resulting in a dominant memory T cell pool that is usually divided in central memory T cells (TCM) and effector memory T cells (TEM).34,35 TCM (CD62L+, CCR7+ and IL7Rα/CD127+) are highly proliferative and can be found in the blood and in the secondary lymphoid organs such as the spleen and lymph nodes.30,31,36,37 TEM (CD62L-, CCR7-, IL7Rα/CD127+) have higher cytotoxic activity and were thought to be the only and main source of surveillance of non-lymphoid organs.31,37,38 However, the characterization of CD8+ T cells is still a complex topic and the fate of these cells is influenced by a variety of factors such as: cell division; priming conditions; antigen presenting cell; co-stimulation and the cytokine milieu. Thus, from activation to differentiation and survival of CD8+ T cells, the extracellular inputs that modulate CD8+ T cells are extremely diverse, which makes the study of these cells intricate as captivating. Tissue-resident memory T cells, a subset of CD8+ T cells During the last decade, attention was brought to a distinct population of memory CD8+ T cells. These cells did not completely fit the central/effector memory paradigm, since they remained for extended periods of time in the non-lymphoid organs without recirculating. Therefore, they were not in 5 equilibrium with the rest of the memory subsets.39,40 These CD8+ T cells were named Tissue-resident memory T cells (TRM).39,41 TRM are, therefore, long-lived memory cells that reside in the non-lymphoid organs and act as firstresponders in case of re-exposure to pathogens within those organs (illustration 1).42–45 TRM were primarily described in organs such as the skin, digestive tract, lungs, and reproductive tract, which elicited the idea that these cells would have a key role in protecting barrier tissues.44,46–50 Interestingly, TRM have also been shown to be present in multiple other organs such as kidneys, liver, heart, brain, and salivary gland.41,44,49,51– 61 |Illustration 1 – Memory CD8+ T cells’ subsets and tissue distribution. Memory CD8+ T cells are divided in multiple subsets. Central memory T cells (TCM) have higher proliferative capacity and preferentially home to secondary lymphoid organs. Effector memory T cells (TEM) present high cytotoxic activity and are frequently found in circulation and in non-lymphoid organs. The tissueresident memory T cells (TRM) is the most recently described subset that mostly remains in the nonlymphoid organs and present limited recirculation [illustration adapted from the works of Smith et al. (2015) and Mueller et al. (2016)59,62]. | 6 The localization and the effector ability of TRM were early indicators that these cells could be crucial for a prompt immune response in sites of pathogen entry and infection. It has been shown that in most tissues TRM have a protective response to a wide variety of infections.45 TRM confer protection from viral, bacterial and parasitic infections, which highlights the essential role of TRM in tissue immunity.39,48,63–66 Besides the local TRM differentiation, Kadoki et al . (2017), suggested that IFN-I production after infection can promote TRM seeding to multiple organs allowing for local and distant protection.67 The protective role of CD8+ T cells relies on their effector function characterized by the cytotoxic elimination of cells as well as cytokine production.7,68 These functions are shared by the TRM and appear to be enhanced in this subset of CD8+ T cells. TRM also have a faster recall response that can be explained by the robust production of granzyme b, TNF-a, and IFN-g.65,68,69 After antigen recognition in the tissues, TRM can also mediate a faster recruitment of multiple unstimulated memory T cells through IFN-g signaling.50 Therefore, it is thought that re-exposure of antigen in the tissue results in an inflammatory milieu and TRM activation that, in turn, leads to a prompt cytotoxic response limiting pathogen replication and spread.50 This initiate an alarm signal that further enhances the local immune response. Due to their immune roles and long persistence in the tissues, it becomes interesting to study TRM in organs that are susceptible to multiple and frequent infections such as the lungs, where these cells might be relevant upon re-stimulation.63,66 Similarly, TRM are fascinating immune mediators in latent viral infections with reactivation potential, such as herpesvirus infections, that can benefit from an early in situ immune response.43,70,71 The fast response within the tissues and the ability to early control and limit infection has driven new efforts to promote these cells upon vaccination.72–74 Zens et al. (2016), demonstrated that vaccineinduced TRM were able to protect and reduce morbidity after an influenza challenge.66 Indeed, understanding the mechanism involved in TRM differentiation in different tissues is necessary to optimize their tissue homing and residence, which is crucial to consider in a vaccine design. Remarkably, cells that share TRM phenotype (CD69+ CD103+ CD49a+) have also been described in tumors and due to their long life span, localization, and cytotoxic activity, TRM have become an exciting topic in antitumor immunity.75–77 In fact, TRM have been associated with increased survival in breast; ovarian and lung cancer.74,78–81 On the other hand, the prompt response of these cells can lead to exacerbated immune responses. Therefore, it is not surprising that TRM have also been linked to autoimmune diseases such as psoriasis and vitiligo.82–84 7 TRM phenotype Maintenance in the tissues is the hallmark of TRM. Initially, to effectively validate TRM residency, parabiosis techniques were used.48,85 However, parabiosis experiments are laborious and challenging.86 As such, with further phenotypic characterization of these cells, the expression of surface molecules as TRM markers in some tissues (e.g. CD69+, CD103+, CD49a+) became a convenient way to define likely TRM populations without the need for parabiosis.45,62,87 Most TRM express CD69, a C lectin type which, although not exclusive of TRM, is an important marker.44,62,88,89 Since it can be found in other immune cells such as NK, dendritic cells (DC) and effector CD8+ T cells, the use of this marker to analyze TRM requires a careful exclusion of such populations.90,91 T cell activation and downregulation of Krüppel-like factor 2 (KLF2) are inducers of CD69 expression.54,90 Upregulation of CD69 expression promotes Sphingosine-1-phosphate (S1P) receptor internalization, resulting in diminished signaling and, thereby, decreased tissue egress.54,92 Actually, the lack of CD69 or the maintenance of the S1P signal results in a diminished TRM population in most organs studied such as skin, lungs and the salivary gland.54,93,94 Another common TRM marker (not exclusive to TRM) is the αE chain of the αEβ7 integrin also known as CD103. Although less universally associated with TRM than the CD69 marker, CD103 is expressed in TRM in the lungs, skin, brain, gut and the salivary glands.46,48,49,53,59–61,63,93,95–97 Most studies have shown that transforming growth factor beta (TGF-b) signaling promotes CD103 expression on CD8+ T cells, however, other stimuli, such as IL-15 in the skin or CCR9 in the gut, have produced the same effect.59,93,98–100 The αEβ7 integrin expressed by T cells can bind to E-cadherin expressed on epithelial cells.101 This allows tethering to occur, which has been described as the main role of CD103 on TRM.101 Indeed, the manipulation of CD103 expression has a significant impact on TRM numbers in the skin, brain, gut and lungs.49,53,94,102,103 Nonetheless, Bergsbaken et al. (2015) have shown that this may not be true for all gut compartments, since CD103TRM can be formed in the lamina propria.104 As in the gut, CD103TRM have also been described in the skin.93,105 Besides the gut, E-cadherin is also widely expressed in the epithelial cells of the salivary glands.61106 However, even though TRM in the salivary glands tend to be CD103+, CD103 knockout (KO) did not significantly impact long-term TRM population in this organ.60 Combined, these data suggest that CD103 is not universally crucial for TRM differentiation even in organs enriched in E-cadherin. Some organs such as the brain have poor E-cadherin expression whilst CD103 is still present on TRM, which suggests a different role for CD103 besides tethering.53,107 Several alternative explanations are possible. For instance, alternative aEb7 ligands may exist but remain unidentified in those organs. Alternatively, CD103 expression might tighten the adhesion with antigen- 8 presenting cells (APC), or simply that CD103 expression may be a bystander effect of the TRM differentiation pathway. Therefore, it is important to note that although expressed by TRM in most organs, CD103 is not a universal TRM marker. Another TRM core marker is the CD49a (VLA-1a / a1 chain of integrin a1b1). Its key function is binding to the collagen IV present in the basal membrane of the mucosal epithelium.45 This interaction allows for cells to adhere but also facilitates migration along the collagen within the tissue.108 CD49a has also been shown to impact CD8+ T cell survival and differentiation within the tissue, being important for T cell localization in organs such as the lungs and intestine.39,68,109–111 CD44 has a similar effect since it helps to stabilize the T cells differentiation and has affinity for extracellular matrix components and selectins. Studies suggest that CD44 can associate with integrins (CD49d/VLA-4; a4b1) on the surface of T cells promoting cell survival and T cell homing to the organs.112,113 Although expressed by TRM, CD44 is also detected on effector and memory T cells since its expression usually indicates T cell activation.112,114 Besides the previous markers, TRM in multiple organs such as the brain and lung, also express programmed cell death protein 1 (PD1), a member of the CD28/CTLA-4 (Cytotoxic T-lymphocyte– associated antigen 4) family of inhibitory receptors.45,79,115 PD1 has been classically described in exhausted CD8+ T cells and its expression is induced by TGF-b signaling.75,116 Most of the TRM (especially the CD103+) express this exhaustion marker, surprisingly however they seem to maintain some of their effector abilities.117,118 Although PD1 expression limits T cell activation in some tumor studies, this effect is not universal and the role of PD1 seems to vary according to the organ/conditions.119,120 Work by Campbell et al . (2008) suggests that Murine Cytomegalovirus (MCMV)-specific CD8+ T cells in the salivary gland express PD1 that does not contribute to exhaustion or viral persistence in this organ.121 Similarly, our unpublished data showed that TRM in the salivary gland retain function even though expressing PD1. It is conceivable that the TGF-b signal in the organs promotes both CD103 and PD1 expression on T cells. More work is required to distinguish the role of PD1 in TRM, however, an interesting hypothesis is that PD1 expression modulates the effector function of TRM, possibly as a safety mechanism to avoid an exaggerated immune response.75 In line with mouse models, human TRM also seem to express CD103, CD69, CD49a, PD1, downregulate CD62L, S1P receptor 1, KLF2 and have a distinct transcriptional profile from the TEM.68,87,93,122 However, making extrapolations from animal models should be done with caution since disparities in TRM properties have been described. As an example, the homolog of B lymphocyte-induced maturation protein -1 (Blimp-1) in T cells (Hobit) is upregulated in TRM in mouse studies, which does not seem to happen in 9 human TRM.87,123,124 CD101, that can be expressed by different lymphoid subsets and limits T cell proliferation and function, has been suggested as an additional marker for human CD8+ TRM, while it is associated with regulatory T cells in mice.87 Altogether, a common core has been described for most TRM even though the markers used should be evaluated according to the subject and tissue of interest. As done previously by multiple groups, in this work TRM were characterized using CD69 and CD103 as markers.59–61,63,125,126 TRM differentiation Although evidence suggests that there are cells committed to become TRM, the precursors have not been fully identified. It is known that TRM precursors cells lack KLRG1. Most studies indicate that TRM arise from KLRG1CD127+ common precursors to memory cells, but it is still debatable if TRM precursors can be KLRG1+ effector cells that subsequently lost the KLRG1 expression.93,96,127–130 The TRM differentiation may rely on specific transcriptional cues. In fact, differentiation of different T cell lineages depends on crucial transcription factors. Even at early differentiation stages the presence of transcription factors, such GATA-3 and Notch1, are crucial for the T cell differentiation.131–134 Similarly, runt-related transcription factor 3 (Runx3) promotes the cytotoxic lineage, whereas, Id3 transcription factor T-cell factor 1, eomesodermin (Eomes) and Bcl6 are linked to TCM, while T-bet, Blimp1 and Id2 are more related to TEM differentiation.127,135–139 Likewise, some transcription factors have been linked to mouse TRM differentiation. Interestingly, as for TEM, TRM require Blimp1. Indeed, both Blimp1 and Hobit are crucial for TRM maintenance in the skin, gut and liver, due to suppression of egress genes, at least in mice.123 Moreover, Milner et al . (2017), demonstrated that Runx3 promotes not only TRM differentiation, but also survival.140 Additionally, T-box transcription factors are also important for T cell differentiation being T-bet associated with effector cells, while Eomes is linked to memory cells.127,141 Interestingly, TRM tend to be negative/low for these T-box transcription factors.142–144 The low expression of T-bet/Eomes allows for upregulation of TGF-b signaling and CD103 expression.145 Although a single master transcription factor has not yet been identified for the TRM lineage, TRM tend to express transcription factors that partially overlap with both TEM and TCM, which is in line with their characteristics and might explain their effector functions and survival abilities. Migration of CD8+ T cells to the tissue Although the TRM precursors have not yet been clearly identified, it is thought that most of TRM differentiate from KLG1CD8+ T cells within the residing tissue. Therefore, migration to and within the 10 organs are essential steps in TRM differentiation. Migration of CD8+ T cells is dependent on the homing receptors expressed on their surface and respective ligands found in the vasculature and tissues. Although CD8+ T cell migration from the blood to the extravascular compartment is a complex process, it can be divided in four essential steps: 1) rolling/tethering, 2) activation, 3) adhesion and 4) diapedesis, in which selectins, chemokines and integrins are the main mediators (Illustration 2).146–148 |Illustration 2 – Transendothelial migration. Representation of the steps involved in leukocyte extravasation into tissues: 1) Rolling/tethering, 2) activation, 3) adhesion and 4) diapedesis. The transendothelial migration process is regulated by different adhesion molecules, both in the leukocytes and endothelial cells. These mediators can change depending on the tissue and the inflammatory condition. Although not limited to each step, for simplification, examples of selectins (represented in blue in the illustration), chemokines (represented in green) and integrins (represented in pink) are shown. | Rolling/tethering of CD8+ T cell The migration process starts with the rolling and tethering of the CD8+ T cells to the endothelial wall. This step is dependent on selectins that can be either expressed by the T cells, as the L-selectin (CD62L), or by the endothelial surface of the vasculature generally induced by inflammatory cytokines.149,150 P and E selectins are examples of this last group and can be recognized by the P selectin glycoprotein-1, E-selectin ligand-1, CD44 and CD43 on T cells.151–154 The skin is representative of this mechanism where inflammation induces the expression of E and P selectins in the post capillary venules that then bind to 11 P selectin glycoprotein-1, CD43 and CD44 facilitating the CD8+ T cell recruitment.152,155–157 In fact, mice deficient in E, P and L selectin-ligands show impaired recruitment of T cells to the skin.158 Activation The resulting rolling motion created by the action of selectins allows for chemokines to bind the seven-transmembrane cell surface G-protein-coupled chemokine receptors expressed by CD8+ T cells allowing for intracellular signaling.159,160 Chemokines were classically divided based on their function: pro-Inflammatory, produced as consequence of infection, inflammation, or by tumor cells (e.g. CCL2-CCL5, CCL11, CCL13, CXCL1CXCL8, CX3CL1), or homeostatic chemokines that regulate cell migration during the development or maintenance of the tissue (e.g. CCL18, CCL19, CCL21, CXCL12, CXCL13). However, due to the mixed function of some chemokines (such as CXCL9-CXCL11, CXCL16, CCL1, CCL17, CCL22, CCL25), chemokines are now more commonly organized by structure, according to their first cysteine residue (C, CC, CXC, CX3C).161–163 Chemokines can be produced by the endothelial cells of the vasculature or within the adjacent tissue.161,164 Infection and inflammatory states lead to an increased and specific production of chemokines by immune cells, such as macrophages and DC in the tissue, which leads to T cell recruitment.163,165–168 Thus, cells within the tissue can also modulate the migration pathways of new immune cells. The interaction of the chemokine receptor with its cognate chemokine ligand promotes transmigration while inducing several signaling pathways that modulate polarization of the cell, actin reorganization, and gene transcription.169–171 Moreover, chemokines are not only important for cellular recruitment but also for localization of the cells once in the organ. The study of homing molecules is essential in understanding TRM differentiation and has been gathering increasing attention. The chemokines involved in T cell homing are widely tissue-specific. Recently, it has been described that CXCL17-CXCR8 interaction mediates migration of TEM and TRM to the vaginal mucosa after herpes infection.172 Similarly, CCL27 expressed by keratenocytes and CCL17 by endothelial cells in the skin, mediate T cell recruitment through CCR10 and CCR4 expression on T cells.173,174 Moreover, CXCR3, CCR5, CCR3, CCR4 and CCR8 have also been implicated in T cell migration to the skin, which demonstrates the promiscuous and complex nature of the chemokine/receptor expression within the different organs and T cells.175–179 This complexity can even occur within the same organ which is further exemplified in the gut where most CD8+ T cells of the small intestine express CCR9, which correlates with the constitutive expression of its ligand (CCL25) by the epithelial cells in this portion 18 CMV is also a major complication of hematopoietic stem cell transplantation (HSCT).251–253 After allogenic HSCT, the rate of CMV recurrence in seropositive patients varies according to the study, but a median value of 37% has been reported.254 HCMV infection also modulates the immune system by altering human leukocyte antigen (HLA) expression, cytokine production, and adherence molecules, which also potentiates the increased risk of secondary bacterial and fungal infections in transplanted patients.253,255–257 Another critical population is pregnant women, in which the vertical HCMV transmission can severely impact the newborn. In fact, HCMV is the leading cause of viral congenital infection (around 0.6% of live births), although these values vary according to the seroprevalence and the characteristics of the population.258,259 Around 10-15% of the infected newborns are symptomatic at birth and similar frequencies of the asymptomatic babies develop neurological sequelae or hearing loss.259–261 In fact, congenital HCMV infection is still the leading congenic cause of hearing loss.259,262,263 Even with asymptomatic infections, HCMV has also been linked to poor response to vaccines, immunosenescence and increased mortality in elderly.264–267 Moreover, the role of HCMV in diseases such as heart disease and atherosclerosis is being studied.268,269 Besides the significant silent burden of infection, HCMV is still an important cause of morbidity and mortality, which is the reason that a HCMV-vaccine is considered a priority. Routes of CMV infection HCMV is most commonly transmitted by contact with body fluids such as breastmilk, sexual contact and saliva but also by placental transfer and solid-organ transplantation (Illustration 3).240,241,270,271 Interestingly, CMV replicates in the epithelial acinar cells of the salivary gland for an extended period and MCMV transcripts can be found in the salivary gland up to 7 weeks after infection.59 In fact, infected infants can shed HCMV in their saliva for over a year, hence promoting horizontal transmission.271–273 The salivary glands are also sites of CMV latency all of which contribute to saliva being a major route of shedding and emphasizes the importance of the salivary gland in CMV infections.274,275 Therefore, understanding the immune response in the target mucosal sites of viral replication and shedding such as the salivary gland is crucial for the development of new therapeutic and preventive strategies. Equally to HCMV, MCMV transmission occurs naturally through the same routes apart from the transplacental vertical transmission in immunocompetent mice.276,277 Regardless of the similarities, most of the MCMV literature uses intravenous (i.v.) or intraperitoneal (i.p.) routes, which poorly represent the natural route of transmission in this model. Both i.p. and i.v. routes result in a significant direct access 19 to the blood and dissemination of the virus and consequently a bypass of the mucosal surfaces, which can modulate the resulting immune response.278 Early after an i.p. infection, the virus infects the subcapsular macrophages of the mediastinal lymph nodes, followed by hematogenous spread to the liver and marginal zone of spleen.278 Infection of the bone marrow was also described at early time points, however, other organs as the salivary gland are infected at later time points partially due to a monocyteassociated viremia.279–281 New efforts have been made to use animal models that mimic more natural routes of transmission. Along those lines, the footpad inoculation (f.p.) mimics transmission through a mouse bite, wound or grooming.282 Differences in MCMV-specific CD8+ T cell accumulation following i.p. or f.p. infection have been seen.283 For instance, after f.p. infection the patrolling monocytes are recruited to the site of infection and play a critical role in viral dissemination to other organs as the salivary gland.282 The limited direct viremia following the f.p. infection, due to a bottleneck effect caused by infection of CD169+ subcapsular sinus macrophages where MCMV viral replication is poorly supported, may also impact the resulting pathogenesis and the immune response.284 As stated before, saliva is a major transmission vehicle for CMV infection, oral and intranasal (i.n.) infections, have also been suggested to resemble inhaled or ingested virions.229,285 However, CMV is an enveloped virus and does not resist the stomach acid.286 Thus, gastrointestinal infection is less likely than oropharyngeal infection.286,287 In fact, a respiratory tract infection is thought to be a predominant mode of transmission in captive mice, resulting in a direct infection of the nasal mucosa and the olfactory neurons.285 Following an i.n. inoculation, the virus is confined to the nasal area for the first 2 days in young Balb/c mice, where olfactory neurons have been shown to be primary targets.231,285 Oduro et al. (2016) showed that the lungs of Balb/c mice can also be infected early after infection and these differences might be partially dependent on the volume, titer, and method of administration.286 Farrell et al. (2017), showed that CD11+ DC are responsible for spreading the virus to the mesenteric lymph nodes and later to the salivary gland.288 As for the i.p. route, monocytes are crucial for viral spread to the salivary gland, where CMV remains detectable for up to a month.231,281,285,286,289,290 Differences between the immune response following the i.n. infection and the “classical” i.p. infection were also reported. The CD8+ T cell response after an i.n. infection was reduced in comparison to an i.p. infection, which may be related to differences in the viral titers that are lower after the i.n. infection in comparison to the i.p. infection.286,291 However, a large number of certain CMV-specific TEM (inflationary responses) were still detected and maintained after an i.n. inoculation in the blood and spleen.286,291,292 An acute presence of antigens in barrier and mucosal 20 tissues are often avoided by the use of systemic infections. Notably however, the infection of these barrier tissues correlates better with natural viral infections. The first line of immune defense is within these barrier tissues, which impacts not only the local but also the following systemic immune response as exemplified by the role of TRM in multiple infections. The route of infection is therefore a crucial determinant for the resulting immune response and protection, especially in barrier tissues. |Illustration 3 – HCMV structure, life cycle and routes of infection. HCMV is a double-stranded DNA virus with an icosahedral capsid surrounded by a proteinaceous tegument and an outer envelope enriched with glycoproteins. These glycoproteins allow membrane fusion and the release of both tegument proteins and nucleocapsids into the host cell. The nucleocapsid is translocated to the cell nucleus, where the genome is released initiating the cascade expression of viral genes. Newly synthetized DNA is encapsulated and transported to the cytoplasm, where it is trafficked to the viral assembly complex for further envelopment. Exocytosis of newly formed infectious virions promotes viral spread and transmission that can occur through multiple routes as the ones represented on the right. [illustration adapted from the work of Crough et al. (2009) and Beltran et al. (2015) 240,293]. | Immune response to CMV CMV infects a broad variety of cells from fibroblast, monocytes, epithelial and endothelial cells to myocytes.294,295 CMV enters the cells either by the endocytic pathway or by direct fusion using mostly the glycoproteins L and H (gL and gH) on its surface.296 Once in the nucleus, viral replication occurs in a Glycoprotein complex Nucleocapsid Tegument Genome Transplants/Transfusions Airborne Sexual contact Urine Saliva Breastfeeding At birth 21 programmed cascade: Immediate early (IE), early and late genes.297–299 IE genes encode proteins in the first 2 hours after infection that are mostly related to the transcription of the subsequent genes.300–302 During the first 24 hours, Early genes give rise to proteins that, in combination with the IE genes, are important for the induction of several promoters involved in DNA synthesis.303,304 Late genes are generally responsible for structural proteins and are involved in the formation of virus particles.304 This cascade of protein expression is relevant not only to understand CMV pathology, but also the resulting immune response. CMV acute infection triggers a robust immune response. The innate immune system functions as the primary response to infection, being important for the acute CMV control especially in neonatal infections due to the reduced CD4+ T cell response and immune system immaturity.305,306 The recognition of the virus can be mediated by pathogen-associated molecular patterns (PAMPs), including glycoproteins from CMV surface, such as glycoprotein b (gB) and gH, and viral DNA. These viral PAMPs are recognized by Tolllike receptors (TLRs) and by stimulator of interferon genes (STING), which leads to cytokine production and activation of some of the main mediators of the innate immune response to CMV, like innate lymphoid cells 1 and NK.307–309 Both group 1 innate lymphoid cells and NK, produce antiviral cytokines such as IFNg acutely after infection.310,311 NK produce perforins and granzymes as well, allowing for their cytotoxic activity.312 Although NK have direct effects on infected cells, they are thought to also mediate a bridge between innate and adaptive immune system.313 The role of NK is clearly highlighted in the MCMV model, in which the susceptibility to the virus is determined. C57BL/6 mice (B6) express the Ly49H receptor that recognizes the MCMV m157 protein, which induces a NK response allowing this to be a MCMVresistant strain.314,315 The lack of Ly49H receptor is the explanation for the susceptibility seen in the Balb/c strain.314 The activating and inhibitory killer immunoglobulin-like receptors (aKIR and iKIR) are expressed by NK in humans and are specific for MHC class I molecule.316 Similarly to Ly49H molecules, killer immunoglobulin-like receptors are also important for destruction of infected cells and virus control.317–319 Although the redundancy is presumed to exist, given the preponderant number of these receptors, rare genetic abnormities that lead to overexpression of these iKIR have been described and can result in recurrent episodes of HCMV disease.320 Moreover, NK are able to confer protection to HCMV after transplantation, while the lack thereof increases the susceptibility to infection.321,322 The importance of the NK response can also be estimated by the immune evasion mechanisms that MCMV has developed to avoid NK recognition. These include downregulation of activating receptors in NK such as NKG2D.323 Moreover, CMV encodes an IL-10 homolog, which results in a decreased pool of NK and multiple deficits in the adaptive immune response.256,324,325 Although NK and group 1 innate lymphoid cells are essential for a rapid response against CMV, help arises from other immune cells. CMV 22 recognition prompts a robust humoral response usually targeted to structural tegument proteins and envelope glycoproteins.326–328 Although antibodies are not crucial for acute viral infection control, MCMVspecific antibodies have been shown to prevent viral dissemination and virus reactivation.329–331 Although the acute immune response to MCMV is a combined effort from several branches of the immune system, T cells are essential for the control of the infection.126 CD4+ T cells are important mediators of acute immune response and accomplish antiviral activity partially due to IFN-g and TNF-a production.332,333 Cytolytic CD4+ T cells specific for CMV are also evident in HCMV-infected people.17,334,335 In fact, reduced CD4+ T cells results in increased HCMV shedding in children possibly through the loss of these direct anti-viral functions of CD4+ T cells.336,337 However, CD4+ T cells also promote the function of other immune cells, as the CD8+ T cells, and it is still unclear whether and how CD4+ T cells help may contribute to CD8+ T cells function after CMV infection.338–340 CMV induces a robust CD8+ T cell response with some CMV-specific T cells remaining active at an increased number even at later time points. This process is called memory inflation and reflects the accumulation of T cells with certain specificities.341–343 In B6 mice, CD8+ T cells specific for the M38316epitope are one of the best studied examples of T cells that undergo memory inflation.344,345 In contrast, in their response to the M45-epitope, among others, T cells behave like classical anti-viral T cells and the population decreases after the first week of infection.343,344 The differences between these responses are correlated with the generation of the epitopes. The M45 epitope is dependent on the immunoproteasome, which is less available after the acute infection.346 In contrast, the M38316-epitope can be processed by the constitutive proteasome and therefore presented during latency.346 Memory inflation is also seen in humans where approximately 10% of the memory T cells are HCMV-specific and can dominate the CD8+ T cell repertoire.347 This increased pool of MCMV-specific CD8+ T cells is a response to the sporadic reactivation events that occur in the endothelial cells of the vasculature.343 Cytotoxic activity and IFN-g production by CD8+ T cells are essential to acute MCMV control.348–350 Indeed, CD8+ T cell transfers prevent pathology and confer protection to lethal MCMV infection in immune compromised mice.351–354 Similarly in humans, HCMV-specific CD8+ T cell transfer conferred protection against reactivation.355–358 Viral control is therefore largely dependent on CD8+ T cells in most organs. However, the salivary gland stands as the exception.121,359,360 As suggested before, the salivary glands are crucial for CMV infection. The acinar glandular epithelial cells of the salivary gland are infected 3-6 days after systemic MCMV infection which results in local viral replication, viral shedding and latency.59,361 23 After MCMV infection, the salivary glands are flooded with DC, gd, CD4+ and CD8+ T cells.361 This migration of immune cells to the salivary gland, especially lymphocytes, results in increase of cytokine production, being that the infected salivary glands are enriched for IL-10, IFN-g, CCL3, CCL4 and CCL5 in comparison to the secondary lymphoid organs.361 However, the immune response in the salivary gland is less efficient than the other infected organs, allowing for most of the acute viral replication to occur for a prolonged time.121 The peculiar immune response within this organ may be the explanation. Besides the low rate recruitment of NK to the salivary gland after MCMV infection, the NK within the salivary gland show a delayed activation and are hyporeactive to the activating receptor link and cytokines´ stimuli in comparison to other NK population seen in spleen and liver, which in the end results in a humble IFN-g response.91 Additionally, in contrast to what happens in the other organs, CD8+ T cells are unable to control viral replication in the salivary glands.360,362 Viral infection of the salivary glands induces a downregulation of MHC-I on the epithelial cells thus preventing CD8+ T cells recognition of the infected cells.360 Indeed, CD8+ T cells are able to control viral infection in the salivary gland in the absence of the immune evasion genes (m04/m06/m152).360 Moreover, there is a lack of cross-presenting APC cells in the salivary gland, although antigen processing and presentation in MHC-II context still happens.360,363 Additionally, CD4+ T cell responses in the gland are also impaired in comparison to the periphery. In contrast to the blood and lymphoid organs, where the CD4+ T cell response can be detected in the first week of infection and is followed by contraction, CD4+ T cells reach the salivary glands with some delay and peak after the first week.364 Moreover, increased levels of IL-10 in the gland also reduce IFN-g production by T cells.365 These mechanisms help explain the immune evasion that leads to the prolonged MCMV replication and latency in the salivary glands.274 Acute viral infection is followed by viral latency. In spite of the fact that, by definition, infectious viruses are not detectable in this latent phase, viral DNA has been detected in cells such as the hematopoietic cells and multiple organs such as the spleen, heart, kidneys, lungs and the salivary glands.280,366–369 Nonetheless, the detection of low levels of DNA can be challenging and therefore the characterization of latent sites remains as an open question. Notably, due to sporadic viral reactivations, continued immune surveillance is required to keep the virus from causing disease.370,371 During latency, viral transcription occurs with IE1 mRNA being one of the first mRNAs detected.371 This does not equal a full reactivation with the production of new infectious viruses since even in the presence of transcripts there are multiple checkpoints that prevent a complete productive cycle.372 An open chromatin structure of the major IE and the differential splicing that allow for IE3 mRNA and the subsequent protein are important for the early gene transcription, but not the only checkpoints.373–375 Moreover, the occurrence of 24 gene desilencing results in viral transcription that allows presentation of antigenic peptides and reexposure of antigen to the T cells, which led to the immune sensing hypothesis of latency control.370,371,376– 380 Contrary to the acute infection, neutralizing antibodies are crucial at preventing viral reactivation both systemically as in the salivary glands at latent times.329,381 Moreover, the use of B-cell depleted animal models allowed for high reactivation rates upon deficiency of NK, CD4+ T cells and CD8+ T cells.370 It is likely that NK and T cells have a redundant role, since the isolated defect of one of these subsets is not enough to produce viral particles even in a B-cell depleted model.370 Interestingly however, CD8+ T cells seem to have a more predominant role in preventing MCMV reactivation and viral shedding than CD4+ T cells, even in the salivary glands.370,376 These data suggest that CMV-specific TRM could have an important role in controlling CMV reactivation in the salivary gland. HCMV vaccine Since the late 1970s, effort has been made to develop a HCMV vaccine.382 A HCMV vaccine could have a tremendous impact, especially in solid organ and hematopoietic stem cell transplant recipients, preventing viremia and disease. Moreover, women of childbearing age can be another target since a primary infection or re-infection/reactivation can have a severe impact during pregnancy. Although the seropositive-status of the mother is not sufficient to confer protection to congenital infection, it seems that maternal antibody and T cell responses are beneficial in limiting transmission and disease.383–385 The immune response that needs to be induced by vaccination to confer protection is still debatable. Neutralizing antibodies are important in the case of solid organ transplants, while T cells have been more associated to protection in hematopoietic stem cell transplants.386,387 In cases of reactivation, while neutralizing antibodies have been frequently associated with virus control, this is not universal.388–393 In fact, neutralizing antibodies seem to have a limited role in cell-to-cell transmission.390 Therefore, the immune response responsible for controlling the viral spread requires further analysis. Furthermore, some vaccination strategies are able to reduce viral shedding in saliva, independently of the neutralizing antibody titers, which also have been shown to be important in preventing mortality in mouse studies and contribute to HCMV protection.394,395 This suggests that another branch of the immune system has impact in shedding. CD4+ T cells have also been associated with protection and studied in vaccine scenarios.383,396 CD8+ T cells may also play a critical role in CMV protection and pp65 and IE1 are commonly studied targets.397 Although several vaccine candidates induced substantial CD8+ T cell responses, the immune 25 dominance of the response does not seem to be essential for protection since subdominant responses have elicited protection in mice.398,399 However, more studies are needed to see if the same applies in humans. It is interesting that some of these findings can change according to the vaccine strategies used, which may suggest that the immune responses, including the TRM, vary according to the specificity, priming conditions and the presence of other immune cells in the gland.400 All of which increase the complexity in developing a successful HCMV vaccine. So far, most of the vaccine strategies used fall under one of these categories: replication-deficient virus/attenuated vaccines; recombinant vaccines; vectored vaccines; DNA vaccines; RNA vaccines; peptide vaccines. Replication deficient/Attenuated vaccines Several strains have been used to develop attenuated vaccines such as AD169, Town and Toledo able to confer protection in specific scenarios. However, some of these approaches failed to elicit enough neutralizing antibody titers.401,402 One of the greatest advantages of this strategy is that it allows the induction of different branches of the immune system from neutralizing antibodies to cytotoxic T cells. However, concern exists about establishment of infection, latency and the development of conditions that may be associated with CMV, such as atherosclerosis. The V160 vaccine developed by Merk is an interesting vaccine candidate that is now starting phase II trials. The V160 is a recombinant vaccine from an attenuated strain (AD169). However, differently from other attenuated vaccines that lack the pentameric complex, its expression was restored in the V160 vaccine which allowed for increased levels of neutralizing antibodies.403,404 In fact, neutralizing antibodies and cellular mediated immune responses are comparable to natural CMV infection.404 Recombinant vaccines Vaccines using the gB subunit are probably the most tested vaccine so far, and demonstrated some efficacy preventing primary HCMV infection in women as well as a reduction of both viremia and time of ganciclovir treatment in transplanted patients.386,405,406 Vaccines expressing gB result in a robust neutralizing antibody response, similarly to the UL128/UL130/UL131a proteins, that interact with the gH/gL heterodimer forming the HCMV pentameric complex needed to enter epithelial cells.407–410 26 Vectored vaccines These vaccines rely on heterologous viral vectors, such as Canarypox, lymphocytic choriomeningitis virus (LCMV), Venezuelan equine encephalitis and Vaccinia virus (VACV) that deliver CMV immunogens, mostly gB, pp65 and/or IE1.411–414 DNA plasmid DNA vaccines consist of plasmid preparations containing DNA sequences that induce an immune response against a pathogen.387,415 An example is the ASP0113 vaccine candidate, which is a DNA-plasmid that targets both gB and pp65.416 This approach reduced CMV viremia and reduced use of antiviral drugs following hematopoietic cell transplantation.416 RNA vaccines These vaccine strategies use mRNA to elicit a robust humoral and T-cell immunity. Interestingly, these mRNA can be used to combine multiple target in a single vaccine. This was tested and surprisingly the combination of epitopes led to competition of the resulting T cell responses, which might be a challenge in designing a T cell-targeted HCMV vaccine.417 However, this can probably be overcome with co-dominant epitopes combined in a single vaccine or a combination of multiple inoculations. |Table 1: HCMV vaccines in enrollment or active development according to the National Institute of Health - clinicaltrials.gov (accessed on 26/04/19). | Type Details Phase Sponsor mRNA vaccines mRNA-1647 (gB) mRNA-1443 (pp65) I ModernaTX, Inc. Replication deficient vaccines V160 I/II Merck Sharp & Dohme Corp Vectored vaccines Multi-antigen CMV-Modified Vaccinia Ankara Vaccine I/ II City of Hope Medical Center HB-101 II Hookipa Biotech Peptide vaccines Multi-peptide CMV-Modified Vaccinia Ankara Vaccine II City of Hope Medical Center Tetanus-CMV fusion peptide vaccine I City of Hope Medical Center CMVpp65-A*0201 peptide vaccine II City of Hope Medical Center DNA vaccines ASP0113 II/III Astellas Pharma Global Development, Inc BD03 I SL VAXiGEN 27 Developing a HCMV vaccine has been a challenging goal. The species-specificity of HCMV represents a limitation to pre-clinical trials in vaccine development, which is one of the many difficulties that has been assumed for the lack/diminished efficacy in some clinical trials.418,419 The failure in inducing enough neutralizing antibodies titers, their limited binding capacity and defining the protective antibody level justify some of the disappointing results.394,420 Moreover, the inability to induce a neutralizingindependent antibody response (e.g. antibody-dependent cellular cytotoxicity/phagocytosis – ADCC/ADCP) can be another limiting factor.395,421,422 The lack of T cell response is another possible justification, which is aggravated by the difficulty in covering the antigen diversity both within and between hosts.423–426 These factors add to the complexity of designing a HCMV vaccine, which will remain intricate until the protective immune response and the immunogens that elicit a robust response are identified and characterized. 34 protection against pulmonary virus infection. 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Limits and patterns of cytomegalovirus genomic diversity in humans. PNAS 112, 4120–4128 (2015). 66 a mechanism for the surprisingly efficient recruitment of activated T cells to salivary glands of mice with no local infection. 67 2.3 Materials and Methods Mice and infections All mice were purchased from the Jackson laboratory and bred in house. B6 C57BL/6 (B6), B6.SJL-PtprcaPepcb/BoyJ (CD45.1), B6.PL-Thy1a/CyJ (Thy1.1) and B6.129S7-Ifngtm1Ts/J (IFN-γ KO) mice were used as recipients in the adoptive transfer experiments and to assess chemokine expression in the salivary gland. OT-Is on a B6 background [C57BL/6-Tg(TcraTcrb)1100Mjb/J] were bred to CD45.1, CXCR3 KO mice (B6.129P2-Cxcr3tm1Dgen/J) and CCR5 KO mice (B6.129P2-Ccr5tm1Kuz/J) to generate congenic CXCR3 KO or CCR5 KO OT-I mice. MCMV-K181 virus (kindly provided by Ed Mocarski) was used in figures 1, 3, 4, 5A-7, figure 9F. MCMV-SL8-015 or MCMV-K181-trf-Ova (MCMV-Ova), have been previously described and are recombinant viruses that express ovalbumin (Ova). 34,35 These viruses were used in figure 8, figure 9A-D and to expand OT-I T cells after the first adoptive transfer on figure 5. OT-I T cells are CD8+ T cells specific for the Ova peptide SIINFEKL. For the experiments in figure 2 MCMV-TK virus was used. MCMV-TK virus is a recombinant virus where the m157 gene was replaced by the thymidine kinase (TK) gene derived from HSV-1, allowing for the replication of this virus to be blocked with Acyclovir or Famcyclovir treatments, since these act as DNA chain terminator once phosphorylated by the TK.36 Briefly 2mg/mL of Famcyclovir was mixed with the drinking water of the treated mice starting 3 days before MCMV-TK intraperitoneal (i.p.) infection. Famcyclovir was replaced every other day until sacrifice. The control groups, with replicative MCMV-TK infection, were given water with no Famcyclovir treatment. Mice were sacrificed at 7, 4 or 28 days after infection, organs were collected and lymphocyte isolation and FACS staining performed as described below. Infections were performed using 2x105 pfu and the i.p. route (100 µl per injection) in all experiments except figure 1, where some of the infections were performed via intranasal (i.n.) or footpad (f.p.) routes in a total volume of 20-25 µL per inoculation after anesthesia with Isoflurane. MCMV-K181, MCMV-Ova and MCMV-SL8-015 were produced as described in [35–38]. All protocols were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. Lymphocyte isolation and FACS Staining For all the experiments, except for the experiment represented in figure 2 and sup. figure 2, intravenous (i.v.) antibody injections were performed, as described in [39,40] without perfusion, to distinguish 68 between vasculature-localized (i.v.+) and parenchyma-localized (i.v.-) CD8+ T cells. In brief, mice were injected intravenously with 3 µg of an anti-CD8α antibody (clone: 53-6.7 conjugated to BV650 or BV421) 3 minutes before sacrifice. Blood was collected from the retro-orbital sinus or from the chest cavity, after cutting the pulmonary vein at sacrifice and the organs were collected in media containing an unlabeled CD8α antibody (clone: 53-6.7). Total CD8+ T cells were identified by CD8β staining (± CD8α of the intravascular portion) during the phenotypic analyses as described below. Lymphocytes from the blood, spleen, inguinal lymph nodes, sub-mandibular salivary glands (referred to as "salivary glands" throughout), kidneys and lungs were isolated as described previously [40] with minimal modifications. Briefly, the mucosal organs were minced using the gentleMACS Dissociator (Miltenyi Biotec) and incubated at 37°C for 1-1.5 hours in digestion media containing 1 mg/ml collagenase type IV, 5 mM CaCl2, 50 mg/ml DNase I, and 10% FBS in RPMI. Salivary glands were suspended in 40% Percoll and overlayed on top of a 75% Percoll layer, while the kidneys and lungs were suspended in 40% Percoll. Suspensions were centrifuged at 600 xg for 25-30 minutes and the lymphocytes were collected from the 75/40 interface (salivary glands) or pellets (kidney and lung). Phenotypic analyses of T cells were performed using the following antibodies: CD8α (clone: 536.7); CD8β (YTS156.7.7); CD69 (clone: H1.2F3); CD103 (clone: 2E7); CD44 (clone: IM7); Killer cell lectin-like receptor subfamily G member 1 (KLRG1) (clone: 2F1); CXCR3 (clone: CXCR3-173); CXCR4 (clone: L276F12); CXCR6 (clone: SA051D1); CX3CR1 (clone: SA011F11); CD4 (clone:RM4-4). OT-Is were identified by their congenic markers with CD45.1 (clone: A20) and/or CD45.2 (clone: 104) and by the T cell receptor (TCR) chains Va2 (clone: B20.1) and Vb5 (clone: MR9-4). In figure 10E (exp. 2) donor cells were identified also by the Thy1.2 marker (clone: 30-H12). All antibodies were purchased from Biolegend or BD Bioscience. All MHC-tetramers, loaded with peptides from M38, were provided by the National Institutes of Health Tetramer Core Facility (http://tetramer.yerkes.emory.edu/) and used as described[41]. A tetramer loaded with the B8R peptide derived from Vaccinia virus was used as a negative control for the tetramer staining following a MCMV-infection. All samples were collected on a BD LSRFortessa or LSR II flow cytometer and analyzed using FlowJo software (TReeStar). The gating strategy is represented in the sup. figure 1. In vitro T cell activation and expansion OT-Is were activated in vitro based on the protocol described [12] with modifications. Briefly, splenocytes from OT-I mice were harvested and 4x106 cells/mL were cultured with 1 µg/mL of the SIINFEKL peptide for 2 days. On the second day the cells were resuspended to 5x105 cells/mL and 69 incubated with 0.03 U/mL of IL-2 that was renewed every 2 days, for a total of 4-5 days, until the adoptive transfer. Adoptive transfers All the adoptive transfers were performed via retro-orbital injections in a volume of 100 µL between congenic donor and recipient mice (differing in CD45.1/.2 or Thy1.1/1.2 expression). In vitro activated T cells: For the adoptive transfers in figures 3, 4, 5(C), 9E-F and 10, in vitro activated, CD8+ CD44+ OT-I T cells were transferred to congenic naïve recipients or recipients infected for 9 weeks (figure 3) or 11 days (figure 4 and 9) with MCMV-K181 (lacking Ova). For treatment with Pertussis Toxin (PTx), OT-Is were suspended at a concentration of 1.5x107 cells/mL and treated, or not, with 50 ng/ml PTx (Sigma-Aldrich) for 1 hour at 37 °C prior to transfer. In figure 9F, wild-type (WT) and CCR5 KO OT-I T cells were mixed and co-transferred to B6 mice that have been previously infected with MCMV-K181 for 11 days. The mixture of donor cells was treated with anti-CXCR3 blocking antibody (clone: CXCR3-173) or isotype control antibody (Polyclonal Armenian Hamster IgG) at a concentration of 30 µg per 4x107 cells for 15 minutes. Recipient mice were also treated with the anti-CXCR3 antibody or isotype control (250 µg/mouse) via i.p. injections on days -2; day 0 (the day of transfer) and day 2. Blocking antibodies and isotype controls were purchased from Bio X cell. In figure 10 the same approach was used but only WT OT-Is were transferred to naïve recipients. In vivo activated T cells: For in vivo activation (figures 5A-B, figure 8, figures 9A-D), naïve OT-I T cells were transferred into naïve congenic recipients, followed by infection with MCMV expressing the cognate SIINFEKL peptide (either MCMV-SL8-015 or K181-MCMV-Ova) 1-3 days after transfer. The number of cells transferred is indicated in each figure legend. In figures 5A-B, 5 x 104 OT-Is were transferred into congenic recipients to produce large numbers of OT-Is for a secondary transfer by day 5 post-infection. In this experiment, OTIs were recovered from the spleen 5 days after infection, treated for 30 minutes with 60 µg/mL of either anti-a4 (clone PS/2), anti-a4b7 (clone DATK32) or the respective isotype controls and transferred to a new group of infection-matched or naïve recipients. The recipients were treated on the day of the transfer with 300 µg of each antibody or isotype control via i.p. injection. Organs were collected 2 days after the secondary transfer and the tissues were processed as described above. 70 CD4 antibody treatment To achieve CD4+ T cell depletion, 100ug of anti-CD4 monoclonal antibody from Bio X Cell® (clone: GK1.5) or the isotype control IgG2b (clone LTF-2) were administered i.p., every other day starting 7 days prior to MCMV infection. The CD4+ T cell depletion was confirmed before infection and the antibody blockade was maintained by repeated administrations (1-2 days interval) untill sacrifice. Cell proliferation assays To assess proliferation of OT-I T cells in naïve or MCMV-infected mice (figures 4E-G), OT-Is were labeled with a cell tracer dye: CellTrace Violet or Carboxyfluorescein succinimidyl ester (CFSE) following manufacturer’s instructions before the adoptive transfer. Briefly, cells were suspended at a concentration of 106 cells/mL and incubated with 5 µM of the Cell Trace Violet dye in for 20 minutes or suspended at a concentration of 1x107 cells/mL and incubated with 1 µM CFSE for 10 minutes. Transwell migration assays For Transwell migration assays, CD8+ T splenocytes were isolated from MCMV-K181 infected mice (7 days) using the EasyStep Biotin selection kit (StemCell Technologies) and biotinylated antibodies against erythrocytes (Ter119), CD19 (6D5), NK1.1(PK136), I-A/I-E (M5/114.15.2) and CD4 (GK1.4) following the manufacturer’s protocol. Typically, CD8+ T cells were 80-90% pure following this protocol. Purified cells were resuspended in RPMI media containing 2% BSA and 25 nM of Hepes buffer (migration media) at a concentration of 5x106 cells/mL and incubated for 1 hour at 37 °C. Subsequently, 5x105 cells, in a total volume of 100 µL were added to the upper chamber of a 6.5 mm Polycarbonate Transwell system (from Corning Inc.) with a pore diameter of 5.0 µm. Chemokines (all from Biolegend) were diluted in migration media and added to the lower chamber at titrating concentrations in a total volume of 600 µL (3-4 replicate wells per concentration). Control (media alone) samples without chemokines in the lower chamber of the Transwell plates, were included on every plate to account for plate-to-plate variations in T cell migration. All tests were run in duplicate, or triplicate on every plate. Chemokine concentrations, shown in figure 7, represent the optimal migration over control (media alone) wells based on replicate titrations of each chemokine. Cells in the Transwell plates were incubated for 1.5 hours at 37°C. At the end of the incubation, 200 µL of cells from the bottom chamber were mixed with counting beads (CountBright Absolute Counting Beads, Invitrogen), while the remaining 71 volume was used for FACS analyses of tetramer-binding cells. Cells were collected by flow cytometry as above. qRT-PCR For assessment of chemokine receptors expressed by T cells in figure 8C-D, OT-I T cells were transferred to B6 mice that were then infected with MCMV-K181-Ova (n=3 mice). 7 days after infection, spleen and salivary glands were collected and CD8+ T cells were enriched using the EasySep Biotin selection kit (Stemcell Technologies), as described above for the Transwell assays. OT-Is were sorted based on the congenic markers CD45.1 and CD45.2. RNA was extracted from the sorted OT-Is (figures 8C-D) and whole salivary glands of naïve B6 or IFN-g KO mice (figure 10F) using the RNeasy Mini Kit (QIAGEN), cDNA was recovered using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). In both cases the β-actin and chemokine receptor transcripts expressed were detected on a StepOnePlus system (Applied Biosystems) using predesigned qPCR assays from Integrated DNA Technologies and 6-carboxyfluorescein for detection. The relative concentration of chemokine receptors on these cells was determined by comparing the chemokine receptor signal to the β-actin signal for the same sample on the same plate and the data is expressed as the 2-ΔCT value (e.g. 2 -(CT value for chemokine A – CT value internal reference control A)). RNA-Seq For the RNA-Seq analysis salivary glands from mice that were infected, with 2x105 pfu of MCMVK181 i.p., for 14 days or naïve mice were collected and RNA was obtained with the miRCURY RNA Isolation kit Tissues (EXIQON). RNA Clean-up and ribosomal RNA (rRNA) depletion for the RNA-Seq analysis Prior to cDNA library preparation, RNA samples were purified using RNA Clean & Concentrator (Zymo research, R1015) and treated with DNase I to remove contaminating genomic DNA. Validation of RNA quality and concentration was determined using the RNA 6000 Pico Kit (Agilent Technologies, catalog number 5067-1513) for the BioAnalyzer 2100 instrument (Agilent Technologies). Four micrograms of total RNA per sample were used as input for the depletion of ribosomal RNA (RiboMinus kit; Ambion, A15020), yielding 6% recovery of input RNA on average. 72 RNA-Seq Library Preparation and Sequencing Library preparation was performed using the Ion Total RNA Seq Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s protocol. The yield and size scatter of the cDNA libraries were evaluated using High Sensitivity DNA Chips (Agilent Technologies, catalog number 5067-4626) on the BioAnalyzer 2100 instrument. The barcoded cDNA libraries were diluted to a final concentration of 100 pM, and used for template preparation on the Ion Chef instrument using the Ion PI Hi-Q Chef Kit (Thermo Fisher Scientific, catalog number A27198). Sequencing was performed on the Ion Proton sequencer system from Thermo Fisher Scientific using Ion Torrent PI v3 chips (Thermo Fisher Scientific, catalog number A26771). Sequenced data were preprocessed on the Ion Torrent server with Torrent Suite version 4.4.3. RNA-seq data Analysis IonTorrent single-end sequence reads were mapped to the mm10 genome using the IonTorrent Torrent Server tmap aligner, version 4.4.11. Gene abundances were estimated by counting strandspecific reads using the Subread package featureCounts tool, where reads with mapq of 0 or multiple mappings were filtered out.42 Reads overlapping more than one gene feature were not counted because the transcript of origin could not be confidently determined, as recommended by the Subread/featureCounts manual. Resulting read counts were then used to analyze differential expression between infected and uninfected samples using the DESeq2 package for R/bioconductor, with default settings.43 For additional data exploration and visualization, reads per kilobase of transcript per million mapped reads (RPKM) estimates were calculated from featureCounts results. Gene Set Enrichment Analyses (GSEA) was also performed to identify upand down-regulated gene groups among available pathway and gene ontology annotations44. Mouse gene symbols were converted to their human orthologs, obtained from MGI on March 3rd 2017, for compatibility with GSEA. GSEA preranked analysis was performed using the list of available human orthologs and the DESeq2 test statistic as the ranking metric, and GSEA enrichment score set to "classic" as described in the GSEA FAQ. Data have been submitted to the NCBI-GEO database (https://www.ncbi.nlm.nih.gov/geo/). The accession number is: GSE107338. ELISAs for chemokine expression Whole salivary glands of naïve B6 and IFN-g KO mice were collected in PBS with 10 μL/mL of the HaltTM Protease Inhibitor Cocktail (Thermo Scientific) and homogenized for 2 cycles of 30 seconds in a mini bead beater. After centrifugation (14000 xg for 15 minutes) the supernatant was stored at -80ºC. 73 The total protein concentration was determined using the Quick StartTM Bradford Protein Assay from BioRad. Samples were normalized to 8 mg/mL of total protein. The concentration of CXCL9 in each sample was quantified in duplicate with the CXCL9 (MIG) ELISA kit (Thermo scientific) according to the manufacturer’s specifications. Statistical analysis Differences in absolute numbers were determined after Log10 transformation and the fold change was determined based on the ratios of the geometric mean. The specific statistical test used for each experiment is indicated in the figure legend. Prism 6 for Mac OS X was used to determine the Log10 transformed values, geometric mean, standard error of the mean (SEM) and to perform the statistical analysis. 74 2.4 Results 2.4.1 I mpact of MCMV infection in TRM differentiation Impact of MCMV infection in the gene expression of salivary glands To understand the modifications in the salivary glands’ gene expression after MCMV infection, salivary glands were extracted from naïve mice or mice that had been infected for 2 weeks with MCMV strain K181 and used for a RNAseq analysis. Table 2 shows the top 50 genes, which expression was increased in MCMV infected mice (complete gene list in sup. tables S1A and S1B). Perhaps not surprisingly, the dominant changes in overall gene expression could be traced back to immune responses, including increases in genes encoding MHC molecules such beta 2 microglobulin (B2m) and genes involved in the antigen processing machinery such as Tap1 and Protease subunit beta (Psmb)8 (Table 2) Interestingly, several IFN-γ-induced guanylate-binding proteins (Gbp) such as Gbp 2, 3, 6, and 7 and interferon gamma inducible protein 47 and IFN-γ-induced GTPases (Iigp) as Iigp 1, immunity-related GTPase family M (Irgm)1, 2 and the interferon-inducible GTPase (Igtp) were among the most expressed genes after infection, suggesting a dominant IFN-g-induced change in gene expression. Gene Set Enrichment Analyses (GSEA) using the Gene Ontology Biological Process database revealed that the top 20 enriched gene sets (over-represented set of genes that are statistically different in two conditions) were all related to innate and adaptive immune responses, cytokine signaling, IFN-I, IFN-γ and leukocyte activation (Table S1C). In contrast, very few genes were significantly downregulated after MCMV infection and many of these were small nucleolar RNAs (SNORDs, Table S1A). |Table 2. Top 50 genes with differential expression between salivary glands of MCMV infected and uninfected mice. Salivary glands from mice that were infected, with MCMV-K181 for 14 days or naïve mice were collected and RNA was obtained. The read counts were then used to analyze differential expression between infected and uninfected samples using the DESeq2 package for R/bioconductor, with default settings. RPKM estimates from infected and uninfected were calculated from featureCounts results and shown in the left panel. log2, mean centered RPKM is shown in the right panel. The top 50 differently expressed genes between naïve and MCMV infected salivary glands are presented. The log2 fold change between infected and uninfected based on the average of the 3 sample’s log-2 mean-centered RPKMs in each group is ranked. The colors display the pattern of the gene expression across the samples (red indicating 75 higher values and blue lower values). This table is a detail of table S1A for all the genes that increased or decreased significantly with infection (FDR<0.05) available in: https://www.jimmunol.org/content/suppl/2017/12/29/jimmunol.1701272.DCSupplemental | Gene log2 Fold Change DK1-R DK1-L DK1-N DK5-R DK5-L DK5-N Igtp 1.613575115 3.16320126 2.78539685 4.80376721 18.2338811 13.2650395 11.2167383 Iigp1 1.59498252 2.47791047 1.74416562 2.61568086 9.07148568 8.92183955 6.80213591 Gbp2 1.529682012 3.63708965 4.13146399 4.81899565 17.9455924 17.1637206 9.57072235 Irgm2 1.270279995 3.43034273 4.008863 4.80959197 10.3655683 10.626312 10.0754461 B2m 1.251512625 69.3788274 74.2119971 90.4467687 178.976127 172.486171 188.129544 Psmb8 1.235543706 13.0200176 10.4434541 11.1705362 22.3136114 34.8272306 28.2914601 CxcL9 1.212842206 0.15368425 0.64657003 0.09696446 2.03005759 2.51576693 1.938944 Psmb9 1.163060538 8.67794849 6.75810079 7.16436263 16.3551348 21.393334 21.1648006 CCL5 1.131996304 2.52233195 3.89098783 1.59142243 8.05733688 14.8385388 14.2444831 Ifi47 1.111818966 1.77427821 2.51931051 1.60921031 4.94013703 5.78619364 6.23582884 Ctss 1.103317736 22.5414871 13.6728168 20.4012164 31.6292104 48.7835938 39.8780901 H2-K1 1.091839072 318.344123 169.837783 239.76888 406.389666 567.934384 485.123789 Oasl2 1.081462425 6.97582429 3.71344798 4.85038762 11.9099733 11.8343354 9.80168342 Apof 1.066056299 6.32281966 3.211577 5.98391324 8.44784861 16.330081 11.5904647 H2-Ab1 1.051072685 62.3711587 64.3938769 58.555808 88.3972417 165.497377 129.501306 Il2rb 1.041490132 0.71759221 2.71710504 1.14929505 4.37486446 4.8709595 5.61114283 Tap1 1.040515845 11.9054068 10.8240207 9.11885051 19.5989275 24.1158513 20.1301823 Cxcr3 1.022376938 0.64743511 0.11673623 0.0000001 2.08413704 2.83883737 2.40048256 Zbp1 0.98894977 0.68833295 0.4343862 0.97715693 2.40680632 2.58807559 2.45641425 Lgals3bp 0.985217966 11.188776 12.3024264 18.6445267 24.5881564 28.2846998 29.8407774 H2-Eb1 0.984557663 47.3469967 37.1380684 32.5731465 59.237142 98.3188971 69.0398826 Irgm1 0.979183699 13.298024 9.78361785 19.3521827 30.7387221 25.9457881 25.7260518 H2-D1 0.975966992 34.2040417 38.0850606 46.5683571 64.8774176 91.4985003 68.2293842 Stat1 0.973509012 10.7528763 8.34637891 10.9390979 18.6749677 16.8218208 19.740955 Irf7 0.971301689 11.367454 6.87054568 18.2585547 19.7180603 30.0376558 24.7588234 CD52 0.963556189 4.73376015 4.85441695 3.08001935 7.81619258 13.8483623 21.1163722 Rnu11 0.943832884 786.804082 1302.62609 1789.20087 2490.40669 4564.51732 3738.69695 Gbp3 0.942197725 2.35344094 2.46477631 2.27468455 5.05800048 5.57128998 4.33196102 Nlrc5 0.934232705 1.47853324 2.08247686 1.25732683 3.52978237 3.53510873 2.98924023 Mir6240 0.928596804 57.2375326 131.640553 80.6524725 188.7695 309.392632 143.050979 Cited2 0.926032554 4.91271864 4.67425146 1.93128689 5.63780222 10.4391072 12.0147566 Ly6a 0.905897585 28.8129855 25.6811551 39.6378538 50.6618585 67.1249754 51.8773949 Laptm5 0.905071857 7.50914419 9.39926244 5.7557993 12.7303126 13.1430594 19.8648775 Cxcr6 0.902946214 0.0000001 0.29766814 0.0000001 1.64929168 1.38743865 1.53026008 Gzma 0.899196608 1.01697666 0.42785557 0.32082206 2.50232321 5.33243914 3.66588501 Slc25a29 0.898340674 6.81954309 5.54687295 4.87636465 8.53710922 14.1286724 11.0620507 Prf1 0.875333012 0.36226286 0.45722637 0.54855262 1.40742055 2.05701686 2.11546909 Hist1h2ai 0.865109761 2.65745687 2.93482179 4.40127765 6.4527719 13.2544556 13.650512 Ifitm3 0.857495228 19.0615116 15.6523829 13.9102109 31.0497824 27.4907228 32.5341637 H2afx 0.843058774 5.03723981 2.90242861 2.90179895 6.12628729 8.90682614 7.81569921 Gbp6 0.84189252 0.48272618 0.87038274 1.37055541 2.57201867 2.27537814 1.64064492 C1qb 0.839781199 35.1725177 17.5094452 21.7494398 42.0288113 53.3327191 35.4596708 Slfn2 0.832163958 5.90262055 5.86964357 7.36367607 10.4237085 11.8721493 12.4761258 Nkg7 0.82699672 0.0000001 0.92412593 0.34647204 1.99123175 5.89922976 3.76102667 Lgals1 0.825345197 5.95270339 5.40007209 10.2109642 13.7314986 17.128835 12.6734227 Selplg 0.825301868 2.05738596 2.30818549 1.13581359 3.06402588 4.51682029 4.44974245 H2-Q4 0.823928738 15.8528492 11.5232266 11.6254607 17.7352258 26.4304388 22.7980019 Stat2 0.812234407 7.24782957 4.70212685 8.81457518 12.6318076 10.4992404 11.6835276 H2-DMa 0.809529553 3.32101968 3.96501858 2.54838754 4.74251492 7.50720954 8.2504523 Rps11 0.795539183 8.15777458 16.2243969 17.3126669 17.2874261 44.1973262 26.7329488 Top 50 genes with differential expression (FDR<0.05) Sample Reads Per Kilobase Per Million Reads (RPKM) uninfected infected DK1-R DK1-L DK1-N DK5-R DK5-L DK5-N -1.1401351 -1.3236375 -0.5373542 1.38702933 0.92803662 0.68606088 -0.7914619 -1.2980489 -0.7133994 1.08075293 1.05675528 0.66540191 -1.0878232 -0.9039546 -0.6818752 1.21494998 1.15068276 0.30802016 Psmb8 -0.9155687 -0.6907283 -0.4280069 0.67980588 0.71564765 0.63885038 Ctss -0.7666671 -0.6695101 -0.3840936 0.60053269 0.54724622 0.67249195 Oasl2 -0.4628578 -0.7809902 -0.6838908 0.31433482 0.95662655 0.65677743 Zbp1 -2.1370104 -0.0641731 -2.8014517 1.58646902 1.89594664 1.5202195 Lgals3bp -0.477507 -0.8382432 -0.7540227 0.43681068 0.82422836 0.80873392 Irgm1 -1.1150957 -0.4897174 -1.779537 0.56044924 1.44142329 1.38247753 Irf7 -0.8934946 -0.3876979 -1.034374 0.58382421 0.81188776 0.91985448 CD52 -0.2600503 -0.9813226 -0.4039779 0.2286245 0.853763 0.56296327 Nlrc5 -0.0799679 -0.9863975 -0.4889106 0.27230873 0.75516928 0.52779794 Ly6a -0.0985126 -1.0081169 -0.6227762 0.67322199 0.6640305 0.39215326 Laptm5 -0.2674069 -1.2446931 -0.3468857 0.15060902 1.10148509 0.60689155 Cxcr6 -0.4866622 -0.4406177 -0.577729 0.01646012 0.92119522 0.56735351 Ifitm3 -1.823063 0.09777123 -1.1435499 0.78493921 0.93990688 1.14399553 Slfn2 -0.3288654 -0.4662458 -0.7135585 0.39029238 0.68949941 0.42887791 Lgals1 3.23135585 0.75987263 -19.394948 4.91799849 5.36384855 5.1218728 Selplg -0.8988956 -1.5630239 -0.3934119 0.90704596 1.01180567 0.93647979 -0.7937841 -0.6568934 -0.0570841 0.34212726 0.54418556 0.62144882 -0.1719363 -0.5223105 -0.7115262 0.15129278 0.88225947 0.37222075 -0.5372774 -0.9800493 0.004007 0.6715679 0.42701107 0.41474077 -0.6565998 -0.5015414 -0.2114166 0.26694978 0.76298148 0.33962658 -0.3296224 -0.6951227 -0.3048513 0.46676073 0.31598885 0.54684677 -0.546126 -1.2725386 0.13753741 0.24848248 0.85573707 0.5769076 -0.6409507 -0.6046392 -1.2609979 0.08252874 0.9077062 1.51635282 -1.3932893 -0.6659426 -0.2080503 0.2690157 1.14309665 0.85516988 -0.5408106 -0.4741256 -0.5899158 0.56298489 0.70242913 0.33943798 -0.6314899 -0.1373561 -0.8652969 0.62392265 0.626098 0.38412226 -1.1956369 0.00593367 -0.7008797 0.52595536 1.23876852 0.12585905 -0.1929141 -0.2647004 -1.5398732 0.00569716 0.89449071 1.09729981 -0.5294748 -0.6954839 -0.0693151 0.28470604 0.69065569 0.31891207 -0.4854824 -0.1615834 -0.8691146 0.27606502 0.32209832 0.91801707 -15.512302 5.99297157 -15.512302 8.46304144 8.21361885 8.35497174 -0.4376389 -1.6867297 -2.1020802 0.86134267 1.95287012 1.41223605 -0.215159 -0.5131594 -0.699028 0.10891358 0.83571995 0.48271289 -1.3239538 -0.9880821 -0.7253607 0.63399082 1.18149095 1.22191494 -1.1383619 -0.9951353 -0.4104858 0.14151082 1.1799973 1.22247495 -0.2107829 -0.4950631 -0.6653011 0.49313773 0.31749941 0.56051004 -0.0245567 -0.8199296 -0.8202426 0.25782286 0.79772127 0.60918478 -1.4618947 -0.6114498 0.043589 0.95172945 0.77493467 0.30309138 0.13842546 -0.8678898 -0.5550449 0.3953555 0.73899773 0.15015599 -0.5365542 -0.5446369 -0.2174836 0.28388695 0.47159944 0.54318832 -20.014962 3.12469639 1.70934587 4.23219617 5.79906163 5.14966157 -0.7431346 -0.8837009 0.03536761 0.4627376 0.78167555 0.34705472 -0.3551468 -0.1892001 -1.2122332 0.21946923 0.77934826 0.75776258 -0.0859745 -0.546172 -0.5334288 0.07590099 0.65148367 0.43819068 -0.2805109 -0.9047465 0.00183107 0.52092923 0.25415308 0.40834406 -0.4795305 -0.2238292 -0.8615721 0.0344955 0.69711994 0.8333164 -1.2185094 -0.2265921 -0.1329289 -0.1350338 1.21920217 0.49386206 Top 50 genes with differential expression (FDR<0.05) Log2, mean-centered RPKM uninfected infected Top 50 with differential expression 82 |Figure 4. CD8+ TRM phenotype cells can form and persist at similar numbers in salivary glands from MCMV infected and naïve mice. A ) Schematic representation of the experimental design. Naïve mice, or mice infected 11 days earlier with MCMV (via the i.p. route) were seeded with 3x106 in vitro activated OT-Is. The recipients were sacrificed at 4 CD69+ 19% ±3.3 63.6% ±8.3 9.3% ±3 % Phenotype of OT-Is Days after transfer # OT-Is Naïve MCMV 31 days after transfer 4 LGSG KDN SPL 43131 103 104 105 107 106 108 MCMV Naïve *** BC D Naïve - SG 21.5% ±3.3 52% ±4.3 11.2% ±1.9 MCMV - SG CD103+ A Figure 3 Naïve PBS (I.P.) SIINFEKL + IL-2 MCMV-K181 (I.P.) D-11 Splenocytes from OT-Is D0 Transfer 6 -7 days 4 weeksD4 Sacrifice Sacrifice 443131 443131 443131 SG LG KDN Naïve MCMV Naïve MCMV 20 40 60 80 100 % Phenotype OT-Is 0 CD69+ CD103+ CD69CD103+ CD69+ CD103E F G 83 or 31 days after transfer. B ) Absolute number of OT-Is in the parenchyma of the salivary gland (SG), lungs (LG), kidneys (KDN) and from the CD8β+ cells of the spleen (SPL). C and D ) Frequency of CD103and CD69expressing OT-Is in the parenchyma of the SG, LG and KDN at 31 days after transfer. Data (A-D) are combined from 2 experiments (n= 7 naïve and 6 infected recipients at day 4; n=6 naïve and 6 infected at day 31). D ) Concatenated FACS plots, of CD103 and CD69 expression of the OT-I T cells in the SG from one representative experiment (n=3 naïve and n=3 infected recipients) with mean ± SEM values considering all the experiments. Error bars represent the SEM and the statistical significance in (B) was measured by unpaired t-test after log10 transformation of absolute numbers (***p<0.001). E - G ) In vitro activated OT-Is dilute cell tracer dye similarly in both naïve and MCMV infected mice. The experiment was performed as described before but the OT-I T cells were labelled with cell tracer violet before transfer to naïve mice or mice infected with MCMV for 11 days. Concatenated FACS plots from the OT-I T cells in the infected (E) or naïve (F) recipients. G ) Data from host CD8β+ T cells, as a control. Plots show host cells in the infected (IDark grey) and naïve (N - Light grey) recipients. Frequencies ± SEM were represented from one experiment (n=2-3). No differences between infected and naïve animals were observed in a second experiment using CFSE labeling (data not shown). | 2.4.2 Mechanisms involved in CD8+ T cell migration to the salivary gland CD8+ T cell homing to the salivary gland is mediated by a4b1 and chemokines at steady state Recent data published by Woyciechowski et al . (2017) have suggested that the integrin a4b1 plays a critical role in T cell recruitment to the salivary gland during systemic inflammation induced by intravenous poly (I:C) treatment.32 We wondered whether this mechanism would also contribute to T cell recruitment to naïve salivary glands. To test this, naïve WT OT-Is were transferred into congenic mice and driven to expand with MCMV-Ova infection. 5 days after infection, splenic T cells containing activated OTIs were transferred to MCMV-K181 infection-matched or naïve recipients in the presence or absence of antibodies to block the a4-integrin or the a4b7 integrin. As expected, the a4 blockade greatly reduced the number of OT-Is in the salivary gland of infected recipients 2 days after transfer (figure 5A).32 Likewise, in naïve recipients, the α4 blockade reduced the numbers of OT-Is in the salivary gland and in the lamina propria of the small intestine compared to the group that received the isotype control antibody, differences that were not observed in the spleens of the same animals (figure 5B). In contrast, the blockade of a4b7 had no significant impact on T cells in the salivary gland, despite inhibiting OT-I migration to the lamina propria of the small intestine (figure 5B), as expected.30,32,54 In addition, pre-incubation of the OT-I T cells with retinoic acid, which strongly induces the a4b7 integrin, had no effect on salivary gland migration 84 (data not shown). 55 The a4 integrin is known to pair with either b1 or b7 chain. Therefore, these data suggest that a4b1 is important for the migration of activated CD8+ T cells to the salivary gland regardless of infection. These data also imply that the salivary gland expresses sufficient VCAM-1, the ligand for a4b1, independently of local MCMV infection. Chemokines are important for activating integrins and promoting lymphocyte transendothelial migration. However, several recent reports have implicated chemokine-independent mechanisms of T cell recruitment (e.g. CD44, IL-33, or antigen).56–58 To test whether chemokines are mediators of CD8+ T cell recruitment to the naïve salivary gland, OT-I T cells were activated in vitro and treated, or not, with PTx, which irreversibly inhibits signalling through G-coupled protein receptors like chemokine receptors.59–61 Untreated, activated OT-I T cells migrated into all organs, including the salivary gland, within 4 days of transfer as expected (figure 5C). In contrast, PTx treatment substantially reduced the numbers of OT-I T cells that migrated into the lymph nodes (figure 5C), as previously shown [60,61] as well as the salivary gland and kidney (figure 5C). These data suggest that activated CD8+ T cells migrate to naïve salivary glands in response to a chemokine signal. |Figure 5. CD8+ T cell accumulation in infected and uninfected salivary glands is dependent on a4 integrin and chemokines. A and B ) Naïve OT-Is (5x104) were transferred into congenic mice that were infected 1 day later, with MCMV expressing Ova via the i.p. route. Splenocytes containing expanded OT-Is were recovered from the spleen 5 days later and transferred to (A) mice infected with WT MCMV (lacking Ova) or (B) naïve mice. Recipients were either treated or not with anti-a4 blocking antibody on the day of the transfer and mice were sacrificed 2 days after the transfer. Data show the absolute number of OT-I T cells recovered from the parenchya of salivary 24.4x **** p=0.27 2.3x AB 104 105 106 102 # OT-Is α4Iso α4Iso SG SPL D5 → D5 MCMV-K181 30x *** SG Small int. - LP SPL 8.9x ** α4Isoα4β7Iso D5 → Naïve 60.3x ** Antibody blockade C PTx SG KDN LN SPL 9.5x **** 9.1x *** 14x ** 1.6x * Vehicle Figure 4 Naïve 103 104 105 106 102 # OT-Is α4Isoα4β7Isoα4Isoα4β7Iso Antibody blockade 103 104 105 106 102 # OT-Is 107 108 103 85 gland (SG), small intestine lamina propria (LP) and from the CD8β+ fraction of the spleen (SPL). Results were combined from 2 independent experiments (n= 5-6 mice per group). C ) Naïve B6 mice were seeded with 8x106 in vitro activated OT-Is that had been treated with PTx or vehicle as a control before the adoptive transfer. Shown are the absolute numbers of OT-Is recovered from the parenchyma of the SG, kidneys (KDN) lymph nodes (LN) and from the overall CD8β+ T cell population of the SPL 4 days after the adoptive transfer. Results from 2 independent experiments were combined (n=7). Error bars represent the SEM and statistical significances in (A-C) were measured by unpaired t-test after log10 conversion of the absolute numbers (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). | Chemokines expressed in the salivary gland with or without MCMV infection To explore the chemokines expressed by the salivary gland in the presence or absence of MCMV infection, we used the RNA-Seq data described before. Interestingly, there were no significant changes in expression of integrins or cell adhesion molecules that survived correction for multiple testing errors (FDR < 0.05), although expression of the a4, aL (CD11a), aX (CD11c) and b2 integrins was increased in the infected salivary glands when the gene set was filtered by a raw p value of lower than 0.05 (table S1B). Of the chemokines, expression of CCL5 and CXCL9 were significantly increased by infection (FDR < 0.05) and CCL7, CCL8, CCL12, and CXCL10 were also detected when we used a raw p-value cutoff of 0.05 (figure 6 and tables S1A and S1B). In contrast, several chemokines were abundantly expressed in the salivary gland, regardless of infection, including CCL28, CXCL12, CXCL14, CXCL16, CXCL17 and CX3CL1 (figure 6 and table S1B). All of these chemokines, with the exception of CXCL17, have been described to recruit T cells in various settings.29,62–74 These data show that MCMV infection of the salivary gland induces a dominant inflammatory response centered on IFN-stimulated gene expression. However, several chemokines were abundantly expressed in the salivary gland regardless of infection. 86 |Figure 6. Chemokine profile of the salivary gland after MCMV infection. The array of chemokines expressed in the salivary glands of uninfected mice, or 14 days after MCMV infection. Shown are the RPKM of chemokines to illustrate the relative abundance of different transcripts. The complete gene list of genes that were significantly differentially expressed is shown in table S1A. The complete gene list sorted by fold change, regardless of significance, is shown in table S1B. Data are from one experiment (n=3 mice per group). Error bars represent the SEM (%=p< 0.05; *=FDR<0.05). | MCMV-specific T cells express multiple chemokine receptors and are able to migrate towards multiple chemokines To test whether MCMV-specific T cells could migrate to some of the chemokines that were present in the salivary gland independently of infection, we conducted Transwell migration assays in vitro with the top 6 most abundantly expressed chemokines, along with CCL5, CXCL9, CXCL10 and CCL19. T cells were harvested from the spleens of mice 7 days after MCMV infection, which represents the peak of T cell clonal expansion and a time at which splenic T cells migrate readily to the salivary gland in adoptive transfer experiments (not shown). 35,75 In multiple experiments, CCL28 and CXCL16 failed to induce any migration of splenic CD8+ T cells (data not shown). In contrast, robust CD8+ T cell migration was induced by CCL19 (approximately 9-fold increased over background, not shown), but most of these cells were CD44low as expected (not shown) and there was no increase in tetramer-binding MCMV-specific T cells Sample Reads per Kilobase Per 106 Reads (RPKM) 60 CCL7 CXCL10 CCL12 CCL8 CCL5 CCL19 CCL20 CCL26 CCL3 CXCL2 CXCL3 CCL1 CXCL15 CXCL11 CCL24 CCL17 CCL27a CCL22 CCL4 CCL25 CXCL1 CCL9 CXCL13 CCL2 CCL21a CCL6 CCL11 CXCL16 CXCL12 CX3CL1 CXCL14 CXCL17 CCL28 Naïve MCMV CXCL9 0 5 10 15 20 30 40 50 * * Salivary gland - RNAseq analysis Figure 5 87 among the migrated cells (figure 7A). All the other tested chemokines induced significant migration of MCMV-specific T cells (figures 7A-B). These included chemokines that were expressed at high levels constitutively (CXCL12, CXCL14, CXCL17 and CX3CL1), as well as chemokines induced in the salivary gland by infection (CXCL9, CXCL10 and CCL5) (figure 7). These data show that MCMV-specific T cells can migrate in vitro , toward several chemokines that are either induced by MCMV infection or constitutively expressed in the salivary gland. Next, we used flow cytometry to explore chemokine receptor expression by OT-I T cells in the spleen and salivary gland, 7-9 days after MCMV-Ova infection. Cells were further differentiated by KLRG1 expression since our previous data showed that most KLRG1 expressing T cells failed to accumulate in the parenchyma of the salivary gland.11 7 to 9 days after MCMV-Ova infection, the KLRG1 expressing splenic OT-Is were mostly CX3CR1+ CXCR3-, which is consistent with recent work. 76,77 Additionally, we found that these KLRG1+ cells expressed CXCR6, but mostly lacked CXCR4 (figure 8A). In contrast, the KLRG1portion of the splenic OT-Is contained subsets expressing or lacking CX3CR1 and a higher frequency of KLRG1cells expressed CXCR3, CXCR4 and CXCR6 (figure 8A). Notably, these phenotypes were consistent: at 7 months after MCMV infection, KLRG1 expression on MCMV-specific T cells still correlated with the expression of CX3CR1 and a lack of CXCR3 (data not shown). Within the parenchyma of the salivary gland, the majority of cells lacked CX3CR1 and KLRG1 (figure 8B, left). In sharp contrast, T cells found in the vasculature of the salivary gland (i.e. stained by the intravascular antibody) were almost entirely CX3CR1+ and most also expressed KLRG1 (figure 8B, right). We have had difficulty getting consistent staining of any of the other chemokine receptors on T cells extracted from the salivary glands. In our hands, CXCR3 has been sensitive to the collagenase used to extract T cells from the salivary gland and attempts at extracting T cells without collagenase have failed to result in sufficient T cell recovery. Therefore, we measured the expression of chemokine receptors on T cells sorted from the spleen and salivary gland using qRT-PCR. Naïve, WT OT-Is were transferred into congenic mice and driven to expand with a MCMVOva infection. 7 days later, OT-I T cells sorted from the spleen and salivary gland expressed CX3CR1, CCR5, CXCR3, CXCR4 and CXCR6 (figure 8C-D). In contrast, CCR10 (receptor for CCL28) and CXCR7 (one receptor for CXCL14) were undetectable on either spleen or salivary gland localized OT-I T cells (data not shown). Interestingly, while salivary gland and spleen-localized T cells expressed comparable amounts of CXCR3 and CCR5, salivary gland-localized T cells expressed more CXCR4 (p=0.016) and CXCR6 (p=0.023) than spleen-localized T cells. In contrast, spleen-localized T cells expressed much more CX3CR1 than salivary gland localized T cells (p<0.0001, figures 8C-D), consistent with our FACS data. 88 |Figure 7. MCMV-specific CD8+ T cells migrate towards multiple chemokines. Mice were sacrificed 7 days after MCMV infection (i.p.). CD8+ T cells were enriched and used to performed transwell migration assays with multiple chemokines. Assays were performed with replicates and media control was included in each assay to account for plate-to-plate variation. A ) Absolute numbers of MCMV-specific T cells (M45-Tetramer+ plus M38-Tetramer+) that migrated towards the indicated chemokine in comparison to control wells with media alone. Data are from a single experiment performed in triplicate or quadruplicate, and representative of 2 to 5 experiments per chemokine. Statistical significance was determined using paired t-test (*p<0.05; **p<0.01; ***p<0.001). B ) Fold change of migration in comparison to media. Pooled data from 2 to 4 independent experiments indicating the fold change of migration of MCMV-specific cells (M45-Tetramer+ plus M38-Tetramer+) in response to the indicated chemokines at the concentrations shown in (A). | 1000 * Figure 6 Fold Change 0 5 10 15 A CXCL17 CCL5 CXCL14 CX3CL1 CXCL12 CXCL9 CXCL10 Migrated cells (Tet+) Migrated cells (Tetramer+) B CCL19 CXCL17 CCL5 CXCL14 CX3CL1 CXCL12 CXCL9 CXCL10 CCL19 0 200 400 600 800 Media 2000 ng/mL 0 50 100 150 200 250 Media 1000 ng/mL 0 50 100 150 200 Media 100 ng/mL Media 300 ng/mL 0 200 400 600 800 1000 Media 100 ng/mL 0 2000 4000 6000 8000 Media 1000 ng/mL 0 1000 2000 3000 4000 5000 Media 1000 ng/mL 1000 0 200 400 600 800 Media 5 ng/mL *** * Migrated cells (Tet+) * 0 50 100 150 200 250 Migrated cells (Tet+) ** ** Migrated cells (Tet+) ** 89 |Figure 8. Most CD8+ T cells in the parenchyma of the salivary gland lack KLRG1 and CX3CR1, but express multiple other chemokine receptors. Naïve OT-Is (2x103) were transferred to naïve B6 mice that were infected via the i.p. route 1 day later with MCMV expressing Ova. Mice were sacrificed 7 or 9 days after infection. A ) Chemokine receptor expression within the KLRG1+ and KLRG1subsets of the OT-I T cells in the spleen. B ) KLRG1 and CX3CR1 expression of OT-Is from the vasculature (i.v.+) and parenchyma (i.v.-) portions of the salivary gland. In (A) and (B), the data show concatenated FACS plots from one representative experiment at day 9 (n= 3) with mean ± SEM values in each quadrant derived from all experiments (total n=8). C and D ) Naïve OT-Is were transferred as in (A) and sorted from the spleen (B) and the salivary gland (C) 7 days after infection with MCMV expressing Ova. Chemokine receptor expression was assessed on sorted T cells by RT-qPCR. Data were combined from 2-5 independent RT-qPCR assays per sample, with cDNA from OT-I T cells sorted from 2-3 independent mice. Error bars represent SEM. | B A KLRG1 CD8b CX3CR1 CXCR3 CXCR6 CXCR4 CXCR4 CXCR3 CXCR6 CXCR3 From CD8β+ OT-I from the SG: 7-9 days after infection KLRG1 CX3CR1 From CD8a (I.V.) + From CD8a (I.V.) - OT-I from the SPL: 7-9days after infection 86.5% ±1.5 11.9% ±1.4 69.1% ±4.5 27.4% ±4.12 3.1% ±0.5 42.2% ±5.4 6.4% ±0.7 4.9% ±0.8 2.63% ±0.15 5.4% ±0.58 28.3% ±4.87 28.7% ±7.49 19.9% ±1.99 12% ±2.89 19.5% ±4.2 54.4% ±4.4 24.4% ±1.7 59.03% ±3.4 19.95% ±3.9 6.6% ±1.03 0.87% ±0.16 19.44% ±4.4 62.04% ±8.18 15.76% ±3.99 62.9% ±4.12 17.47% ±3.2 7.1% ±1.4 80% ±1.2 8.1% ±1.2 26.7% ±4.3 10% ±1 7.6% ±1.7 Figure 7 0 0.1 0.2 0.3 D 2 -∆CT T cells from the spleen T cells from the salivary gland CX3CR1 CCR5 CXCR3 CXCR4 CXCR6 C CX3CR1 CCR5 CXCR3 CXCR4 CXCR6 0 0.1 0.2 0.3 90 CXCR3 is critical for T cell migration to uninfected salivary glands, but is dispensable after MCMV infection Since MCMV infection induced chemokines that bind CCR5 and CXCR3, we directly tested whether these receptors were critical for the accumulation of MCMV-specific T cells in the salivary gland. To this end, OT-I T cells that expressed or lacked either receptor were mixed with their WT counterparts and co-transferred to congenic B6 recipients. Recipient mice were infected on the following day with MCMV virus expressing Ova (figure 9A-D). Two weeks after the infection, there were only small reductions in the numbers of CXCR3 KO or CCR5 KO OT-Is in the salivary gland (figures 9A and 9C) and these small differences were mirrored in the spleens of the same animals, implying no impairment in the recruitment of T cells lacking either CXCR3 (figure 9A) or CCR5 (figure 9C) receptor. Moreover, there were no differences in the absolute numbers of OT-Is that expressed the tissue-resident markers CD69 and CD103 in the gland (figures 9B and 9D). It was possible that CCR5 and CXCR3 played redundant roles in migration of T cells to the salivary gland during MCMV infection. To address this possibility, WT and CCR5 KO OT-I T cells were activated in vitro and transferred into infected mice, with or without an antibody specific for CXCR3 that has been reported to block CXCR3-dependent cell migration.78,79 For infection, we used the K181 strain of MCMV lacking Ova, again to avoid the influence of antigen and additional T cell expansion after adoptive transfer (figure 9E). However, donor OT-I T cells still migrated to the salivary gland in all cases and the absolute number of OT-Is in the gland was similar in both groups independently of the CXCR3 blockade (figure 9F). There was a subtle difference on the overall number between the WT OT-Is in the unblocked group and the CCR5KO OT-Is in the CXCR3 blocked group, possibly suggesting a combined impact of CXCR3 and CCR5 on T cell migration. However, the effect was subtle and the CXCR3 blockade had an impact in the spleen in all mice. Therefore, it is difficult to distinguish if this effect was partially due to differences on T cells in circulation (figure 9F). There was no impact of CCR5 deficiency with or without CXCR3 blockade on T cell migration to lungs or kidneys. Although it is possible that the CXCR3 blockade was poorly effective in vivo in infected mice, these data suggest that CCR5 and CXCR3 are not required for T cell accumulation in infected salivary glands. As a control for these experiments, we had transferred in vitro activated WT OT-Is to naïve recipients, with or without CXCR3 blockade (figure 10A). Surprisingly, in naïve mice, the CXCR3 blockade had a striking impact on T cell accumulation to the salivary gland resulting in approximately a 9-fold reduction in the numbers of OT-Is that reached the parenchyma (figure 10B). Although the blockade had an impact on the numbers of OT-I T cells in the spleen, as in infected mice (figure 9F), it was lower than the impact on the salivary gland in naïve mice and there was no effect on the T cells recovered from the 91 lungs or kidneys (figure 10B). These data imply that the CXCR3 blockade was effective in naïve mice and suggest that the CXCR3 blockade was having a specific impact on T cell accumulation in the salivary glands of naïve mice, despite the absence of an effect in infected mice. To confirm these surprising results, and determine whether CXCR3 was needed on T cells specifically, CXCR3 KO and WT OT-Is were activated in vitro , mixed together, and co-transferred to naïve mice. At 4 days after transfer, the absolute number of each OT-I population was assessed and the ratio of KO to WT cells was calculated within the parenchyma and the circulation (figure 10C). In line with our blocking antibody data, accumulation of CXCR3 KO OT-Is in the salivary gland, but not the kidneys or spleen, was markedly impaired, in two separate experiments (figures 10D-E). Together, these data strongly imply that T cell migration to and accumulation in uninfected salivary glands depends critically on CXCR3. CXCL9 is expressed in the salivary gland at steady state even in the absence of IFNγ Because the ligands for CXCR3 (CXCL9 and CXCL10 in B6 mice) are strongly induced by IFN-γ and upregulated by MCMV infection (figure 6), we wished to confirm that these ligands were present in naïve mice and test whether IFN-γ was required for T cell migration to the salivary gland. The chemokine CXCL9 was readily detectable by ELISA and qRT-PCR in the salivary glands of naïve B6 and was also evident in IFN-γ KO mice (figures 10F-G). 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A great effort has been done to manipulate these cells and take advantage of their unique characteristics to promote a better immune response to infection and tumors. From prime-boost to repeated vaccination, multiple strategies are able to differentiate TRM in different organs.1–5 However, the requirements for TRM differentiation and maintenance vary according to the organ and the strategy used.6 Therefore, to successfully promote and modulate TRM it is crucial to understand the cascade of events that leads to TRM differentiation, its characteristics and modifiers. Consequently, this work was focused on further characterizing the TRM differentiation in mucosal tissues with clinical relevance. Salivary glands are essential for replication and shedding of herpesviruses.7–10 Human Cytomegalovirus (HCMV)'s high prevalence and morbidity/mortality worldwide makes it an interesting target and tool to study TRM. Briefly, our results showed that Murine Cytomegalovirus (MCMV) infection alters the gene expression profile in the salivary glands, however activated CD8+ T cells could differentiate in TRM, regardless of viral infection or viral replication. Moreover, CD8+ T cell homing to the salivary gland seems to be promoted by CXCR3 (in naïve mice) and a4b1 expression (in both naïve and MCMV infected mice). The succeeding discussion will be divided into 3 main parts. Initially, the influence of MCMV infection in the salivary gland environment and its impact in CD8+ T cell recruitment and TRM differentiation will be discussed (Part 3.1). This will be followed by a reflection about the mechanisms involved in TRM differentiation in naïve salivary glands (Part 3.2). Finally, the possible implications of the prompt ability for TRM to differentiate in the salivary glands will be discussed (Part 3.3). 106 PART 3.1 What does MCMV do to the salivary glands? 3.1.1 Differences in gene expression between salivary glands from naïve and MCMV infected mice MCMV infection of the salivary gland induces, in the mouse model, tissue inflammation, which recreates some of the Sjögren’s syndrome clinical findings.11,12 Although commonly accepted that Cytomegalovirus (CMV) infection promotes an inflammatory status of the glands, few studies have addressed the specific inflammatory changes that occur after MCMV infection. Cavanaugh et al. (2003) have shown that interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), CCL1, CCL3, CCL4 and CCL5 were enriched in the salivary gland of MCMV infected mice compared to the secondary lymphoid organs at 2 weeks after infection.13 In line with previous findings, our data (Table 2 and Table S1A-B) show that, after MCMV infection, an inflammatory state is seen in the salivary gland, with those molecules being detected in infected salivary glands along with up regulation of IFN-g related genes, such as CXCL9 chemokine. Additionally, the RNAseq analysis performed identified novel changes between naïve and infected salivary glands 2 weeks after MCMV infection. Looking at the top 50 differently expressed genes, MCMV infection mainly promoted interferon (IFN)-induced genes as well as genes related to antigen processing/presentation/recognition. Nonetheless, it is important to note that these results should be interpreted carefully since post transcriptional and (post) translational modification, as well as protein interactions may occur and are not considered within our analysis, therefore limiting the strength of transcript comparisons. However, some of these genes are interesting to consider in the context of MCMV infection, thus motivating a brief discussion. Interferon induced genes Following infection, IFN signaling is mediated by Janus and tyrosine-specific kinases that phosphorylate signal transducers and activators of transcription (STAT) proteins.14,15 The translocation of STAT proteins to the cell nucleus results in expression of multiple IFN-associated genes. As described before, both type I and II IFN (IFN-I and IFN-II) are induced after MCMV infection and are mediators in the antiviral response to CMV.14,16–18 Therefore, it is not surprising that multiple IFN-induced genes were increased in the salivary gland following MCMV infection. Multiple IFN-induced GTPases were identified in our RNAseq analysis. IFN can induce four families of GTPases: the guanylate-binding proteins (Gbp), the immunity-related GTPases (Irg), the Mx-proteins and the very large inducible GTPases.19,20 Although most of these GTPases have a low expression profile in mice, their transcription is promoted in response to interferon alpha (IFN-a), interferon beta (IFN-b) 107 and/or by IFN-g.20 Therefore, it was not surprising that the transcription of GTPases such as Interferon gamma-induced GTPase (Igtp); Gbp2; Gbp3; Interferon gamma inducible protein 47; Immunity-related GTPase family M (Irgm) 1 and 2 were significantly increased in the salivary glands by MCMV infection. These GTPases play an important role in resistance to intracellular pathogens such as Toxoplasma gondii , rabies virus and Mycobacterium tuberculosis through different mechanisms from lysosome fusion and acidification and autophagy.21–26 Although the role of these GTPases in viral infection is not as striking, some reports using human samples suggest that they can be linked to protection against hepatitis C virus and Influenza virus.27–29 Previous reports suggest that GTPases such as Irgm1 and Irgm3 are not critical for CMV control.30,31 Although the expression of these GTPases may only reflect the increased IFN-signal in the glands without significant antiviral role, this is still an interesting question to test since most of the functions of GTPases in CMV infection remain to be determined. For further studies it is important to note that the GTPases family and function differs in different species, being more extensive in mice in comparison to humans.26 IFN signal also induces other immune mediators such as IFN-induced transmembrane proteins (IFITM). IFITM proteins are constitutively expressed in multiple tissues, especially barrier epithelial cells and its expression is significantly promoted by both type I and type II interferons.32 Due to the increased IFN expression following MCMV infection, Not surprisingly, IFITM3 was significantly increased in infected salivary glands. IFITM proteins are antiviral mediators preventing entry, endosomal fusion and/or viral replication of several viruses such as: Influenza A virus, Vesicular stomatitis virus, West Nile virus, Dengue virus and Human immunodeficiency virus (HIV), but this effect is not universal.32–36 Most of the viruses, which entry is restricted by IFITM are thought to fuse depending on pH or cathepsin mechanisms, whereas nonsensitive viruses tend to fuse at the plasma membrane.37,38 Since HCMV entry in epithelial cells depend on both endocytosis and pH dependent fusion, it was surprising that IFITM3 expression cannot prevent viral entry in epithelial cells.39 Although HCMV is able to bypass IFITM3 to enter the cells and the fact that neither MCMV nor HCMV replication impair its expression, it is important to note that IFITM3 has other antiviral roles. IFITM3 expression in mice limits MCMV induced lymphopenia and the production of cytokines such as TNF-a and IL-6, which modulates T cell and Natural killer cell (NK) responses.40 Interestingly, following influenza virus infection IFITM3 expression in TRM in the lungs was antigen-driven and prevented these cells from being infected.41 Remarkably, IFITM3 promoted TRM survival and increased protection following subsequent viral challenges. Notably, the same protection conferred by IFITM3 expression in TRM occurs in the brain following vesicular stomatitis virus infection.42 As a parallel, MCMV- 114 Finally, it is relevant to mention that the changes caused by MCMV infection detected by the RNAseq and their potential roles will continue to be discussed in future sections. Besides the impact on the local gene expression, MCMV infection also modulates the cellular composition of the salivary glands. Subsequently, we will discuss the impact that MCMV infection has in T cell recruitment to the salivary gland and TRM differentiation. 3.1.2 Impact of MCMV infection in CD8+ T cell recruitment to the salivary glands and TRM differentiation For most tissues, inflammation and/or antigen are important for CD8+ T cell homing and TRM differentiation.110–115 Even in organs such as the skin, where TRM can develop in response to inflammation, antigen tend to promote enhanced TRM differentiation.113 Infection and the resulting inflammatory environment impact CD8+ T cells in many ways since antigen sensitivity, proliferative capacity, and their trafficking abilities.116–118 All of these can promote CD8+ T cell maintenance in the tissue and TRM differentiation. Variances in the infection, such as the different routes, resulted in different CD8+ T cell numbers in the salivary gland, which can indicate a role for the inflammatory status or for the presence of local antigen in the recruitment of CD8+ T cells. Therefore, based on what happens in most organs, it was tempting to think that local CMV viral infection and the resulting inflammation, could promote TRM differentiation in the salivary glands. 3.1.2.1 CD4+ T cells are not required for the differentiation of M38-specific TRM One key difference upon MCMV infection is the arrival of many different immune cells in the salivary gland. As referred, CD4+ T cell are crucial in controlling MCMV infection in this organ. Therefore, it is not unexpected that CD4+ T cells are present in significant numbers in the gland after MCMV infection.17,104,119 CD4+ T cells are key in licensing APC cells, promoting APC-CD8+ T cell-interaction and thus CD8+ T cell activation.120,121 CD4+ T cells also improve CD8+ T cell proliferation, cytotoxic activity and survival.122– 124 Naturally, CD4+ T cells impact multiple CD8+ T cell subsets and have been shown to be crucial in CD8+ TRM differentiation, maintenance and antiviral activity in the brain in the context of CMV infection.125,126 Help provided from CD4+ T cells additionally promotes CD8+ T cell migration to the female reproductive tract and TRM differentiation in the lungs.127,128 Therefore, a role for CD4+ T cells in promoting TRM could also be expected in the salivary gland. Surprisingly however, CD4+ T cell depletion did not impact the number of TRM in the salivary gland after MCMV infection (sup. figure 2). Although still not completely understood, it 115 seems that the dependence of CD4+ T cell diverges according to the organ studied. In fact, CD4+ T cells were also not required for TRM accumulation in the skin.129 It is likely that other homing molecules overcome the help provided by CD4+ T in migration of CD8+ T cells to other organs. Moreover, other immune cells such as DC and macrophages could provide differentiation signals such as transforming growth factor beta (TGF-b) and TNF-a, that would promote CD8+ T differentiation and retention, as happens in the intestine, and thus reducing the impact of CD4+ T cells.130 Even though our studies focused on a specific CD8+ T cell population (M38-specific), our data suggest that CD4+ T cell help is not crucial for MCMVspecific TRM differentiation in the salivary gland. Interestingly, CD4+ TRM were found in the salivary gland after MCMV infection and the interaction and dependence between these two subsets would also be a relevant topic to explore both in acute and latent times of infection. It is also curious to consider that reduced CD4+ T cells increase the MCMV viral load in the salivary gland and therefore the antigen burden in the organ.119,131 Intriguingly, neither significantly influenced the overall number of TRM. 3.1.2.2 MCMV replication impacts CD8+ T cell accumulation in the salivary glands but not the TRM differentiation Using Famcyclovir to prevent MCMV-TK virus replication, we further asserted that viral replication in the salivary gland was unnecessary for TRM differentiation (figure 2). Curiously, viral replication promoted early MCMV-specific CD8+ T cell accumulation in the salivary gland. Surprisingly however, the number of TRM in the salivary gland was comparable between mice infected with replicative and non-replicative virus (figure 2), which suggests that viral replication impacts CD8+ T cell accumulation in the salivary gland but did not significantly contribute to TRM differentiation. It is important to reinforce that these experiments did not exclude a role for antigen since viral DNA was still detectable in the salivary gland of some mice that were infected with a replicative-deficient virus (data not shown). This can possibly be explained by the migration of infected cells to the salivary gland. DC have been previously described as MCMV carriers, allowing for viral spread to the salivary glands, therefore it is possible that DC were initially infected with MCMV-TK and then migrated to the salivary glands.132,133 Even though the Famcyclovir treatment has been shown to avert viral replication, the presence of viral DNA in the salivary gland could provide antigen to drive TRM differentiation in this organ.134 For this reason, we chose the OT-I system to further investigate the role of tissue inflammation in TRM differentiation independently of antigen. 116 3.1.2.3 MCMV infection promotes early OT-I T cell recruitment to the salivary gland When we compared OT-I T cell recruitment in the salivary gland of naïve and MCMV infected mice in an antigen-independent system, OT-I T cell migration and early accumulation was promoted by MCMV infection (figure 4). This result is an interesting parallel with the increased accumulation of MCMV-specific CD8+ T cells in the salivary glands in the presence of virus replication (figure 2). Combined, these results suggest that MCMV viral replication transiently promotes CD8+ T cell accumulation in the salivary gland regardless of antigen recognition. Importantly, the augmented number of OT-I T cells in infected salivary glands at acute time points after infection was not due to cell proliferation (figure 4). Instead, the increased recruitment of OT-I T cells to infected salivary glands may reflect an enhanced ability of T cells to enter the salivary gland when MCMV infection is present. Woyciechowski, S. et al. (2017) have suggested that inflammation can increase the expression of vascular cell adhesion molecule (VCAM)-1 on the salivary gland´s vasculature, enhancing the recruitment of T cells expressing the corresponding receptor,+a4b1 integrin.135 The mechanism by which infection induces VCAM-1 expression was not explored but it is possibly mediated by IFN-g since it induces the expression of integrin ligands such as VCAM-1 in endothelial cell in other infections.136 This is a possible explanation for how infection promotes recruitment of immune cells in an antigen-independent way. Another factor that may help justify the increased recruitment of OT-I T cell to infected salivary glands is the tissue damage and the resulting production of inflammatory cytokines such as IL-1 and TNFa. These cytokines can induce Pand E-selectin expression by the vascular endothelium, both in human as in mice.137–140 Curiously however, no differences in integrin ligands or selectins were detected by the RNAseq between MCMV infected and naïve glands (table S1A-S1B), which makes these factors less likely to explain the difference in OT-I T cell recruitment between naïve and infected recipients. Nonetheless, it would be interesting to determine the levels of integrin ligands (such as VCAM-1) and selectins on the vasculature of the salivary glands early following MCMV infection using other techniques such as immunofluorescence. Alternatively, the environment that the OT-I T cells were exposed to before reaching the salivary gland differs in infected and naïve mice and can also contribute to the expression of selectin ligands by the T cells. Activated CD8+ T cells express enzymes required to fully produce the core 2 O-glycans, which allow for Pand E-selectin binding and the initial contact between T cells and the endothelium.141 Interestingly, cytokines such as IL-12 and IL-15, that have been detected in different tissues after MCMV infection, promote O-glycans expression in CD8+ T cells in vitro and thus enhance the ability of T cells to 117 bind to the vasculature.18,142,143 These different mechanisms, help hypothesize that the resulting cytokine production following MCMV infection may promote the vasculature-binding activity of CD8+ T cells and explain the initial increase of OT-I T cells seen in infected salivary glands. Besides integrins and selectins, the inflammatory status following MCMV infection also changes the chemokine profile in the salivary gland which can modulate CD8+ T cells recruitment in an antigenindependent way. CXCL9 and CXCL10 mRNA levels were increased in infected salivary glands in comparison to naïve salivary glands (figure 6) and therefore likely candidates to contribute to the improved recruitment of activated OT-Is. Surprisingly, the main chemokine receptor for CXCL9 and CXCL10 (CXCR3) had no significant impact in CD8+ T cell migration to infected salivary glands. This result proves even more astounding since all these chemokines have been implied in the trafficking and maintenance of T cells to different infected organs such as the liver, lymph nodes, genital mucosa and the skin in melanoma models.144–148 Nonetheless, the absence of a significant effect of CXCR3 deficiency in CD8+ T cell accumulation in infected mice can have several explanations. It is possible that MCMV infection of the glands triggers a broader expression of chemokines, which can diminish the singular importance of each chemokine/receptor in CD8+ T cell homing during infection. This hypothesis is even more likely if we consider that both MCMV-specific CD8+ T cells and OT-I T cell express multiple chemokine receptors after activation that were able to induce migration in vitro . Additionally, MCMV infection may not only alter chemokine expression in the organs, but also the expression of these chemokine receptors by the CD8+ T cells. In fact, IFN-g is important for the induction of CXCR3 on T cells.149 Therefore, it is plausible that MCMV infection and the increased levels of IFN-g in multiple tissues induced changes in CXCR3 expression by the OT-I T cells occurred after the adoptive transfer. This may have limited the effectiveness degree of the CXCR3 blocking antibody in the infected mice (figure 9). Consequently, it is important to note that early CD8+ T cell recruitment to infected salivary glands was not tested using CXCR3 KO OT-I T cells (experiments were performed using blocking antibodies). Therefore, a more significant difference in CD8+ T cell recruitment to infected salivary glands could have been seen comparing WT or CXCR3 KO OT-I T cells at early time-points after the adoptive transfer. Although the role of CXCR3 in T cell recruitment in naïve and infected salivary glands were not compared directly, it is unlikely that CXCL9 and CXCL10 explain the increased recruitment in infected salivary glands. Nonetheless, it is important to note that the chemokine receptor was the variable tested, thus the role of these specific chemokines in promoting CD8+ T cell recruitment was not directly verified. This becomes relevant since dimerization and promiscuous recognition of ligands by other chemokine 118 receptors has been described.150,151 If so, other chemokine receptor(s) besides CXCR3 may alternatively recognize CXCL9 and CXCL10. Therefore and although unlikely, we cannot exclude a minor role for these chemokines in CD8+ T cell recruitment to MCMV infected salivary glands. Another facilitator of CD8+ T cell recruitment and differentiation in the tissues is the presence of antigen.152–154 Although using the OT-I system makes it unlikely for OT-I T cell to recognize antigen in the recipient mice, since OT-I T cells express RAG genes we cannot exclude the impact of antigen in both OTI recruitment and TRM differentiation. However, this is very unlikely as the recipient mice do not express ovalbumin. Additionally, the presence of OT-I T cell clones that express a self-reactive endogenous achain are unlikely to have interfered with the results due to the rarity of these cells and due to the low variability among experiments. Additionally, prior to the transfers, over 80% of the OT-I T cells presented the classic Vα2+ Vβ5+ phenotype. Moreover, other groups have also shown the differentiation of TRM in RAG-/- models using other antigen-independent models.155,156 New data from the Snyder´s group (not shown - personal communication with Corinne Smith) replicated the differentiation of TRM in naïve mice after adoptive transfer of RAG-/- OT-I T cells. Thus, although it would be interesting to use RAG-/- OT-I T cells to further compare the cell accumulation and TRM differentiation in the gland of naïve and MCMV infected mice, we believe that the differences seen between these groups are more likely due to the virus-induced inflammatory environment than due to antigen recognition. Although we were unable to define which mediator (integrins, selectins and/or chemokines) promotes OT-I migration to the salivary gland early after MCMV infection, it is possible that these mechanisms have a synergic contribution. Further work will be needed to clarify the impact of MCMV in promoting the expression of homing molecules both in the CD8+ T cells and in the vasculature of the salivary glands. Defining these mechanisms would highlight important clues about CD8+ T cell trafficking and possibly allow us to improve CD8+ T cell migration and immune surveillance in the salivary gland. 3.1.2.4 MCMV infection does not impact the differentiation of a non-cognate TRM population It was surprising that the greater initial accumulation of OT-I T cells in infected salivary glands was not maintained in later time points of infection and that no differences were seen in the overall OT-I TRM between naïve and MCMV infected mice (figure 4). It is conceivable that after the peak of viral replication in the salivary gland, the inflammatory cues and factors that promoted the early OT-I T cell accumulation in the infected salivary glands are no longer present explaining the lack of differences in 119 OT-I T cell numbers at a later time after adoptive transfer. This loss of the early advantage may also imply differences in retention and survival cues for CD8+ T cells. CD69 expression promotes CD8+ T cell retention and maintenance within non-lymphoid organs by decreasing the S1P signal.157,158 According to our RNAseq data, CD69 inducers such as TNF-a and IL33 are expressed in the salivary glands of both naïve and infected mice. These cytokines could promote CD69 expression in both groups and contribute to similar late accumulation of OT-I T cells and TRM differentiation. However, the RNAseq data does not exclude local expression niches within the infected glands that may promote this process. Another possible explanation for the similar number of TRM in both naïve and infected glands relies on the expression of survival modulators. An interesting factor is the P2X purinoceptor 7 (P2RX7) that has been described in Lymphocytic choriomeningitis virus (LCMV)-specific TRM in the salivary gland and is induced by IL-12.159 The lack of P2RX7 did not significantly alter the frequency of LCMV-specific CD8+ T cells in the gland (most of which presented a TRM phenotype). However, by recognizing NAD+ and extracellular nucleotides ATP produced upon tissue damage and inflammation, P2RX7 was able to decrease TRM survival in vitro. 159 Therefore, it is conceivable that the inflammation caused by MCMV increases local NAD+ and extracellular nucleotide, which could represent a disadvantage for P2RX7 expressing TRM. This could help to explain the similar number of TRM in the infected and naïve mice. It is possible that this effect is intensified in an antigen-independent model, since TCR signal and antigen recognition reduces the expression of P2RX7.159 The restriction of P2RX7 expression caused by TCR signaling may function as a safety mechanism preventing the establishment of unrelated TRM that could compete with the differentiation of cognate-specific TRM within the tissue. Interestingly, and contrarily to what was described above, CD8+ P2RX7-/- cells were reduced in the salivary gland in comparison to WT cells after LCMV infection.160 Similarly, after LCMV infection P2RX7 was important for the generation of CD103hi TRM in organs such as the small intestine.160 Due to multiple and distinct described roles of this receptor in TRM, it would be interesting to measure the P2RX7 expression in TRM over time in the salivary gland in both naïve and MCMV infected mice (in the presence and absence of cognate antigen). Moreover, survival cues can be dependent on the nutrition and blood supply available to the salivary gland. It is likely that acute MCMV infection increases the vessel permeability allowing for extra blood supply and nutrients to the glands. As for the epidermis, it is possible that the OT-I survival and the TRM differentiation in the salivary gland are shaped by the blood supply and nutrients available.161 If that is the case, it is possible that MCMV infection increases the recruitment of OT-I cells to the salivary glands 120 initially in infected recipients, though, at later times with less inflammation, the cells that remain in the gland are the limited cells that are able to survive under more hypoxic conditions and differentiate into TRM. It is important to mention that this plateau in OT-I T cells and TRM in the salivary glands can also represent a limitation of our model since a restricted number of OT-I T cells were transferred. Within the transferred cells, it is plausible that a certain proportion of TRM precursors were transferred limiting the resulting TRM number. The transfer of subsequent populations of activated OT-I cells might help us determine the limit of TRM differentiation. Nonetheless, it is crucial to understand which precursor cells become TRM under certain conditions. This might be accomplished by further in vitro characterization of the cells that can become CD103+ and CD69+ associated with subsequent in vivo depletion studies. Our data suggest that MCMV replication and the resulting inflammatory environment may promote CD8+ T cell recruitment to the salivary gland but does not seem to impact TRM differentiation. It is important to note that, although antigen is not required for TRM differentiation, our work cannot exclude a role for antigen in promoting this process. One way to further test the importance of antigen would be to use OT-Is from RAG -/- mice as explained before. It would be interesting to compare naïve recipients as well as MCMV and MCMV-Ova infected mice and the resulting differentiation of TRM with the proper normalization for transferred cells, expansion and proliferation. 121 PART 3.2 Mechanisms involved in TRM differentiation in naïve salivary glands 3.2.1 Mediators of CD8+ T cell homing to naive salivary gland Neither a certain route of immunization (figure 1) or MCMV infection (figure 3 and 4) significantly increased the ability of the activated CD8+ T cells that reached the salivary glands to differentiate into TRM. Then, the migration of CD8+ T cells and specifically TRM precursors to the salivary glands seems to be an important determining factor for TRM differentiation and therefore worth exploring and characterizing. a4b1 mediates CD8+ T cell recruitment to salivary gland Our data demonstrated that, as happens in infected salivary glands, α4β1 expression also contributes to CD8+ T cell migration to naive salivary glands (figure 5). This suggests that α4β1 ligands (as VCAM-1) are expressed in the salivary gland at sufficient levels at a steady state to promote CD8+ T cell entry. Although it was already shown that LCMV infection and inflammation induce VCAM-1 expression in the salivary glands’ vasculature, previous reports fail to identify VCAM-1 in the salivary gland of control C57BL/6 (B6) mice.135,162 To further complement our results, the VCAM-1 expression in the vasculature of naïve mice needs to be assessed. This could be achieved using immunofluorescence and its function tested using blocking antibodies or mouse models with VCAM-1 conditional deletion. The same evaluation should be performed to other α4β1 ligands, especially because epithelial cells in the salivary glands produce other ligands under steady state conditions such as fibronectin, which have not been tested in the context of T cell homing to the salivary glands. Role of chemokines in CD8+ T cell migration to the salivary gland Chemokines are other crucial mediators of CD8+ T cell migration to the organs, as seen by the crucial role of CCR5 in a rapid recruitment of memory CD8+T cell to the airways following some viral infections.163–166 Besides recruitment, chemokines are also critical for the localization within the tissue. This is important for T cells to receive the appropriate signals and resident cues that mediate TRM differentiation. These two chemokine roles are evident by the CXCL17-CXCR8 interaction that promotes mobilization of CD8+ T cells to and within the female genital tract, leading to protection against HSV-1.167 Inflammation and infection is thought to promote chemokine expression in the affected tissues allowing for an increased recruitment of immune cells and consequent protection.168–171 CMV is thought to induce chemokines in different organs such as the spleen and liver where CXCR3-CXCL9/CXCL10 122 interactions promote CD8+ T cell infiltration and IFN-g responses.147 Surprisingly however, our RNA-Seq data showed minor changes in chemokine expression between the salivary glands of naïve and MCMV infected mice (figure 6). Several chemokines were indeed constitutively expressed in the salivary gland of naïve mice at a steady state (figure 6). Interestingly, these chemokines expressed in naïve glands can be recognized by receptors expressed by CD8+ T cell and had the potential to induce CD8+ T cell migration in vitro (figure 7 and figure 8). Since chemokines are usually prominent upon infection or tissue damage, it is fascinating to think of explanations for the lack of more striking differences in chemokine expression between naïve and MCMV infected salivary glands. We believe that the presence of food antigens and microbiota in the salivary glands might contribute to this surprising basal expression of chemokines. The impact of food antigens in the inflammatory and chemokine profile in the tissues is still not fully characterized. In fact, the recognition of food antigen tends to be associated with food allergies and increased IgE and Th2 responses.172,173 However, multiple changes occur in response to food antigens, especially in the gastrointestinal (GI) tract.174 For example, dietary nucleotides increase the proportion of γδ+ IEL in the gut, while alkylamine antigens found in mushrooms, apples and edible plants promote γδ+ T cells expansion.175,176 γδ+ T cells have also been described in the salivary glands and if a similar mechanism promotes their presence and expansion, γδ T cells can shape the gland environment and indirectly induce CD8+ T cell recruitment, by producing CCL3, CCL4 and CXCL10.177–180Therefore, it is possible that, under certain conditions, food antigens can be a sufficient trigger to promote local immune responses and chemokine production. This is appealing to consider in organs of the GI tract that have contact with food-derived antigens such as the salivary gland. Nevertheless, food is not the only source of antigens that can promote an immune response in the gland. Salivary glands are in communication with the oral cavity and retrograde migration of bacteria from the oral cavity through the salivary ducts has been proposed (retrograde theory of sialolithiasis formation).181,182 Therefore, it is tempting to think that the contact with microbiota may contribute to the basal chemokine and pro-inflammatory levels in naïve salivary glands.183–185 Microbiota has indeed been linked to T cell homing modulation.186 Microbial association to germ-free mice resulted in increased CD8+ T cells in the intestine.187 Microbiota was also associated with the differentiation of residing γδT cells in the intestine.188,189 Furthermore, some reports suggest that probiotics such as Lactobacillus acidophilus can modify the inflammatory status of the intestinal epithelial cells, leading to cytokine and chemokine production.185 The mechanisms by which microbiota induce immune cell recruitment are not totally clear but might be due to the antimicrobial function of some chemokines such as CXCL9, CXCL10 and CXCL11.190,191 Therefore, it is possible that organs that contact regularly with microorganisms, such as the 123 GI tract and the salivary glands, constitutively express chemokines with anti-microbial properties that further promote the recruitment and maintenance of immune cells. This hypothesis possibly helps in explaining the constitutive expression of chemokines in the salivary gland (figure 6) and the surprising role of CXCR3 in CD8+ T cell migration to the salivary gland in naïve mice (figure 10). The characterization of the ligand present in naïve salivary glands that promotes CXCR3-dependent CD8+ T cell migration still needs to be performed. Although only CXCR9 and CXCL10 are classical CXCR3 receptor ligands in B6 mice, chemokine receptors have been shown to heterodimerize with other chemokine receptors as referred before. CXCR3, in particular, can heterodimerize with other receptors such as CXCR4.151,192,193 Since CXCR4 was also expressed by CD8+ T cells in the salivary gland, it is still formally possible that nonclassical receptor/ligand combinations are responsible for recruiting T cells to the salivary glands. Thus, due to heterodimerization of chemokines and their receptors, there may be some variability in the ability/affinity of ligands to bind CXCR3, which shapes the OT-I T cell recruitment. Our results are also limited since the role of CXCR3 receptor in CD8+ T cell localization within the salivary gland was not systematically done. Although our preliminary results using immunohistochemistry did not show differences in location between CXCR3 WT or KO cells in the salivary gland parenchyma (data not shown), additional experiments comparing several sections of the gland and different times after transfer are needed to surely answer this question. Although CXCR3 expression was not required for TRM differentiation in MCMV-infected salivary glands (figure 9), it is still exciting to question if CXCR3 overexpression could promote CD8+ T cell migration, especially to naive salivary glands and thus TRM differentiation. This would be an advantage for the development of vaccines against infections that target the gland. To test this hypothesis CXCR3 expression could be achieved by either modulating the in vitro activation conditions (antigen and IFN-g levels); by using a vector systems or preferentially using an inducible set-up such as Cre-inducible mice, which will allow for a controlled CXCR3 expression.149 That way, it would be possible to determine the role of this chemokine in CD8+ T cell migration but also in different steps of TRM differentiation. 3.2.2 Cues for TRM differentiation in naïve salivary glands More surprising than activated CD8+ T cells being recruited to naïve salivary glands, was the fact that similar proportions of TRM differentiated in salivary glands of naïve and MCMV-infected mice (figure 4). Although this study does not define the in vivo inducers of TRM differentiation, according to our results, the salivary gland at a steady state must have the necessary cues for TRM differentiation. The signals that promote TRM differentiation are not clear for all the organs but TNF-a,+IL-33 and TGF-b have been implied 130 3.4 Final considerations In sum, our results provide further evidence that a portion of activated CD8+ T cells differentiate into TRM in both uninfected and MCMV infected salivary glands. Although differences in gene expression were seen in the salivary glands after MCMV infection, these differences did not correlate with changes in TRM differentiation. Moreover, the characterization of homing receptors and mediators of CD8+ T cell migration to the salivary glands was conducted. CD8+ T cell migration to the gland was promoted by the expression of CXCR3 (in naïve mice) and a4b1 (both naïve and MCMV infected mice). 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