Glycans as key checkpoints of T cell activity and function

REVIEW published: 27 No embe 2018 doi: 10.3389/ immu.2018.02754 F on ie s in Immunology | www. on ie sin.o g 1No embe 2018 | Volume 9 | A icle 2754 Edi ed by: Jasmeen S. Me zaban, King Abdullah Uni e si y o Science and Technology, Saudi A abia Re iewed by: Ma in J. Riche , McGill Uni e si y, Canada Y e e Van Kooyk, VU Uni e si y Medical Cen e , Ne he lands *Co espondence: Salomé S. Pinho [email p o ec ed] †These au ho s ha e con ibu ed equally o his wo k Special y sec ion: This a icle was submi ed o T Cell Biology, a sec ion o he jou nal F on ie s in Immunology Recei ed: 31 July 2018 Accep ed: 08 No embe 2018 Published: 27 No embe 2018 Ci a ion: Pe ei a MS, Al es I, Vicen e M, Campa A, Sil a MC, Pad ão NA, Pin o V, Fe nandes Â, Dias AM and Pinho SS (2018) Glycans as Key Checkpoin s o T Cell Ac i i y and Func ion. F on . Immunol. 9:2754. doi: 10.3389/ immu.2018.02754 Glycans as Key Checkpoin s o T Cell Ac i i y and Func ion Má cia S. Pe ei a1,2,3†, Inês Al es1,2,4†, Manuel Vicen e1,2,3, Ana Campa 1,2,3,5, Ma iana C. Sil a1,2, Nuno A. Pad ão1,2,4, Vanda Pin o1,2, Ângela Fe nandes1,2, Ana M. Dias1,2 and Salomé S. Pinho1,2,4* 1Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o (IPATIMUP), Po o, Po ugal, 2Ins i u e o Resea ch and Inno a ion in Heal h (I3S), Po o, Po ugal, 3Ins i u e o Biomedical Sciences o Abel Salaza , Uni e si y o Po o, Po o, Po ugal, 4Medical Facul y, Uni e si y o Po o, Po o, Po ugal, 5Cen o Hospi ala do Po o, Po o, Po ugal The immune sys em is highly con olled and ine- uned by glycosyla ion, h ough he addi ion o a di e si y o ca bohyd a es s uc u es (glycans) o i ually all immune cell ecep o s. Despi e a ela i e backlog in unde s anding he impo ance o glycans in he immune sys em, due o i s inhe en complexi y, ema kable indings ha e been highligh ing he essen ial con ibu ions o glycosyla ion in he egula ion o bo h inna e and adap i e immune esponses wi h impo an implica ions in he pa hogenesis o majo diseases such as au oimmuni y and cance . Glycans a e implica ed in undamen al cellula and molecula p ocesses ha egula e bo h s imula o y and inhibi o y immune pa hways. Besides being ac i ely in ol ed in pa hogen ecogni ion h ough in e ac ion wi h glycan-binding p o eins (such as C- ype lec ins), glycans ha e been also shown o egula e key pa hophysiological s eps wi hin T cell biology such as T cell de elopmen and hymocy e selec ion; T cell ac i i y and signaling as well as T cell di e en ia ion and p oli e a ion. These e ec s o glycans in T cells unc ions highligh hei impo ance as de e minan s o ei he sel - ole ance o T cell hype - esponsi eness which ul ima ely migh be implica ed in he c ea ion o ole ogenic pa hways in cance o loss o immunological ole ance in au oimmuni y. This e iew discusses how speci ic glycans (wi h a ocus on N-linked glycans) ac as egula o s o T cell biology and hei implica ions in disease. Keywo ds: N-glycosyla ion, glycans, T cells, immune esponse, au oimmuni y, sel - ole ance INTRODUCTION The immune sys em is highly egula ed by a se ies o s imula o y and inhibi o y pa hways ha a e c ucial o main ain a heal hy and balanced sys em. Dis up ion o he con ol o his immunological balance can esul in abno mal s imula o y signals associa ed wi h he loss o immune ole ance in au oimmuni y o in he c ea ion o abe an immunosupp essi e ne wo ks ha occu in cance . Accumula ing e idences ha e been demons a ing he impo ance o glycans and glycans binding p o eins [including galec ins (1,2), C- ype lec ins (3), and sialic acid-binding immunoglobulin- ype lec ins (siglecs) (4,5)] in he egula ion o bo h inna e and adap i e immune esponses. In ac , all cells a e co e ed wi h a dense coa o glycans ha cons i u e a majo molecula in e ace be ween cells and hei en i onmen . The di e si y o glycans p esen a ion a cell su ace is eno mous, encoding a my iad o impo an biological in o ma ion ha emains o be ully cha ac e ized. Glycosyla ion is he enzyma ic p ocess esponsible o he a achmen o glycans (ca bohyd a es) o Pe ei a e al. Glycans as Checkpoin s in T Cells Biology p o eins o lipids (p edominan ly ia ni ogen (N) and oxygen (O) linkages), a p ocess ha occu s in he Endoplasmic Re iculum/Golgi compa men o essen ially all cells being media ed by he coo dina ed ac ion o a po olio o di e en glycosyl ans e ases and glycosidases enzymes (6). The p ope de elopmen and unc ion o he immune sys em elies bo h on he dynamic egula ion o he exp ession o glycan-s uc u es and glycan-binding p o eins, and he in e ac ions be ween hem (7). This e iew discusses he ole o glycans (wi h a ocus on N-linked glycans) on T cells biology and unc ion, including T cell de elopmen , ac i a ion, di e en ia ion, and signaling. This dynamic in e play be ween glycans and T cells ac i i y con olling bo h au o- eac i i y and sel - ole ance will be p esen ed and discussed (Figu e 1). GLYCANS IN T CELL DEVELOPMENT AND THYMUS SELECTION T cells a e de eloped in he hymus whe e a mic oen i onmen is se , which enables he selec ion o T cell ecep o s (TCRs) o gene a e a di e se epe oi e o po en ial an igen ecogni ion (8). Lymphoid p ogeni o s om he bone ma ow en e in o he co ical issue o he hymus, whe e hey s a o expand and de elop (9,10). Despi e he ac ha he ole o glycosyla ion in T cell de elopmen and hymus selec ions s ill emains o be ully unde s ood, some impo an indings highligh he ele ance o glycans in his p ocess (Figu e 2). Role o Glycans in Thymus Seeding and T Cell Lineage Commi men The ini ial s ep o T cell de elopmen , he a icking o hymus-seeding p ogeni o s (TSPs) o he hymus, is an ac i e p ocess ha elies on he exp ession o P-selec in in he hymic epi helium and i s pa ne , P-selec in glycop o ein ligand-1 (PSGL-1), exp essed by ci cula ing TSPs-de i ed om he bone ma ow (11). The exp ession and pos - ansla ional modi ica ions o PSGL-1 a e egula ed in bone ma ow p ogeni o s. The de iciency o α1,3 ucosyla ion on PSGL-1, equi ed o i s binding o P-selec in, was shown o be associa ed wi h he impai men o TSPs homing in o he hymus (12). Once TSPs en e he hymus, hey de elop in o ea ly hymocy e p ogeni o s (ETPs), a subse o he CD4−CD8−double nega i e 1 (DN1) popula ion, which gi e ise o mul iple lymphoid lineages (8). The conse ed No ch signaling pa hway is esponsible o he commi men o DN1 hymocy es o he T cell lineage (13). The glycosyla ion p o ile o No ch ecep o s (and ligands) was shown o egula e No ch-dependen in acellula signal ansduc ion. The luna ic, manic, and adical F inge a e he glycosyl ans e ases ha modi y No ch ecep o s by ans e ing N-ace ylglucosamine (GlcNAc) o O-linked ucose glycans o epide mal g ow h ac o -like (EGF-like) epea s, p esen in he ex acellula domain o No ch, and desc ibed o egula e i s cell- su ace signaling and unc ion (14,15). Loss o he h ee F inge glycosyl ans e ases leads o a educed binding o No ch o Del a- like ligands (DLL), namely DLL4, al e ing he equencies o se e al T cell subse s in he hymus (16). The i s indica ion ha F inge-media ed No ch glycosyla ion was in ol ed in T cell de elopmen was shown when he luna ic F inge gene, L ng, was misexp essed unde a lck-p oximal p omo e (17). This al e a ion o he No ch glycosyla ion p o ile (lack o GlcNAc in he EGF-like epea s) esul ed in a la ge B cell popula ion de eloped om lymphoid p ogeni o s in he hymus. In ac , u he wo k showed ha L ng is poo ly exp essed in CD4+CD8+ double posi i e (DP) hymocy es, bu when ec opically exp essed in ha popula ion (unde lck-p oximal p omo e ), led o an inc eased binding o No ch o i s ligands on s omal cells, blocking DN de elopmen , and enabling B cell di e en ia ion (18). These s udies also e ealed ha changes in he glycosyla ion o No ch ac oss T cell de elopmen also impac s on i s signaling pa hway. A DN s ages, he eac ions ha d i e de elopmen a e dependen on No ch in e ac ions wi h DLLs, which exis a unc ionally limi ing concen a ions. The high le els o L ng exp ession in DNs acili a e No ch in e ac ions wi h DLLs and he d ama ic down egula ion o L ng in DPs coincides wi h No ch-independen eac ions o T cell de elopmen . The inal commi men o he T cell lineage occu s a he DN3 s age, whe e a ecombina ion-ac i a ing genes (RAG)-media ed p oduc i e ea angemen o he Tc b leads o he exp ession o he ß chain o he TCR (TCRß) and he o ma ion o a p e-TCR signaling complex (13,19). Role o Glycans in Thymocy e ß Selec ion Toge he wi h No ch and In e leukin (IL)-7, he p e-TCR signaling ini ia es ß-selec ion, by inducing he down egula ion o he RAG complex exp ession (Rag1 and Rag2) in quiescen DN3 (DN3a), becoming la ge cycling DN3 hymocy es (DN3b), which di e en ia e in o DN4 cells. A de icien p e-TCR signaling in lck- null cells is escued by L ng o e exp ession, bu no in a Rag2−/− backg ound, indica ing a p e-TCR dependency o de elopmen (20). Upon ß-selec ion, i was ecen ly demons a ed ha DN4 cells up egula e glucose and glu amine me aboli es ha en e in o he hexosamine pa hway, inc easing he p oduc ion o UDP-GlcNAc, which is needed o unde go clonal expansion (8,21). The UDP-GlcNAc is also he subs a e o he O- GlcNAc ans e ase (OGT) in he p ocess o O-GlcNAcyla ion o in acellula p o eins on se ine and h eonine esidues (22). Recen e idences showed ha O-GlcNAcyla ion egula es he p ocess o T cell de elopmen (23). Using a condi ional knockou mouse model o OGT in he DN s age, i was shown a educed popula ion o DPs, indica ing ei he a de iciency on ß-selec ion o in clonal expansion o DN4s. The absence o OGT appea ed no o impac sel - enewal o DNs, o hei di e en ia ion in o DPs, bu o p omo e he ailu e o he clonal expansion o DN4, in esponse o No ch ligands. A eedback mechanism was p oposed in which he me abolic changes ( he shi o glycolysis) ha suppo he DN- o-DP s age o hymocy e di e en ia ion, con olled by No ch, induces c-Myc exp ession, which in u n con ols he a e o T cell nu ien up ake as well as he exp ession o OGT and consequen ly he abundance o O-GlcNAc (15). The O-GlcNAcyla ion o c-Myc was also shown o inc ease i s s abili y (24), u he con ibu ing o he eedback loop. In he s age o pos -βselec ed DN4 hymocy es, i was seen a 10- old inc ease in exp ession o ST6 β-Galac oside F on ie s in Immunology | www. on ie sin.o g 2No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology FIGURE 1 | Glycans as a majo connec i e chain ha con ols T cell esponse in ei he a ole ogenic o immunos imula o y scena io. Glycosyla ion appea s o be cen al in egula ing se e al s eps o a T cell’s li e. Du ing T cell de elopmen , di e en popula ion o T cells (ETP, ea ly hymocy e p ogeni o ; DN1, 2, 3, and 4, double nega i e; DP, double posi i e; SP, single posi i e) display speci ic glycosyla ion pa e ns. The no mal glycosyla ion o SP popula ion esul s in an educa ed T cell unc ion. Howe e , by gene ic, en i onmen al o me abolic cons ains, T cell glycosyla ion can be comp omised e-di ec ing immune sys em owa d an immunos imula o y o ole ogenic esponse. Glycans a e p oposed he e as key playe s in immune-unbalanced diseases, such as au oimmuni y and cance . α2,6-Sialyl ans e ase 1 (ST6Gal I) when compa ing o he DN3 popula ion, which esul ed in an inc ease in α2,6-linked sialic acid (25). Acco dingly, in ST6Gal1 de icien mice, he DN popula ions we e dec eased, beginning a he DN1 subse . Mic oa ay da a showed a down egula ion o CD96 ( ecep o molecule o nec in-1, ha plays a pu a i e ole in cell mig a ion) in he DN2 and DN3 popula ions in he ST6Gal1 de iciency backg ound, and a dis up ion o hymopoiesis in hese mice was p oposed. Mo eo e , ST3 β-Galac oside α2,3-Sialyl ans e ase 1 (ST3Gal I) exp ession is dec eased in mos DN and in all DP, only inc easing in single-posi i e (SP) hymocy es (26). In ST3Gal1−/−mice, he TCR epe oi e was signi ican ly al e ed, indica ing a ole o sialyla ion in hymocy e selec ion (27). Role o Glycans in Posi i e and Nega i e Selec ion in he Thymus The ß-selec ed DN4 cells unde go apid sel - enewal, gi ing ise o a clonally expanded popula ion, ha di e en ia e in o DP CD4+CD8+ hymocy es (8). In his de elopmen al s age, ma u e TCRαß ecep o s a e o med (28) and he exp ession o he co- ecep o s CD4 and CD8 con e a MHC class II and class I es ic ion o TCR ac i a ion, espec i ely. The newly o med ma u e TCRs a e hen sc eened by hymic epi helial cells (TECs) by he speci ici y and binding s eng h o he MHC ligands p esen ed. The nex de elopmen al p ocess is named posi i e selec ion, whe e he DP popula ion is en iched o cells ha exp ess an immunocompe en TCR (8,29). The selec ed DPs hen commi o he SP CD4+o CD8+lineage and go h ough a p ocess called nega i e selec ion, which elimina es au o eac i e T cells (8,29). The a ini y o he co ec ly assembled TCRαβ o he MHC-an igen complexes de e mines cell su i al and di e en ia ion. Glycosyla ion modi ica ions o he TCR may p o ide an al e na i e mechanism o con ol posi i e and nega i e selec ion by di ec ly a ec ing he TCR-MHC-an igen binding, TCR in e ac ion wi h i s co- ecep o s and he h eshold o ac i a ion (30), an issue ha is a om being ully elucida ed. F on ie s in Immunology | www. on ie sin.o g 3No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology FIGURE 2 | The hallma ks o glycans in T cell biology. N-glycans ha e a b oad e ec on he mul iple T cell unc ions wi h impac bo h in au o eac i i y and in immune ole ance. Pa icula ly, he complex b anched N-glycans ca alyzed by be a 1,6-N-ace ylglucosaminyl ans e ase V (GnT-V) (encoded by MGAT5 gene) ha e been demons a ed o con ol di e en T cells unc ions by a ge ing di e en T cells ecep o s (such as TCR, CD25, and CD4) and he e o e egula ing T cell p oli e a ion, T cell di e en ia ion, T cell signaling as well as he p oduc ion o in lamma o y cy okines. Al e a ions on GnT-V ac i i y bu also in alpha-mannosidase II (α-MII) as well as in N-ace ylglucosaminyl ans e ase I (GnT-I, MGAT1 gene) and II (GnT-II, MGAT2 gene) ac i i y we e shown o comp omise T cell homeos asis being associa ed wi h he de elopmen o se e al au oimmune diso de s in humans and mouse models (such as EAE, IBD, SLE, TID). The FUT8-media ed co e ucosyla ion o TCR was associa ed wi h hype ac i a ion o CD4+T cells (T cells au o eac i i y) whe eas he modi ica ion o he co-inhibi o y ecep o s (CTLA-4 and PD-1) by FUT8-media ed co e ucose esul s in immune ole ance. The T cell de elopmen and T cell sel - enewal a e con olled by GnT-I-media ed glycosyla ion and by O-GlcNAcyla ion h ough OGT (O-GlcNAc ans e ase), espec i ely. The subuni s o he TCRαβ con ain a leas 7 po en ial si es o N-linked glycosyla ion and he TCR-CD3 complex is es ima ed o ha e 12 N-glycan addi ion si es ha con ibu e o TCR olding and unc ions (31,32). Indeed, selec i e emo al o conse ed N- glycosyla ion si es o he cons an egions o he TCR, enhanced i s unc ional a idi y ( he sensi i i y o he T cell esponse o o he cell which ca ies he espec i e MHC-pep ide) (32). Howe e , whe he N-glycosyla ion in he a iable egions o he TCR a ec i s selec ion emains o be add essed. Mo eo e , low le els o sialyla ion in DPs a e associa ed wi h binding o Majo His ocompa ibili y Complex (MHC) class I (common o all nuclea ed cells) and he inc eased exp ession o sialic-acid linkages on di e en ia ed SP CD8+ hymic T cells was shown o dec ease he binding a idi y o CD8 o MHC class I molecules, which ac s as a egula ion o a TCR a ini y dependen nega i e selec ion (33). Fu he mo e he de iciency o he Mga 5 gene, ha encodes o a Golgi b anching enzyme N-ace ylglucosaminyl ans e ase V (GnT-V) was shown o ma kedly inc eases TCR clus e ing and signaling a he immune synapse, esul ing in a lowe T cell ac i a ion h eshold and inc eased incidence o au oimmune disease in i o and in human (30). In a model o posi i e selec ion, i was demons a ed ha b anching N-glycosyla ion dynamically expands he a ini y spec um o posi i e selec ion by di e en ially con olling bo h he lowe and uppe limi s o posi i ely selec ed TCR-MHC-an igen in e ac ions (34). The in acellula domains o CD4 and CD8 co- ecep o s bind Lck, enhancing TCR esponses o low-a ini y MHC-an igen complexes when coupled o he TCR (35). Bo h co- ecep o s ha e N-glycosyla ion si es and i was shown ha he b anching de iciency in Mga 1 / Lck-C e+T cells esul ed in dec eased su ace exp ession o CD4 and CD8 ecep o s (34). The lack o b anched N-glycans in he same gene ic backg ound also dec eased TCR h eshold signaling (30). These e idences suppo ed ha b anching N-glycans display an impo an ole in he ma u a ion o DN cells and/o TCR selec ion. Changes in he exp ession o O-linked glycans also impac T cell de elopmen by modula ing galec in binding. Galec in-1 was shown o induce apop osis o imma u e hymocy es h ough binding o co e 2 O-glycans exp essed in CD43 and CD45 (36). In con as , CD45 on ma u e hymocy es bea s co e 1 O-glycans as well as N-glycans capped wi h α2,6-linked sialic acid, which inhibi s galec in-1 binding (36). O e all, glycosyla ion appea s o play a c i ical ole in he di e en s ages o hymocy e de elopmen and in he gene a ion o an e icien immune sys em. Ne e heless, u he esea ch is F on ie s in Immunology | www. on ie sin.o g 4No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology needed in o de o unde s and how glycans con ol each s age o hymocy es de elopmen , di e en ia ion and selec ion, which migh e eal no els insigh s on he in luence o he glycome in majo diseases, such as au oimmuni y and cance . GLYCANS IN THE REGULATION OF T CELL ACTIVITY AND FUNCTIONS The p ope unc ion o T lymphocy es is highly dependen on hei su ace ecep o s, which in u n a e highly media ed by glycosyla ion. Al hough O-glycan s uc u es ha e been shown o play impo an oles on immune-associa ed molecules (37), he p ominen ole o N-linked glycans is emphasized in his sec ion (Figu e 2). As p e iously men ioned, MHC I is exp essed by almos all nuclea ed cells and in e ac s wi h TCRs on CD8+T cells; in u n, MHC II is exp essed by p o essional an igen p esen ing cells (APCs) (dend i ic cells - DC, mac ophages, B cells and TECs) and is ecognized by CD4+T cells (7,38). Mo e han 3 decades ago, i was demons a ed ha blocking MHC1a N-glycosyla ion, h ough accep o si e mu a ion, esul s in signi ican inc eases in in acellula mis olded p o ein along wi h dec eases in cell su ace exp ession (39). MHC II is assembled by wo glycop o eins, αand βchains. The αchain con ains N-linked high-mannose and complex glycans whe eas he βchain is only cons i u ed by complex N-glycans (40). In con as o he ole o MHC I, MHC II glycosyla ion was shown o ha e a pa icula impac on he e ec i e an igen binding, as well as in he p esen a ion o mic obial ca bohyd a e an igens, which consequen ly in luences downs eam T cell esponses. This was demons a ed by he deple ion o he Mga 2 gene, which comp omises N-glycan b anching, dec easing ca bohyd a e an igen p esen a ion by MHC class II and leading o loss o T cell s imula o y ac i i y (41). Du ing TCR signal ansduc ion, glycans play a key ole in s abilizing indi idual molecules in he complexes a he immunological synapse and by p o ec ing hem om he ac ion o p o eases du ing T cell engagemen (31). Addi ionally, glycans can also es ic nonspeci ic p o ein-p o ein in e ac ions, like agg ega ion o TCRs on he memb ane, helping o o ien he in e ac ions o he p o eins in he cen al clus e s (31). Deme iou e al. demons a ed ha β1,6-GlcNAc b anched N- glycans s uc u es (ca alyzed by GnT-V) egula e T cell ac i i y, namely in CD4+T cells by inc easing he h eshold o T cell ac i a ion, supp essing T cell g ow h and signaling (30,42). Mo eo e , co e- ucosyla ion, which e e s o ucose a ached o he inne mos N-ace ylglucosamine o N-linked glycans, ca alyzed by α1-6 ucosyl ans e ase (FUT8), was also shown o a ec T cell ac i i y in immune media ed diso de s (42,43). The T cell ac i i y is also dependen on glycosyla ion o co- ecep o s, such as he complex o ma ion be ween TCR and CD45. Galec in-3 is a key media o o his complex, by es ablishing a molecula la ice h ough binding o polylac osamine s uc u es in b anched N- glycans. Consequen ly, CD45 phospha ase ac i i y induces down egula ion o T cell signaling, p e en ing T cell ac i a ion (44). Fu he mo e, CD45 is al e na i ely spliced in o i e di e en iso o ms on human leukocy es (CD45ABC, CD45AB, CD45BC, CD45B, and CD45RO) (45–47), all deco a ed wi h up o 11 N-glycans in he memb ane p oximal egion. Impo an ly, all iso o ms p esen di e en glycosyla ion p o iles (48,49), ha change du ing T cell di e en ia ion and ac i a ion (50,51), as e iewed in (36). CD28 is ano he T cell su ace glycop o ein ac ing as a seconda y signaling molecule o T cell ac i a ion. In e es ingly, nea ly 50% o he molecula mass o CD28 is cons i u ed by N-glycans (52). P e ious s udies epo ed ha N-glycosyla ion o human CD28 can nega i ely egula e CD28-media ed T cell adhesion and co-s imula ion, namely he in e ac ion be ween CD28/CD80. Mu a ion o all po en ial N-linked glycosyla ion si es o CD28 as well as ea men o Ju ka cells wi h inhibi o s o N-glycosyla ion pa hway esul ed in a de ec i e CD28 glycosyla ion wi h enhancemen o he binding o CD80 exp essed on APCs (52). The b anching N-glycosyla ion o CD25 ecep o also modula es i s cell su ace e en ion con olling T di e en ia ion wi h impac in immune ole ance. Recen ly, i was demons a ed ha a dec eased UDP-GlcNAc and complex b anching N-glycosyla ion induces a dec eased cell su ace e en ion o CD25 and IL-2 signaling, p omo ing a T helpe (TH) 17 o e induced egula o y T cell (iT eg) di e en ia ion (53) (Figu e 2). Impo an ly, he co-inhibi o y ecep o s a e likewise modula ed by N-glycosyla ion. One o he majo nega i e egula o s o T cell esponse is he cy o oxic T-lymphocy e p o ein 4 (CTLA-4), ha comp ises wo N-glycosyla ion si es desc ibed o modula e i s cell su ace e en ion on T cells and he eby i s a ini y o CD80/CD28 on APCs (54–56). The impac o N-glycosyla ion in he modula ion o he inhibi o y unc ions o CTLA-4 and p og ammed cell dea h p o ein-1 (PD-1) is discussed in mo e de ail in sec ion “Glycans in ole ogenic/immunosupp essi e esponses”. None heless, o he co-inhibi o y ecep o s like Lymphocy e-ac i a ion gene 3 (Lag-3), mucin-domain-con aining molecule-3 (Tim-3), and T cell immuno ecep o wi h Ig and ITIM domains (TIGIT) may also unde go glycans-media ed egula ion, as hey exhibi N-glycan-binding si es, howe e he ole o glycans on hese molecules emains o be explo ed (57). Taken oge he , N-glycosyla ion plays an ins umen al ole in he egula ion o T cell ac i a ion and unc ions by a ge ing no only TCR bu also i s co- ecep o s (Figu e 2). GLYCANS AS MODULATORS OF HYPER-REACTIVE/AUTOIMMUNE RESPONSES Au oimmuni y is cha ac e ized by he loss o sel - ole ance and de elopmen o an au o eac i e immune esponse owa d he indi idual’s own o ganism. Glycan mo i s play a c ucial ole in he de e mina ion o sel /non-sel an igens. Speci ic glycan s uc u es, exp essed by mic obial pa hogens, a e commonly esponsible o he p ima y ac i a ion o he inna e immune sys em; howe e , he mechanisms in ol ed in he sel /non-sel disc imina ion, media ed by glycans a e a om being ully F on ie s in Immunology | www. on ie sin.o g 5No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology elucida ed. Abno mal le els o b anched N-glycans ha e been associa ed wi h exace ba ed immune esponses in mu ine models (58). Pa icula ly, he dys egula ion o he N-glycosyla ion pa hway has been associa ed wi h au oimmune-like pheno ypes. The inabili y o syn he ize β1,6-GlcNAc an ennae, in Mga 5−/− mice has been associa ed wi h an inc eased suscep ibili y o immune-media ed diso de s such as an inc eased delayed- ype hype sensi i i y esponses, as well as inc eased suscep ibili y o de elop expe imen al au oimmune encephalomyeli is (EAE) (30, 59) and se e e o ms o coli is (60). The lack o β1,6 b anching N-glycans a o s TCR clus e ing, leading o a dec ease o he TCR h eshold and consequen ly inc eased T cell ac i a ion (30) associa ed wi h he hype immune esponse obse ed in hese mice (Figu e 2). This hype immune pheno ype is also due o an abno mal o ma ion o la ices be ween TCR-b anched glycosyla ion and galec ins (61,62). Acco dingly, β3 GnT2- de icien mice show T cell hype sensi i i y due o he educ ion o polylac osamine on he N-glycans (ligands o galec ins), simila ly o wha is obse ed in Mga 5 de icien mice (30,61). Fu he mo e, absence o α-mannosidase II (which ca alyses he las hyd olysis o he α-mannose), was shown o esul in signs o glome uloneph i is, deposi s o glome ula IgM immunocomplexes and complemen componen 3 as well as high le els o an i-nuclea an ibodies (63,64), which is consis en wi h a Lupus-like synd ome (Figu e 2). Taken oge he , hese e idences suppo he ole o N-glycosyla ion in he pe spec i e o T cell biology. The ole o N-glycans in an igen p esen a ion and ecogni ion is s ill elusi e, and in ac abno mal glycoan igen p esen a ion migh also impac T cell ac i i y. Abno mal accumula ion o high-mannose, paucimannose, and agalac osyl bi-an enna y glycans, ha e been de ec ed in kidney issue om MRL-lp mouse (a well-s ablished mu ine model o SLE) (65). Mo eo e , e idences ha e been showing ha Mga 1 / Syn1-C e mice, wi h Mga 1 dele ion a he Synapsin I-exp essing cells (abundan in neu al issues), p esen ed neu ological de ec s, wi h high le els o neu onal apop osis and caspase 3 ac i a ion (66). These high le els o apop osis a e obse ed in se e al au oimmune diseases, which esul s in ac i a ion o immune sys em (67) (Figu e 2). Al hough highly unexplo ed, a e au oimmune diseases a e also associa ed wi h N-glycosyla ion dys unc ions. As example, idiopa hic in lamma o y myopa hies (IIM) a e a g oup o a e diseases o au oimmune na u e, whose e iopa hogenesis is a om being o ally unde s ood (68). Muscle cells su ace is en iched wi h glycop o eins and se e al lines o e idence p o ide suppo o a undamen al ole o glycosyla ion in muscle homeos asis and unc ion (69,70). Glucosamine (UDP- N-Ace yl)-2-Epime ase/N-Ace ylmannosamine Kinase (GNE) gene ic mu a ions (a gene ha encodes N-ace ylmannosamine (ManNAc) kinase enzyme, esponsible o he biosyn hesis o N-ace ylneu aminic acid) esul s in hypo-sialyla ion o muscle glycop o eins; he p ophylac ic supplemen a ion wi h sialic acid p ecu so (ManNAc) was shown o p e en he muscle pheno ype in mice wi h gene mu a ions ha cause he edi a y inclusion-body myosi is (hIBM), a muscle pheno ype ha esembles one ype o IIM (71). Al oge he , hese indings highligh he impo ance o u he s udies add essing he ole o N-glycosyla ion in he pe spec i e o neoau oan igens, since au oan igens con ain a signi ican amoun o glycoan igens due o he inc eased numbe o N-glycosyla ion si es compa ing wi h o he p o eins (72). The Glycan binding p o eins (GBPs) a e exp essed in he APCs being cha ac e ized by a ca bohyd a e ecogni ion domain which speci ically ecognizes glycan s uc u es p esen a he cell su ace ecep o s. This glycan-GBPs engagemen esul s in ei he an an i- o p o-in lamma o y esponse (73). C- ype lec ins, siglecs, and galec ins a e examples o GBPs, ha a e ins uc o s o immune esponses (5,73). As example, SIGN1R (exp essed by APCs and he analogous o he human dend i ic cell-speci ic ICAM-g abbing non-in eg in - DC-SIGN) signaling was shown o esul in he expansion o IL-10-sec e ing T eg cells, p e en ing he de elopmen o au oimmune diseases such as EAE and ype 1 diabe es (T1D) (74). Galec in-1 also plays an impo an immune- egula o y ole in EAE (75) as mice de icien in galec in-1 (Lgals1−/−) ha e inc eased TH1 and TH17 esponses being mo e suscep ible o EAE when compa ed wi h wild ype mice (76). Mo e ecen ly, Galec in-1 was shown o modula e he cy oly ic ac i i y o CD8+T cell. The in e ac ion o Galec in-1 and Fas ligand seems o be esponsible o he e en ion o his glycop o ein a he su ace o cy o oxic T lymphocy es hampe ing he cy oly ic abili y o hese cells (77). O e all, GBPs-glycop o ein in e ac ion is essen ial o ins uc a T cell—media ed immune esponse. No ably, one o he i s e idences add essing he ela ionship be ween he dys egula ion o N-glycosyla ion and human au oimmuni y was obse ed in mul iple scle osis (MS) pa ien s. Du ing ac i e, elapse o in e y ea ly s ages o emission, pe iphe al blood mononuclea cells om MS pa ien s display a signi ican dec ease o he enzyma ic ac i i y o Golgi β1,6 N- ace ylglucosaminyl ans e ase (co e 2 GlcNAc-T), compa ed o heal hy subjec s (78). Mo eo e , MGAT5 polymo phisms we e associa ed wi h MS se e i y (79) oge he wi h MGAT1,IL2R, and IL7R Single Nucleo ide Polymo phisms (80–82). Addi ionally, in In lamma o y Bowel Disease (IBD), i was also demons a ed ha lamina p op ia T lymphocy es om ulce a i e coli is (UC) pa ien s exhibi ed a de iciency in β1,6-GlcNAc b anching N- glycans due o dec eased le els o MGAT5 gene exp ession (83). Impo an ly, low le els o b anched N-glycans in lamina p op ia ea ly a diagnosis we e shown o p edic UC pa ien s ha will ail he esponse o s anda d he apy, hus displaying a bad disease cou se (84). The supplemen a ion o in es inal T cells om UC pa ien s and mouse models wi h coli is wi h GlcNAc p omo ed he enhancemen o β1,6 b anching N-glycans on T cells, supp essing TCR signaling and educing he p oduc ion o p o-in lamma o y cy okines such as umo nec osis ac o alpha (TNFα) and in e e on gamma (IFNγ). P e-clinical s udies bo h in IBD and MS demons a ed he immunomodula o y p ope ies o N-glycans in he con ol o T cell-media ed immune esponse (60,85), pa ing he way o he de elopmen o human clinical ials, ha a e cu en ly on going (53,60). Less explo ed bu o u mos impo ance is he s udy o N-glycosyla ion p o ile in a e au oimmune diso de s, since i s e iopa hogenesis is s ill e y elusi e. Glycosyla ion changes in muscle-associa ed human disease ha e ocused in muscula dys ophies (86) and congeni al F on ie s in Immunology | www. on ie sin.o g 6No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology diso de s o glycosyla ion (87). Recen s udies ha e shown ha muscle cell su ace glycosyla ion is inely egula ed and subjec ed o al e a ions unde in lamma o y condi ions (88), poin ing o a possible in e ac ion be ween muscle glycocalyx and he ex acellula milieu, which is pa icula ly en iched in immune cells and an ibodies in IIM pa ien s (89). O e all, glycans a e c i ical de e minan s in au o eac i e esponses bo h by di ec ly egula ing T cell ac i i y and also h ough he c ea ion o abno mal glycoan igens ha may unleash an au o eac i e immune esponse. GLYCANS IN TOLEROGENIC/IMMUNOSUPPRESSIVE RESPONSES Recen s udies ha e been highligh ed ha al e a ions on he glycosyla ion pa e n o T cells’ ecep o s, as well as he al e a ions o he glycosyla ion p o ile o umo cells ( umo glyco-code), a e implica ed in he modula ion o he immune esponse leading o immunosupp essi e pa hways, known o occu in he umo mic oen i onmen associa ed wi h umo immunoescape (90). Role o Glycans in he Modula ion o Inhibi o y T Cell Recep o s PD-1, as al eady in oduced, is a cell su ace inhibi o y T cell ecep o esponsible o immune-inhibi o y esponses associa ed wi h he so-called “T cell exhaus ion” (91). The exp ession o his cell su ace ecep o , as well as Tim-3, was desc ibed o be posi i ely egula ed by he co e ucosyla ion pa hway, ca alyzed by FUT8 enzyme (92). The inhibi ion o co e ucosyla ion in PD-1 was demons a ed o lead o an an i- umo immune esponse media ed by T cells ac i a ion, being a new a ac i e a ge o enhancing an i- umo immuni y in u u e clinical se ings (Figu e 2). This was a pionee s udy ha suppo ed he impo ance o PD-1 pos - ansla ional modi ica ions by glycosyla ion on T cell-media ed immunosupp ession (92). Addi ionally, he glycosyla ion o p og ammed dea h ligand-1 (PD-L1), a PD-1 ligand, was desc ibed o ha e an impo an ole in i s cellula s abiliza ion. The in e ac ion o non-glycosyla ed PD-L1 wi h glycogen syn hase kinase 3β(GSK3β), a key enzyme on glycogenesis, leads o he deg ada ion o his molecule (93). In iple-nega i e b eas cance cells, i was u he shown ha he β1,3-N-ace ylglucosaminyl ans e ase (B3GNT3), in ol ed in he biosyn hesis o poly-N-ace yllac osamine chains, is impo an o he in e ac ion be ween PD-1 and i s ligand PD-L1 (94). The use o an an ibody a ge ing he glycosyla ed o m o PD-L1 esul ed in i s deg ada ion and in e naliza ion, wi h he blockage o PD-L1/PD-1 in e ac ion and consequen ly he inducemen o an i- umo ac i i y in iple-nega i e b eas cance in i o and in i o models (94). In acco dance, T egs om heal hy humans and mice we e shown o display an inc eased a iabili y on i s N-glycosyla ion pa e n when compa ed wi h CD4+T cells. The le els o he complex b anched N-glycans we e shown o be co ela ed wi h he exp ession o p o eins in ol ed in T eg supp essi e unc ions, including PD-1, PD-L1, and also o he nega i e egula o s o T cell esponse, namely CTLA-4 (95). In ac , he CTLA-4 p o ein, comp ises mul iple N- and O-glycosyla ion si es known o modula e i s e en ion a T cell su ace and consequen ly a ec ing i s unc ion (56). The TCR ac i a ion is associa ed wi h an inc eased β1,6-GlcNAc b anched N-glycosyla ion o CTLA-4, which enhances CTLA-4 e en ion a he T cell su ace and he eby supp esses T cell ac i a ion p omo ing immune ole ance (96) (Figu e 2). Acco dingly, he p esence o Th 17Ala polymo phism in human CTLA-4 was shown o esul in he educ ion o he N-glycosyla ion si es om one o wo si es, which limi ed CTLA-4 e en ion a T cell su ace (80). Supplemen a ions wi h GlcNAc and Vi amin D p omo ed an enhancemen o N-glycans b anching exp ession, inc easing he cell su ace e en ion o CTLA-4, culmina ing in immunosupp ession (80). Glycans as Ins uc o s o Immunosupp essi e Responses Tumo cells abe an ly exp ess di e en ypes o glycans s uc u es when compa ed wi h no mal coun e pa s, such as an inc eased sialyla ion, an exp ession o unca ed glycans and an o e exp ession o b anched N-glycans (97). This al e a ion in he cellula glycosyla ion p o ile go e ns se e al s eps o umo de elopmen and p og ession, such as umo cell dissocia ion, p oli e a ion, in asion, me as asis, angiogenesis, wi h ecen e idences poin ing owa d i s e ec s in umo immunoedi ing and immunosu eillance (98). GBPs exp essed on immune cells a e able o ecognize al e ed glycan s uc u es exp essed a umo cell su aces ins uc ing ei he immunos imula o y o immunoinhibi o y esponses. The exp ession o sialyla ed glycans, such as Tn an igen and Lewis an igens, abe an ly exp essed in umo cells, we e desc ibed o be ecognized by DC-SIGN, exp essed by mac ophages and imma u e DCs, which lead o immunosupp ession (99). The ucose esidues p esen in Lewis s uc u es (Lewis x and Lewis y), a ached o ca cinoemb yonic an igen (CEA) (100), we e desc ibed o igge he up egula ion o he an i-in lamma o y cy okines IL-10 and IL-27 by APCs and he induc ion o TH2, ollicula (TH ), and T eg immune esponses (101,102). Besides, an igen-con aining liposomes modi ied wi h DC-SIGN-binding Lewis b and x esul ed in glycans ecogni ion and in e naliza ion h ough DCs wi h consequen ac i a ion o CD4+and CD8+T cells (103). Fu he mo e, mac ophage galac ose binding lec in (MGL) was ound o be able o ecognize Tn an igen and N- ace ylgalac osamine (GalNAc) esidues, esul ing in an inc eased ecogni ion by Toll-like ecep o 2, ul ima ely esul ing in he sec e ion o cy okines (IL-10 and TNF-α). (104). I s in e ac ion wi h e minal GalNAc esidues on CD45 glycop o ein nega i ely egula es TCR signaling, wi h consequen dec ease o T cell p oli e a ion and inc eased T cell dea h (105). Mo eo e , by blocking he umo -in il a ed mac ophages ( esponsible o he high le els o IL-10), i was obse ed an e ec i e CD8+ T cells esponse, highligh ing he impo ance o combining an i- umo immune he apy wi h con en ional chemo he apy (106). Fu he mo e, i was ecen ly demons a ed in ch onic in ec ion ha IL-10 induces he up egula ion o he Mga 5 F on ie s in Immunology | www. on ie sin.o g 7No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology gene inc easing b anched N-glycans on CD8+T cells, which in u n dec eases T cell ac i i y and allows i al pe sis ence (107). Despi e he di e en con ex in which his hypo hesis was s udied, Mga 5-media ed b anching glycosyla ion can cons i u e a po en ial mechanism by which IL-10 is supp essing CD8+T cells in cance . In addi ion, sialyla ed glycans also play a ole in immunosupp ession, media ed by siglecs, a amily o lec in ecep o s ha p edominan ly exhibi immune-inhibi o y unc ions. In in i o and in i o s udies, he binding o sialyla ed an igens by siglec-E exp essed on DCs p omo ed an inc ease o an igen-speci ic T eg esponse and a educed numbe s o an igen-speci ic Te cell esponse, associa ed wi h umo g ow h (108,109). Indeed, he sialyla ed umo an igens, such as Sialyl-Tn (sTn) and Sialyl-T (sT) exp essed in mucins, namely MUC1, we e associa ed wi h umo immune ole ance. The ecogni ion o MUC1-ST by siglec-9 on umo - in il a ing mac ophages was shown o ini ia e inhibi o y immune pa hways media ed by MEK-ERK signaling (110). Mo eo e , siglec-binding o sTn-exp essing mucins, led o he ma u a ion o DCs and DC-media ed induc ion o FOXP3+ T eg cells and educed INFγ-p oducing T cells (111,112). A ecen s udy also demons a es ha siglec-9 exp essed by CD8+ umo in il a ing lymphocy es (TILs) in non-small cell lung cance (NSCLC) pa ien s was associa ed wi h educed su i al. Acco dingly, siglec-9 polymo phisms we e associa ed wi h he isk o de eloping lung and colo ec al cance . Addi ionally, he cha ac e iza ion o siglec-9+CD8+TILs e ealed ha hese cells concomi an ly exp ess se e al inhibi o y ecep o s, including PD-1, TIM-3, Lag3, and o he s. In addi ion, he same s udy u he e eals ha lack o sialic acid-con aining glycans in umo cells led o a delay o umo g ow h and an inc eased in il a ion o CD3+and CD8+T cells (113). Ano he impo an GBP ha ha e been poin ed ou as a c ucial checkpoin in T cell iabili y and ac i i y a e galec ins. Galec in-1, 3, and 9 we e p edominan ly desc ibed in T cell immunosupp ession. Galec in-1, was demons a ed o be exp essed by ole ogenic DCs (75) and CD4+CD25+ T cells (114), igge ing T cell apop osis h ough binding o N-glycans and O-glycans on CD45, CD43, and CD7 o by sensi izing es ing T cells o FAS-induced dea h (115,116). The TH1 and TH17 ac i a ed cells a e suscep ible o galec in- 1-induced cell dea h once hese cells exp ess he epe oi e o glycans equi ed o galec in-1 binding, while TH2 cells a e p o ec ed ia α2,6-sialyla ion on cell su ace glycop o eins, which was desc ibed o p eclude galec in-1 ecogni ion and binding (76). In addi ion, se e al umo s ha e he capaci y o sec e e galec in-1 in o de o p omo e immunosupp ession, h ough a mechanism ha in ol es a bias owa d a TH2 cy okine p o ile and ac i a ion o ole ogenic ci cui s media ed by IL-27-p oducing DCs and IL-10-p oducing ype 1 T eg cells (117). On o he hand, galec in-3 has an ambiguous ole in T cell iabili y: when i is localized a in acellula le el, his p o ein p esen s a p o ec i e ole h ough a cell dea h inhibi ion pa hway ha in ol es B-cell lymphoma 2 (Bcl-2) (118), whe eas ex acellula galec in-3 induces cell dea h in ac i a ed T cells, by binding o glycosyla ed ecep o s o T cells h ough a dis inc way han galec in-1 (115). Mo eo e , galec in-3 has he capaci y o bind o N-glycans on CTLA-4 p olonging he inhibi o y signals (119), as well as o Lag-3 on he su ace o CD8+T cells, supp essing i s unc ion (120). Finally, galec in-9 ab oga es TH1, TH17, and CD8+T cells h ough glycosyla ion- dependen binding o Tim-3 (121–123), whe eas may egula e p o-in lamma o y cy okine p oduc ion by binding wi h o he ecep o s (124). Al oge he , hese indings suppo he ele ance o glycans on T cells-media ed immunosupp essi e/ ole ogenic pa hways which ha e ele an implica ions in umo p og ession. Ta ge ing he abno mal glycosyla ion pa e n o cance cells cons i u es a p omising s a egy o ins uc an e ec i e an i- umo immune esponse, an issue ha needs o be u he explo ed. GLYCANS AS METABOLIC REGULATORS OF T CELL FUNCTION The impac o glycosyla ion on T cell de elopmen and unc ions is eno mous, as e ealed by he c i ical oles o glycans in he de elopmen and p og ession o majo diseases such as au o-immuni y and cance , as desc ibed he ein. In o de o accompany he bioene ge ic and biosyn he ic demands equi ed o T cell p oli e a ion and ac i a ion, a shi in he T cell me abolism is equi ed. While naï e T cells a e in a me abolic quiescen s a e, mainly using oxida i e phospho yla ion o maximize ATP p oduc ion, T cells unde clonal expansion o unde di e en ia ion, ep og am hei me abolic s a us o ae obic glycolysis and glu aminolysis in o de o inc ease he a ailabili y o glycoly ic p ecu so s o he biosyn hesis o nucleo ides, amino acids and lipids (125–127). Du ing T cell ac i a ion, he hexosamine biosyn he ic pa hway (HBP—a b anch o he glucose me abolism) is up egula ed in o de o gene a e he nucleo ide suga -dono subs a e UDP-GlcNAc, equi ed o N-glycosyla ion, O-GlcNAcyla ion, and glycosaminoglycans p oduc ion ha a e needed o a p ope T cell unc ion (128). Media o s om he glycoly ic pa hway such as glucose (Glc), glu amine (Gln), ace yl CoA a e known o in e e e wi h he a ailabili y o he UDP-GlcNAc in he cell (129–131). Toge he Glc and Gln we e shown o inc ease UDP-GlcNAc in nu ien - s a ed T cells. In he same se up, he supplemen a ion o bo h Glc and glucosamine (GlcN–a me aboli e o he HBP) u he inc eased he UDP-GlcNAc cellula con en , demons a ing he sensi i i y o he HBP o nu ien s ha en e di ec ly (GlcN) o h ough a p ecu so pa hway (Glc in glycolysis) (130). Despi e he gene al use o he UDP-GlcNAc as a subs a e dono o HBP, he e a e some glycosyl ans e ases ha a e mo e suscep ible o nu ien changing han o he s, such as he case o OGT (132). In ac , he supply wi h Glc and Gln a e c ucial o p o ein O-GlcNAcyla ion, ha is impo an du ing T cell de elopmen , being associa ed wi h T cell malignan ans o ma ion (23). Among he N- ace ylglucosaminyl ans e ases (GnTs) ha pa icipa e in he HBP, he less sensi i e o nu ien changing (and hus subs a e a ailabili y) a e GnT1, GnT2, and GnT3, due o lowe Michaelis Cons an (Km)le els, meaning ha hese enzymes equi e low F on ie s in Immunology | www. on ie sin.o g 8No embe 2018 | Volume 9 | A icle 2754 Pe ei a e al. Glycans as Checkpoin s in T Cells Biology le els o he subs a e o syn he ize he speci ic glycans. In con as , GnT4 and GnT5 p esen highe Kmand he e o e hei ac i i y is highly dependen on he a ailabili y o he UDP- GlcNAc subs a e (119,133). The e o e, hese wo enzymes a e sensi i e o al e a ions in glucose and HBP me abolism (as he GlcN o N-ace yl glucosamine–GlcNAc) (62), which ul ima ely will in e e e in he N-glycan b anching biosyn hesis on T cells wi h impac in hei ac i i y, as de ailed in sec ion “Glycans in he egula ion o T cell ac i i y and unc ions” (60). In ac , supplemen a ion wi h Glc, Gln, and GlcNAc inc eases b anching N-glycans on Ju ka cells and es ing T cells om mice (85,119,130). Mo eo e , CD4+T cells om MGAT5+/+o MGAT5+/−− mice supplemen ed wi h o al GlcNAc also esul s in up o 40% inc ease o b anching N-glycans, de ec ed by L-PHA (130). This enhancemen o b anching N-glycosyla ion upon GlcNAc supplemen a ion was shown o unc ionally impac on T cells ac i i y by educing T cell ac i a ion, dec easing TH1 di e en ia ion, and inc easing e en ion o he g ow h inhibi o y ecep o CTLA-4 a T cell su ace (85,130). Impo an ly, e idences sugges ha he glycolysis and glu aminolysis compe e wi h HBP pa hway o he same me aboli es. Recen ly, A aujo e al showed ha , du ing TH17 di e en ia ion he exis ence o common media o s sha ed be ween HBP, glycolysis ( uc ose-6-phospha e) and glu aminolysis (Gln) esul s in a s a a ion o he HBP media o s, ansla ed in a educ ion o N-glycan b anching due o he limi a ion on he UDP-GlcNAc a ailabili y (53). Fueling HBP wi h GlcNAc swi ched he cell a e om TH17 o iT eg di e en ia ion, h ough s imula ion o IL2-Rαsignaling (53). This in e play be ween me abolic pa hways was u he demons a ed by he inc ease on Glc, Gln, a y-acids up ake, and lipid s o age upon s imula ion o he HBP wi h GlcNAc supplemen a ion, sugges ing a ep og amming o he cellula me abolism upon GlcNAc lux (53,134). The impac o glycans as me abolic egula o s o T cells is also es i ied by i s e ec s in ex i o and in i o models o au oimmune diseases. The me abolic supplemen a ion wi h GlcNAc in ex i o human colonic T cells om IBD pa ien s esul ed in an enhancemen o he b anching N-glycosyla ion pa hway ha was accompanied by a signi ican educ ion o T cell p oli e a ion, sup ession o TH1/TH17 immune esponse ( h ough dec eased p oduc ion o IFN-γand IL-17A p o- in lamma o y cy okines) and dec eased TCR signaling (60). Acco dingly, he GlcNAc supplemen a ion o mice models wi h au o-immune diseases such as EAE, TID, and IBD esul s in inhibi ion o TH1, TH17 immune esponse concomi an ly wi h a signi ican imp o emen o he clinical symp oms (60,85). T ea men wi h GlcNAc a e disease onse also demons a e inhibi o y e ec s on he de elopmen o he EAE, by educing he sec e ion o INF-γ, TNF-α, IL-17, and IL-22 (85). In e es ingly, a dual ole o GlcN ( he p ecu so o GlcNAc) on he p og ession o au oimmune diso de s was shown, by demons a ing i s impac in p e en ing TH1-media ed Type I diabe es ( h ough he educ ion o IFN-γp oducing CD4+T cells), bu also he GlcN e ec s in exace ba ing TH1/TH17–media ed EAE symp oms ( ough s imula ion o TH17 esponse) (135). In con as , ano he s udy showed ha GlcN supp esses acu e EAE h ough he blockage o TH1 and induc ion o TH2 esponse (136). GlcN supplemen a ion was u he shown o media e T cell ac i a ion by dec easing he N-glycosyla ion o CD25 (IL-2Rα) om CD4+ T cell (135). This down- egula ion o N-glycosyla ion migh be explained by he compe i ion be ween GlcN and Glc o he same glucose anspo e which migh impac in he educ ion o he GlcNAc concen a ion. Al oge he , al e a ions on he glucose me abolism and pa ially changes in he me abolic lux o HBP ha e a di ec impac on T cells N-glycosyla ion p o ile wi h majo consequences in hei unc ion and ac i i y. Ul ima ely, he modula ion o he HBP cons i u es an impo an me abolic a ge able o con ol bo h au o eac i e and immunosupp essi e esponses known o occu , espec i ely, in au oimmuni y and cance . CONCLUDING REMARKS The con ibu ion o he glycome as a majo egula o o he immune sys em is clea . Glycans ac i ely pa icipa e in he cellula and molecula mechanisms unde lying he genesis o he loss o immunological ole ance associa ed wi h (au o)immuni y, om one hand, pa icipa ing also in he c ea ion o ole ogenic pa hways associa ed wi h cance p og ession, om he o he . The impo ance o glycans in immune esponse spans om i s ole in he modula ion he T cell de elopmen ; hei impo ance as a sou ce o glycoan igens p esen a ion; as well as hei ole as ine une s o T cell esponse. In his con ex , glycans can exe a dual ole, ac ing ei he as immune inhibi o y checkpoin s o as immune s imula o y signals. Unde s anding in dep h he in luence o glycans in he immune egula o y ci cui s ha media e he pa hophysiology o au oimmuni y and cance will gene a e a pla o m wi h ex ao dina y po en ial o illumina e he iden i ica ion o no el bioma ke s and a ge s o he de elopmen o e icien immunomodula o y s a egies wi h applica ions in he clinical se ing. AUTHOR CONTRIBUTIONS All he au ho s w o e he manusc ip . AD and NP c ea ed he igu es. SP pe o med he c i ical e iew o he manusc ip . FUNDING The Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o in eg a es he i3S esea ch uni , which is pa ially suppo ed by he Po uguese Founda ion o Science and Technology (FCT). This a icle is a esul o he p ojec NORTE-01-0145-FEDER-000029, suppo ed by he No e Po ugal Regional P og amme (NORTE 2020) unde he PORTUGAL 2020 Pa ne ship Ag eemen h ough he Eu opean Regional De elopmen Fund. This wo k was also unded by Fundo Eu opeu de Desen ol imen o Regional (FEDER) unds h ough he COMPETE 2020—Ope acional P og amme o Compe i i eness and In e na ionaliza ion (POCI), Po ugal F on ie s in Immunology | www. on ie sin.o g 9No embe 2018 | Volume 9 | A icle 2754