Multidimensional Analysis Integrating Human T-Cell Signatures in Lymphatic Tissues with Sex of Humanized Mice for Prediction of Responses after Dendritic Cell Immunization.

Decembe 2017 | Volume 8 | A icle 17091 Me hods published: 08 Decembe 2017 doi: 10.3389/ immu.2017.01709 F on ie s in Immunology | www. on ie sin.o g Edi ed by: Ramesh Akkina, Colo ado S a e Uni e si y, Uni ed S a es Re iewed by: San hi Go an la, Uni e si y o Neb aska Medical Cen e , Uni ed S a es Michael Scho sae , Icahn School o Medicine a Moun Sinai, Uni ed S a es *Co espondence: Ha alampos Ha ziki ou ha alampos.ha ziki ou@ helmhol z-hzi.de; Michael Meye -He mann mmh@ heo e ical-biology.de; Rena a S ipecke s ipecke. ena a@mh-hanno e .de †Sha ed i s co-au ho ship. Special y sec ion: This a icle was submi ed o Vaccines and Molecula The apeu ics, a sec ion o he jou nal F on ie s in Immunology Recei ed: 24Augus 2017 Accep ed: 20No embe 2017 Published: 08Decembe 2017 Ci a ion: VolkV, ReppasAI, Robe PA, SpineliLM, Sunda ase yBS, TheobaldSJ, Schneide A, Ge aschL, De esRo hC, KlössS, KoehlU, Kaisenbe gC , Figuei edoC, Ha ziki ouH, Meye -He mannM and S ipeckeR (2017) Mul idimensional Analysis In eg a ing Human T-Cell Signa u es in Lympha ic Tissues wi h Sex o Humanized Mice o P edic ion o Responses a e Dend i ic Cell Immuniza ion. F on . Immunol. 8:1709. doi: 10.3389/ immu.2017.01709 Mul idimensional Analysis In eg a ing human -Cell signa u es in Lympha ic issues wi h sex o humanized Mice o P edic ion o Responses a e dend i ic Cell Immuniza ion Vale y Volk1†, And eas I. Reppas2†, Philippe A. Robe 2, Loukia M. Spineli3, Bala Sai Sunda ase y1, Sebas ian J. Theobald1, And eas Schneide 1, Lau a Ge asch1, Candida De es Ro h1, S ephan Klöss4, Ul ike Koehl4, Cons an in on Kaisenbe g5, Cons anca Figuei edo6, Ha alampos Ha ziki ou2*, Michael Meye -He mann2* and Rena a S ipecke1*† 1 Depa men o Hema ology, Hemos asis, Oncology and S em Cell T ansplan a ion, Hanno e Medical School, Hanno e , Ge many, 2 Depa men o Sys ems Immunology, B aunschweig In eg a ed Cen e o Sys ems Biology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 3 Ins i u e o Bios a is ics, Hanno e Medical School, Hanno e , Ge many, 4 Ins i u e o Cellula The apeu ics and GMP Co e Facili y IFB-Tx, Hanno e Medical School, Hanno e , Ge many, 5 Clinic o Gynecology and Obs e ics, Hanno e Medical School, Hanno e , Ge many, 6 Depa men o T ans usion Medicine, Hanno e Medical School, Hanno e , Ge many Mice ansplan ed wi h human co d blood-de i ed hema opoie ic s em cells (HSCs) became a powe ul expe imen al ool o s udying he he e ogenei y o human immune econs i u ion and immune esponses in i o. Ye , analyses o human Tcell ma u a ion in humanized models ha e been hampe ed by an o e all low immune eac- i i y and lack o me hods o de ine p edic i e ma ke s o esponsi eness. Long-li ed human len i i al induced dend i ic cells exp essing he cy omegalo i us pp65 p o ein (iDCpp65) p omo ed he de elopmen o pp65-speci ic human CD8+ Tcell esponses in NOD.Cg-Rag1 m1Mom-Il2 γ m1Wj humanized mice h ough he p esen a ion o immune- dominan an igenic epi opes (signal 1), exp ession o co-s imula o y molecules (signal 2), and in lamma o y cy okines (signal 3). We exploi ed his alida ed sys em o e alua e he e ec s o mouse sex in he dynamics o Tcell homing and ma u a ion s a us in hymus, blood, bone ma ow, spleen, and lymph nodes. S a is ical analyses o cell ela i e equencies and absolu e numbe s demons a ed highe CD8+ memo y Tcell eac i i y in spleen and lymph nodes o immunized emale mice. In o de o unde s and o which ex en he mul idimensional ela ion be ween o gan-speci ic ma ke s p edic ed he immuniza ion s a us, he immunopheno ypic p o iles o indi idual mice we e used o ain an a i icial neu al ne wo k designed o disc imina e immunized and non- immunized mice. The highes accu acy o immune eac i i y p edic ion could be ob ained om lymph node ma ke s o emale mice (77.3%). P incipal componen analyses u he iden i ied clus e s o ma ke s bes sui ed o desc ibe he he e ogenei y o immuniza ion esponses in i o. A co ela ion analysis o hese ma ke s e lec ed a issue-speci ic 2 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 impac o immuniza ion. This allowed o an o gan- esol ed cha ac e iza ion o he immuniza ion s a us o indi idual mice based on he iden i ied se o ma ke s. This new modali y o mul idimensional analyses can be used as a amewo k o de ining minimal bu p edic i e signa u es o human immune esponses in mice and sugges s c i ical ma ke s o cha ac e ize esponses o immuniza ion a e HSC ansplan a ion. Keywo ds: hema opoie ic s em cell ansplan a ion, co d blood, dend i ic cell,  cell ma u a ion, lympha ic, humanized mice, gende , a i icial neu al ne wo k IN RodUC IoN Humanized mice ansplan ed wi h human hema opoie ic s em cells (HSCs) became a b oadly used expe imen al and p eclinical pla o m o cha ac e ize he c i ical s eps o he econs i u ion o he human immune sys em (1–3). In his con ex , humanized mice a e cu en ly used o s udy human-speci ic in ec ions and o es d ugs, accines, and cell he apies (2, 3). Eng a men o human HSCs in he mouse bone ma ow (BM) and subsequen ea ly Tcell de elopmen in hymus (Thy) could be con enien ly s udied in sho - e m models las ing 10–16weeks (4). Ye , ull ma u a ion o Tcells owa d memo y cells in HSC- ansplan ed humanized mice was shown o be conside ably mo e he - e ogeneous and challenging and equi ed pe iods o analyses o 20weeks o longe (5, 6). Thus, his lymphopenia coincides wi h he delayed Tcell immune econs i u ion in pa ien s a e hema opoie ic s em cell ansplan a ion (HSCT) (5, 6). Mul iple complemen a y app oaches we e ied o suppo he de elop- men o human cells in immune-de icien mice such as, o example, he adminis a ion o human cy okines (7) and he gene a ion o new ansgenic mouse s ains exp essing human cy okines (8) o human leukocy e an igens (HLA) molecules (9, 10). Mo e complex and demanding s a egies explo ing co- ansplan a ion wi h human e al hymus and li e issues (bone ma ow, li e , hymus model) in o mice showed an o e all imp o ed Tcell de elopmen and ma u a ion (11–14). No ably, since Tcell esponses depend on he s eng h o he signals deli - e ed by he an igen/HLA o he Tcell ecep o (TCR) (signal 1), co-s imula ion (signal 2), and p o-in lamma o y cy okines (signal 3), s udies demons a ing he p esence o human den- d i ic cells (DCs) in humanized mice elucida ed hei ole in ac i- a ion o he cogna e Tcells (15). Thus, as po en ial al e na i e Abb e ia ions: aAPC, a i icial an igen-p esen ing cell; Ab, An ibody; ANN, a i icial neu al ne wo k; ANOVA, analysis o a iance; B. D., below de ec ion; BM, bone ma ow; BLT, bone ma ow, li e , hymus (mouse model); CB, co d blood; CM, cen al memo y; CTL, cy o oxic lymphocy es; DN, double nega i e; DP, double posi i e; DC, dend i ic cell; ELISPOT, enzyme-linked immuno spo assay; EM, e ec o memo y; FBS, e al bo ine se um; HCMV, human cy omeg- alo i us; HIV, human immunode iciency i us; HLA, human leukocy e an igen; HSCs, hema opoie ic s em cells; HSCT, hema opoie ic s em cell ansplan a ion; iDCpp65, induced dend i ic cells exp essing pp65; IDLV-G2a-pp65, in eg a ion- de icien len i i al ec o co-exp essing GM-CSF/IFN-α and he HCMV-pp65; IL, in e leukin; MCP-1, monocy e chemoa ac an p o ein 1; MLNs, mesen e ic lymph nodes; N, naï e; NRG, NOD.Cg-Rag1 m1Mom-Il2 γ m1Wj; PB, pe iphe al blood; PBS, phospha e-bu e ed saline; PCA, p incipal componen analysis; Pp65, phosphop o ein 65; PLN, pe iphe al lymph nodes; SPL, spleen; TCRαβ, Tcell ecep o αβ; TE, e minal e ec o ; Th1, T helpe ype 1; Thy, hymus; WT1, Wilms umo 1 p o ein. app oaches o imp o ing Tcell econs i u ion in humanized mice and ul ima ely in humans, adop i e au ologous DCs, such as hose explo ed clinically o cance immuno he apy (16) and human immunode iciency i us (17), o in i o ac i a ed DCs, as p e iously shown o be e ec i e in humanized mice (18), could ep esen aluable op ions. Likewise, we ha e p e i- ously desc ibed he p eclinical es ing o long-li ed gene ically enginee ed induced DC (iDCs) in humanized mice. These cells we e gene a ed a e a as o e nigh ansduc ion o monocy es wi h len i i al ec o s encoding g anulocy e-mac ophage colony s imula ing ac o (GM-CSF), in e e on-α (IFN-α), and he human cy omegalo i us (HCMV) phosphop o ein (pp) 65 (19, 20). iDCs exp essing pp65 (iDCpp65) accines a e cu - en ly in clinical de elopmen o p o ec ion o pos ansplan pa ien s (21), since pp65 has been long known o be a majo immune-dominan CD8+ cy o oxic Tlymphocy e a ge an igen in heal hy se oposi i e adul s (22). Fu he mo e, non-exhaus ed, long-li ed CD8+ e ec o memo y (EM) Tcells a e conside ed o be c ucial o main ain li elong p o ec ion om HCMV eac i a- ion in pos ansplan pa ien s (23). We p e iously demons a ed ha mul iple adminis a ions o iDCpp65 in o NOD.Cg-Rag1 m1Mom-Il2 γ m1Wj (NRG) mice ansplan ed wi h human HSCs p omo ed a po en de elopmen o CD8+ an igen-speci ic memo y esponses in sho (16weeks) (20) and long (20–36 weeks) models (19, 24). We ha e also demons a ed ha ano he impo an ac o o be conside ed ega ding he analyses o human Tcells in mice humanized wi h co d blood (CB)-HSCs is he gende o he ecipien mouse. Fo he ini ial 10–15weeks a e HSCT, emales showed a mo e obus Tcell de elopmen and ma u a ion, whe eas male’s Tcells ma ched he emale’s Tcell ma u a ion s a us only 20weeks pos - ansplan (25). In his cu en wo k, we sough o e alua e whe he human- ized emale and male mice would show di e en ial pa e ns o Tcell esponses o iDCpp65. We cha ac e ized he CD4+/ CD8+ Tcells and hei subse s [naï e (N), EM, cen al memo y (CM), and e minal e ec o (TE)] in di e en lympha ic issues and con i med a dis inc beha io be ween emales and males, suppo ed by s a is ical me hods. In o de o in eg a e he da a ob ained om di e en issues and e alua e he immuniza ion esponsi eness among hem, we adop ed a classi ica ion machine lea ning algo i hm based on an a i icial neu al ne wo k (ANN). A P incipal Componen Analysis (PCA) (26, 27) was u he used o educe he c i ical in o ma ion equi ed o p edic esponsi eness om he ANN (28). The ma ke s pinpoin ed by he PCA e ealed ha he co ela ion s uc u e o o gan-speci ic ma ke s is s ongly impac ed by immuniza ion and, he e o e, 3 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 iabili y (by ypan blue exclusion), len i i al copies pe cell (by RT-q-PCR), and DC immunopheno ype (by low cy ome y) we e pe o med as p e iously desc ibed (20, 21, 24). Fo immu- niza ion, IDLV- ansduced cells we e hawed, washed wice wi h PBS, and e-suspended in PBS a concen a ion 5.0×106cell/ml. Cells we e kep on ice un il injec ion. A e CB-HSCT, mice we e andomly dis ibu ed in o wo g oups, a non- ea ed con ol and a g oup immunized wi h 5.0 × 105 iDCpp65 cells. Cells we e adminis e ed subcu aneously in he le hind lank a weeks 6 and 10 and in igh hind lank a weeks 7 and 11 pos ansplan a- ion (Figu e1A). Weekly weigh and gene al heal h moni o ing we e pe o med un il he end o he expe imen a week 20 pos ansplan a ion. s ep 3: Longi udinal Cha ac e iza ion o human Cell de elopmen in PB Samples o PB we e collec ed om all mice a weeks 10, 16, and 20 a e HSCT o e alua e he le el o eng a men o human hema opoie ic cells and expansion o T and Bcells. Two ounds o lysis we e pe o med o emo e e y h ocy es (0.83% ammo- nium chlo ide/20mM HEPES, pH 7.2, o 5min a oom em- pe a u e, ollowed by s abiliza ion wi h cold PBS and washing). Cells we e labeled o low cy ome y analyses wi h he ollowing an ibodies as p e iously desc ibed (24): paci ic blue an i- CD45, Alexa700 (AF700) an i-CD19, allophycocyanin (APC) an i-CD3, phycoe y h in-cyanine7 (PC7) an i-CD8 (BioLegend, Fell, Ge many); allophycocyanin-H7 (APC-H7) an i-CD4, Phycoe y h in (PE) an i-CD14 (BD Biosciences, San Jose, CA, USA); and phycoe y h in-cyanine5 (PC5) an i-CD62L, luo es- cein iso hiocyana e (FITC) an i-CD45RA (Beckman Coul e , K e eld, Ge many) o 15min a oom empe a u e, washed, and analyzed by LSR II low cy ome e (BD Biosciences, Heidelbe g, Ge many). s ep 4: e alua ion o he human Cell Responses agains pp65 in Immunized Mice Th ee mice immunized wi h iDCpp65 and showing well- de e loped lymph nodes we e used o ob aining human memo y Tcells in high pu i y as p e iously desc ibed (20, 24). C yop ese ed single cell suspensions gene a ed om lymph nodes we e hawed, pooled, and e-suspended in X-VIVO 15 medium. Ac i a ion o Tcells was pe o med by MACS magne ic beads conjuga ed wi h an i-CD2/CD3/CD28 mono- clonal an ibodies (Mil enyi Bio ec, Ge many) in a bead- o-cell a io o 1:2, in p esence o 25 IU/ml o human IL-2, 5 ng/ ml IL-7, and 5ng/ml IL-15 (Cellgenix, Ge many) o 48 h. Cy okines we e e eshed e e y 2 days un il he end o he homeos a ic expansion (day 9 pos -ac i a ion wi h magne ic beads). Ac i a ed cells we e e-s imula ed a e cocul u e ei he wi h au ologous iDC ( o APC-media ed homeos a ic s imula ion bu lacking an igens) o wi h iDCpp65 o 7days using a T/DC cell a io o 10:1. Fo in acellula IFN-γ de ec- ion, expanded T cells we e i s seeded in iplica es wells (3×105cells/well) o a 96-well ound-bo om pla e and hen, o 16 h, he cells we e u he ac i a ed wi h 10 µg/ml o ha hese ma ke s can be used as bioma ke s o e ie e he in o ma ion o he immuniza ion s a us. MA eRIALs ANd Me hods s ep 1: Gene a ion o humanized Mice ansplan ed wi h human CB-hsC S udy p o ocols we e app o ed by he E hics Commi ee o he Hanno e Medical School o acquisi ion and banking o human HSCs ob ained om umbilical co d issues a e in o med consen om dono s (mo he s a e m). The HSCs we e labeled acco ding o a nume ical code ha could no be aced back o he dono ’s pe sonal in o ma ion, hus keeping he dono ’s anonymi y. All expe imen s in ol ing mice we e pe o med in acco dance wi h he egula ions and guidelines o he animal wel a e o he S a e o Lowe Saxony (Nds. Landesam ü Ve b auche schu z und Lebensmi elsiche hei , Deze na 33/ Tie schu z). 5-week-old NRG mice we e o iginally ob ained om The Jackson Labo a o y (JAX, Ba Ha bo , ME, USA) and b ed in-house unde pa hogen- ee condi ions. P io o HSCT, mice we e suble hally i adia ed (450 cGy) using a [137Cs] column i adia o (Gammacell 3000 Elan; Bes The a onics, O awa, ON, Canada). 4h a e i adia ion, 1.5–2.0×105 human CD34+ hema opoie ic cells isola ed om emale dono umbili- cal CB we e adminis a ed o each mouse ough he ail ein as desc ibed (20, 24). We had p e iously shown ha immune econs i u ion in emale mice ecipien s was as e han in males (25) and we, he e o e, used emale dono s o a oid any pu a i e immune esponses agains an igens exp essed in he Y ch omosome o male ecipien s. S em cells om HLA*A02.01 posi i e (CB1, CB3) o nega i e (CB2) uni s we e used o gene - a e humanized mice (CB1: n=11, CB2: n=10, CB3: n=9). S a ing a week 10 pos ansplan a ion, he human immune econs i u ion in mouse pe iphe al blood (PB) was assessed by low cy ome y e alua ing he equency o human CD45+ cells. s ep 2: Immuniza ion o Mice wi h idCs exp essing he pp65 An igen CD14+ monocy es we e isola ed a high pu i y ( om he same CB uni s used as sou ce o CD34+ HSCs) by immune-magne ic beads (Mil enyi Bio ec, Be gisch Gladbach, Ge many) and c yo- p ese ed. CD14+ cells we e used o he gene a ion o iDCpp65 a e ansduc ion wi h a icis onic in eg ase-de ec i e len- i i al ec o co-exp essing human cy okines GM-CSF/IFN-α and he HCMV-pp65 p o ein as desc ibed (IDLV-G2a-pp65) (20, 24). In sho , monocy es we e p e-condi ioned wi h ecom- binan human GM-CSF and IL-4 (bo h 50 ng/ml; Cellgenix, F eibu g, Ge many) o 8 h p io o len i i al gene ans e . T ansduc ion o monocy es wi h IDLV-G2a-pp65 was pe o med a a mul iplici y o in ec ion o 5 (2.5mg/ml p24 equi alen ) in he p esence o 5µg/ml p o amine sul a e (Valean , Duesseldo , Ge many) o 16h. A e wa d, cells we e ha es ed by esuspen- sion in phospha e-bu e ed saline (PBS), washed wice, and c yop ese ed. Fo ansduc ion quali y assessmen , a sample o ozen cells was hawed and main ained in he X-VIVO 15 medium (Lonza, Basel, Swi ze land) o 7days. Analyses o cell FIGURe 1 | Gene a ion o induced dend i ic cells exp essing pp65 (iDCpp65) o immuniza ion o humanized mice. (A) Scheme o expe imen al design. Pu i ied CD34+ hema opoie ic s em cells ob ained om h ee co d blood (CB) uni s and de oid o con amina ing Tcells we e used o ansplan a ion o h ee mice coho s. Pu i ied CD14+ cells om he same CB we e ansduced wi h a icis onic in eg ase-de ec i e len i i al ec o co-exp essing huGM-CSF, huIFN-α, and HCMV-pp65. C yop ese ed cells we e hawed, analyzed o iabili y, iden i y, and po ency cha ac e is ics in i o, and used o p ime/boos immuniza ions [a weeks 6, 7, 10, and 11 a e hema opoie ic s em cell ansplan a ion (HSCT), n=17]. Longi udinal analyses o pe iphe al blood we e pe o med on weeks 10, 16, and 20 a e HSCT. Mice we e sac i iced a week 20 a e HSCT and bone ma ow, SPL, Thy, pe iphe al lymph node (PLN), and MLN we e isola ed and analyzed. Non-immunized humanized mice (n=11) om he same co esponding CB uni s we e used as con ols. (B) Pe cen age o iable iDCpp65 cells a e c yop ese a ion and hawing (whi e ba s) and 7days a e he in i o cul u e (black ba s) o each CB uni . (C) iDCpp65 gene a ed wi h CB 1 (whi e), 2 (g ay), and 3 (black ba s) we e main ained o 7days in i o and he ex ac ed DNA was analyzed by RT-q-PCR o LV copy numbe pe cell. (d) Concen a ion o huIFN-α (g ay ba s) and huGM-CSF (black ba s) de e mined o cell supe na an s collec ed a day 7 o in i o di e en ia ion o iDCpp65 gene a ed om CB dono 1 and 3 and measu ed by ELISA. (e) Rep esen a i e do plo s o low cy ome y analyses o iDCpp65 (CB1) a haw and a day 7 o di e en ia ion in i o, showing high iabili y (7AAD nega i e popula ion), down egula ion o CD14, up egula ion o CD45 and CD11c (used as ga es o u he analyses), and up egula ion o HLA-DR, CD80, CD86, and pp65 upon iDCpp65 di e en ia ion. 4 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 CMV PepTi a o (pp65 o e lapping pep ide pool, Mil enyi Bio ec, Be gisch Gladbach, Ge many) o 10µg/ml o Wilms Tumo 1 (WT1) nega i e con ol o e lapping pep ide pool (Mil enyi Bio ec, Ge many). A p o ein anspo inhibi o cock ail (eBioscience, F ank u , Ge many) was added o he cells 2h a e pep ide s imula ion. A he end o s imula ion, he su ace s aining wi h APC an i-CD3, APC-H7 an i-CD4, and PC7 an i-CD8 an ibodies (BioLegend, Ge many) was 5 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 pe o med. Subsequen ly, cells we e pe meabilized and ix- a ed o in acellula s aining wi h PE an i-IFN-γ an ibodies (eBioscience, San Diego, CA, USA). Samples we e acqui ed by LSR II low cy ome e and da a we e analyzed using FlowJo so wa e e sion 7.6.4 (T ee S a Inc., Ashland, OR, USA). As a ge s o enzyme-linked immuno spo (ELISPOT) assays, K562 cells exp essing HLA*A02.01 (also known as a i icial an igen-p esen ing cells o “aAPCs”) and aAPCs exp essing pp65 endogenously (aAPC/pp65) we e cul u ed in RPMI 1640 (Lonza, Swi ze land) con aining 10% e al bo ine se um. Fo IFN-γ de ec ion by ELISPOT, Mul iSc een HTS pla es (Me k Millipo e, Da ms ad , Ge many) we e coa ed wi h an i-IFN-γ an ibodies (Mab ech, Nacka S and, Sweden) a 4°C o e nigh . Then, 2.5×104 Tcells we e mixed wi h 7.5×104 aAPCs ei he no ca ying an an igen, o wi h aAPCs pulsed wi h WT1 pep ides (aAPC+WT1), o wi h aAPCs pulsed wi h pp65 pep- ides (aAPC+pp65) o exp essing pp65 endogenously (aAPC/ pp65). The Tcell/aAPC cocul u es we e incuba ed o e nigh , washed, and incuba ed wi h bio in-conjuga ed an i-human IFN-γ monoclonal Ab, ollowed by incuba ion wi h alkaline phospha ase-conjuga ed s ep a idin. Colo de elopmen was pe o med using NBT/5-b omo-4-chlo o-3-indolyl phospha e liquid subs a e, and he pla es we e analyzed in an ELISPOT eade (AELVIS, Hanno e , Ge many). The mean ELISPOT coun s o ac i a ion wi h iDC (4 eplica e cul u es) and iDCpp65 (6 eplica e cul u es) we e ob ained. s ep 5: Analyses o human Cy okines in Mouse Plasma A sac i ice, PB samples we e collec ed by hea punc u e and cells we e subsequen ly sedimen ed by cen i uga ion. The supe - na an con aining plasma was s o ed a −80°C un il he analysis. A e hawing, plasma samples we e cen i uged a 2,000g o 10 min a oom empe a u e p io he analysis o emo e emaining cell deb is. 25µl o plasma we e used o analysis o each sample. The concen a ion in plasma o human GM-CSF, IFN-γ, monocy e chemoa ac an p o ein (MCP-1), TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, and IL-12 (p70) was analyzed by a 14-plex Luminex ki (Milliplex Millipo e, MA, USA) acco ding o manu ac u e ’s p o ocol. s ep 6: Cha ac e iza ion o he e minal human Cell Responses in Lympha ic issues Spleen (SPL), pe iphe al lymph nodes (PLN), mesen e ic lymph nodes (MLNs), BM, and Thy cells we e isola ed a e sac i ice and homogenized. Fo mice ansplan ed wi h CB1-3, cell suspen- sions gene a ed wi h PLN and MLN we e combined, whe eas o CB2 and CB3, he e was an addi ional cell suspension ac ion o MLN analyzed sepa a ely. Cell suspensions we e washed and e-suspended in PBS o subsequen s aining wi h luo och ome- conjuga ed monoclonal an ibodies as desc ibed abo e o PB (24). Thymocy es we e s ained 15 min a oom empe a u e wi h he ollowing an ibodies: paci ic blue an i-CD45, AF700 an i-CD4, APC an i-CD3, PC7 an i-CD8, FITC an i-TCR αβ (BioLegend, Ge many). A e washing, cells we e e-suspended in PBS con aining 1% human se um and analyzed by an LSR II low cy ome e . s ep 7: es ablishing he da abase and s a is ical Analyses All a iables consis ing o cell pheno ypes de e mined as ela i e equency (PB and lympha ic issues) and coun s (lympha ic issues) and cy okines concen a ions (plasma) we e o ganized in a Pi o Table using Excel so wa e 2010 (Mic oso , Redmond, WA, USA). Each sample was coded acco ding o he CB uni (1, 2, o 3) cell ype, issue, ime-poin o analyses, in e en- ion g oup (con ol o iDCpp65), and mouse gende ( emale “F” o male “M”). Be a eg ession analysis was employed o model he associa ion be ween he in e en ion g oups and he cell pheno ypes measu ed as “ ela i e equency,” which is exp essed as odds a io (i.e., he odds o e en be ween iDCpp65 and con ol), whe eas nega i e binomial eg ession was used o model he associa ion be ween he in e en ion g oups and he coun cell pheno ypes exp essed as a e a io (i.e., he incidence a e be ween iDCpp65 and con ol). Bo h models whe e implemen ed wi h and wi hou s a i ica ion by gende . Pa ame e es ima ion was pe o med by leas squa e means. The p- alues calcula ed a signi icance le els 0.05 and 0.01 using he wo-sided z- es s a is ic we e conside ed signi ican . All analyses we e implemen ed using he SAS 9.3 so wa e (SAS, Ca y, NC, USA). PROC GLIMMIX was used o Be a eg ession analysis, oge he wi h PROC GENMOD o he nega i e bino- mial eg ession analysis. A wo-way analysis o a iance was pe o med o analyze he esul s o he IFN-γ ELISPOT assay using G aphPad P ism e sion 5 so wa e (G aphPad So wa e, Inc., La Jolla, CA, USA). s ep 8: ANN Classi ica ion App oach The compu a ional analysis was ca ied ou in Ma Lab e sion 7.11.0, 2010 (Ma hWo ks, Inc., Na ick, MA, USA) using he Neu al Ne wo k Pa e n ecogni ion applica ion. The inpu s (15 ma ke s ep esen ing cell pheno ypes o PB, SPL, PLN+MLN, o MLN, BM and 8 ma ke s o Thy) and hei co esponding ou pu s [immuniza ion s a us o each mice, “yes” (1) o “no” (0)] we e used o aining he ANN. To ind he app op ia e numbe o neu ons in he hidden laye , we pe o med a k- old c oss alida ion (29) (wi h k being equal o 3, 4, 5) esul ing in 12 neu ons (8 o Thy). The ou pu laye consis ed o wo neu ons. The classi ica ion accu acy (pe cen - age o co ec classi ica ions) was es ima ed by a e aging o e 2000 ANN. Fo each o hose ANN, he inpu da ase was andomly di ided in o aining (70% samples/ issue), alida- ion (15% samples) and es ing (15% samples). The Le enbe g– Ma qua d back-p opaga ion ( ainlm) algo i hm was used o he aining s ep, as i is bes sui ed o small ne wo ks (30). Fo he hidden laye , we used sigmoid ans e unc ions. The da a we e i s s anda dized (i.e., each ma ke was no malized o 0 mean alue and uni a iance) in o de all he ma ke s o be a he same scale. We also pe o med he same classi ica ion analysis o immu- nized (1) and non-immunized mice (0), bu by di iding ou 6 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 samples in o male and emale mice da a se s. In his case, we used he same pa ame e se ing o ANN as abo e and es ed he classi ica ion accu acy o each subpopula ion. In addi ion o he classi ica ion accu acy (de ined as he ac ion o co ec ly classi ied samples), we measu ed he sensi i i y and speci ici y o he classi ica ion, applied inside each g oup. The sensi i i y, de ined as sensi i i y= numbe o ue posi i es numbe o ue posi i ees + numbe o alse nega i es , ep esen s he p obabili y o a sample classi ied as immunized (1) o belong o he immunized g oup. The speci ici y p o ides he p obabili y o a sample classi ied as non-immunized (0) o belong o he non-immunized g oup. Speci ici y is de ined as sp eci ici y= numbe o ue nega i numbe o ue nega i es ees + numbe o alse posi i es . The 15 ma ke s co espond o he equencies o human lym- phocy e lineages analyzed pe issue (PB, BM, SPL, MLN+PLN, MLN) o each mouse included cells de e mined as equencies o CD45, CD19/CD45, CD3/CD45, CD14/CD45 (in BM CD34/ CD45 ins ead o CD14/CD45), CD4/CD45, CD8/CD45 and a equency o o he non-de e mined CD45+ cells, equencies o CD4 sub ypes (N, CM, EM, and TE), and equencies o CD8 sub ypes (N, CM, EM, and TE). Eigh ma ke s we e used o Thy: equencies o CD45, CD3/CD45, CD4/CD45, CD8/CD45, CD4−/CD8− (double nega i e, DN), CD4+/CD8+ (double posi- i e, DP), TCRαβ+/CD3, and CD4 o CD8 a io alues. s ep 9: PCA P incipal componen analysis (31) was used o ecognize clus e s o in e ela ed ma ke s o con ol and iDCpp65 mice in he di e en issues. PCA cons uc s speci ic di ec ions, which a e called p incipal componen s, along which he da a a e mos dispe sed and hus bes dis inguishable. In his way, a da a se can be ep esen ed by he p incipal componen s, which inco po a e a speci ic amoun o he a iance (o dispe sion). Since he componen s a e unco ela ed o each o he , he ma k- e s, which a e s ongly co ela ed wi h a componen , compose a clus e o ma ke s, which a y oge he . He e, we c ea ed hese clus e s by selec ing he pheno ypic ma ke s ha we e s ongly co ela ed o an i-co ela ed (mo e han 80%) wi h any componen o each g oup. The i s ou componen s we e conside ed o analysis since hey we e able o inco po a e mo e han 80% o he o al a iance in bo h con ol and iDCpp65 mice in all issues. These componen s we e he basis o a iance- dis ibu ion compa isons and co ela ion hea -maps o bo h mouse g oups. The ma ke s ha we e s ongly co ela ed o an i-co ela ed wi h he i s go e ning componen we e used o co ela ion compa isons be ween he con ol and iDCpp65 mice. The da a o con ol and iDCpp65 mice om BM and SPL we e ep esen ed by 30 a iables (pe cen ages and coun s o each o he 15 ma ke s); om combined PLN, PB, and MLN by 15 a iables (only pe cen ages); om Thy by 14 a iables (pe cen ages and coun s). As in he aining o ANN, he da a we e i s s anda dized (i.e., each ma ke was no malized o 0 mean alue and uni a iance) in o de eno malize all ma ke s o he same scale. ResUL s C yop ese ed idCpp65 Remained Viabili y and Cha ac e is ics a e hawing We ha e shown be o e ha human T cell esponses agains HCMV-pp65 we e consis en ly s imula ed in humanized NRG mice immunized wi h iDCpp65 (19, 20, 24). He e, iDCpp65 we e gene a ed and c yop ese ed immedia ely a e ansduc ion, and subsequen ly used o p ime-boos immuniza ions o emale and male mice (Figu e1A). E ec s o immuniza ions in lympha ic issues we e analyzed 20 weeks a e HSCT co esponding o 9weeks a e he las immuniza ion. The cell accine, iDCpp65, showed high iabili y di ec ly a e hawing (66–88%) and in i o cul u e o 7days (42–63%) ela i e o s a ing numbe o cells (Figu e1B). E icien ansduc ion wi h IDLV and pe sis ency o episomal i al copies we e con i med o cells main ained in cul- u e o 7days and showing in a e age i e len i i al copies/cell (Figu e1C). T ansgenic cy okines ha accumula ed on he cell supe na an o iDCpp65 de i ed om CB1 and CB3 o 7days we e de ec ed in he ange o 500–1,000pg/ml o IFN-α and 50–100pg/ml o GM-CSF (Figu e1D). The iable cells showed a ypical DC immuno pheno ype wi h co-exp ession o HLA-DR (61.60–91.85%), CD86 (93.20–98.97%), and CD80 (29.1–97.7%) su ace ma ke s (Figu e 1E, ep esen a i e da a o a ba ch o iDCpp65, Figu e S1A in Supplemen a y Ma e ial). In acellula immunos aining o de ec ion o pp65-posi i e iDCpp65 cells by low cy ome y assay showed a iable esul s among di e en CB dono s (CB1: 49.20%, CB2: 2.49%, CB3: 16.40% when calcula ed o o al iable cells in suspension 7days a e in i o cul u e, Figu e S1B in Supplemen a y Ma e ial). idCpp65 Immuniza ions A ec ed Lymphocy es Coun s in PB, Plasma Cy okines P o iles, and Lymph Node de elopmen Th ee independen coho s o NRG mice a e CD34+ HSCT we e gene a ed. 5.0×105 hawed and iable au ologous iDCpp65 we e injec ed a weeks 6, 7, 10, and 11 a e HSCT. Body-weigh and gene al heal h condi ions we e moni o ed weekly. Al hough emales om bo h con ol and iDCpp65-immunized coho s we e ligh e han males, mice o bo h gende s gained weigh no mally o he 20weeks a e HSCT (Figu e2A) and showed no signs o g a - e sus-hos disease o o gan pa hologies a sac i ice (da a no shown). The equencies o human CD45+ cells de ec ed in PB a weeks 10, 16, and 20 pos -HSCT we e consis en ly highe in emales, pa icula ly o emales immu- nized wi h iDCpp65 (Figu e2B). The expansion o human CD8+ and CD4+ Tcell de ec able in PB was supe io in he iDCpp65 coho compa ed o con ols, especially in he male g oup (Figu e2B; Table S1 in Supplemen a y Ma e ial). Analyses o human cells in PB 20weeks a e HSCT by assessmen o he FIGURe 2 | Longi udinal analyses o emale and male humanized mice. (A) Weigh moni o ing o con ol (n=11, uppe panel) and immunized mice (n=17, lowe panel). A ows indica e he weeks o induced dend i ic cells exp essing pp65 (iDCpp65) immuniza ions a e hema opoie ic s em cell ansplan a ion. Bi-weekly weigh (g) de e mined o emales (F) indica ed in g ay and o males (M) in black. The numbe o F and M mice pe g oup is indica ed. (B) Mean ela i e equencies o human CD45+, CD8+ in CD45+, and CD4+ in CD45+ cells de e mined in blood by low cy ome y o con ol (dashed line) and iDCpp65-immunized (solid line) F (uppe panel) and M (lowe panel) a weeks 10, 16, and 20 pos ansplan a ion. E o ba s ep esen SEs. *p<0.05 a e indica ed on he g aph. (C) Rela i e equencies o human cell ypes measu ed by low cy ome y in PBL o emale (F) and male (M) mice a week 20 pos ansplan a ion. Mean ela i e equencies o CD19+ (whi e), CD4+ (ligh g ay), CD8+ (da k g ay), and o he CD45+ cells (black) we e measu ed in con ol and iDCpp65-immunized mice, *p<0.05 a e indica ed on he g aph. (d) Pheno ypes o dis inc CD8+ and (e) CD4+ Tcells we e de e mined as naï e (N, whi e, CD45RA+/CD62L+), cen al memo y (CM, ligh g ay, CD45RA−/CD62L+), e ec o memo y (EM, da k g ay, CD45RA−/CD62L−) and e minal e ec o (TE, black, CD45RA+/CD62L−). Mean ela i e equencies a e shown o con ol and iDCpp65-immunized mice. (F) Concen a ion o human cy okines measu ed in plasma o F (uppe panel) and M (lowe panel) mice 20 weeks pos ansplan a ion. Mean concen a ions o cy okines we e de e mined o iDCpp65-immunized (g ay) and con ol (whi e) g oups. Ba s and ci cles e lec he SE o he es ima ed mean concen a ions and he obse ed concen a ions o indi idual samples, espec i ely. Concen a ion alues below he de ec ion limi o he assay a e indica ed (b.d.), *p<0.05, **p<0.01 indica ed on he g aph. (G) The a e age equency o lymph nodes ound in F ( op panel) and in M (bo om panel) mice o iDCpp65-immunized and con ol g oups. Values o inguinal (uppe -), axilla y (middle-), and iliac (lowe panel) lymph nodes ound 20weeks a e hema opoie ic s em cell ansplan a ion. Numbe o samples: emale, n=5/9; male, n=6/8, con ol/immunized, espec i ely. 7 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 FIGURe 3 | Func ional memo y T esponses agains pp65 a e induced dend i ic cells exp essing pp65 (iDCpp65) immuniza ion. (A) Tcells isola ed om lymph nodes o immunized mice (n=3) we e e-s imula ed in i o wi h ei he induced DC (iDC) o iDCpp65. Absolu e Tcell numbe s be o e (whi e ba s) and 7days a e (black ba s) s imula ion a e shown. (B) Rela i e old inc ease o cells popula ions be o e and 7days a e cocul u e wi h iDC (g ay ba s) and iDCpp65 g oup (black ba ). (C) CD8+ and (d) CD4+ Tlymphocy es expanded a e cocul u e wi h iDC (n=3) o iDCpp65 (n=3) we e le uns imula ed (whi e ba s) o e-s imula ed wi h a Wilms Tumo 1 (WT1) pep ide pool (g ay ba s) o pp65-pep ide pool (black ba s). F equency o cells p oducing IFN-γ is shown. (e) Tcells expanded wi h iDC ( ou cul u es pe g oup) o iDCpp65 (six cul u es pe g oup) we e cocul u ed wi h an a i icial an igen-p esen ing cell (aAPC) on an ELISPOT pla e. The aAPCs we e e he no loaded (whi e ba s), loaded wi h WT1 pep ides (ligh g ay ba s), loaded wi h pp65-pep ides (da k g ay ba s), o ansduced o endogenous pp65 exp ession and loading (black ba s). Mean absolu e numbe o IFN-γ-spo s and *p<0.05 (analysis o a iance), †p<0.05 (F- es ) a e indica ed. 8 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 mean ela i e equencies showed ha CD8+ Tcells we e highe in he immunized coho (p=0.03) (Table S1 in Supplemen a y Ma e ial), wi h high signi icance o he male g oup (p=0.01 CD8+; p=0.09 CD4+) (Figu e2C; Table S1 in Supplemen a y Ma e ial). This was associa ed wi h a highe accumula ion o EM and TE CD8+ cells in he g oup o iDCpp65-immunized males whe eas he g oup o emales showed highe accumula ion o CM CD8+ cells in immunized e sus con ol g oup (Figu e2D; Table S1 in Supplemen a y Ma e ial). The ela i e equencies o CD4+ Tcell sub ypes we e only sligh ly al e ed, showing an inc ease in he ela i e equencies o CM cells o males and EM cells o emales (Figu e 2E; Table S1 in Supplemen a y Ma e ial). A luo escen -based bead assay was used o measu e he concen a ion o 12 human cy okines in mouse plasma (GM-CSF, MCP-1, IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and IL-12p70). IL-1β, IL-2, IL-4, and IL-12p70 we e below he de ec ion limi . Fo he g oup o non-immunized mice, he baseline concen a ions o human cy okines in he plasma we e consis en ly highe o males. Rema kably, IL-5, MCP-1, and IL-8 we e only de ec able in non-immunized males. Upon immuniza ions, bo h gende s showed inc eased IFN-γ ( a e a io 2.74, p= 0.15) and GM-CSF concen a ions ( a e a io 2.96, p=0.092) (Table S2 in Supplemen a y Ma e ial). The inc ease o IFN-γ concen a ion a e immuniza ion was pa icula ly high o immunized emales (p=0.003) (Figu e2F). De ec ion o IL-5, MCP-1, IL-6, and IL-8, in plasma o emales was only pos- sible a e iDCpp65 immuniza ion. The e o e, o e all, iDCpp65 immuniza ion ha nessed he ma u a ion o human T cells in PB, which was in gene al associa ed wi h an inc ease o human cy okines in plasma (in pa icula o emales). The unde sized and incomple ely de eloped lymph nodes in humanized mice a e di icul o be de ec ed. They can be, none heless, de ec ed as qui e small “ a y” s uc u es in he expec ed ana omical egions (inguinal, axilla y, iliac). Upon immuniza ion wi h iDCpp65, hese d aining LNs become mac oscopically mo e no iceable. Al hough hese egene a ed LNs a e no ully “no mal” in ela- ionship o lymph nodes ound in immune compe en mice, hey con ain a high densi y o human Tcells (19). In his cu en s udy, we con i med a highe equency o de eloped d aining lymph nodes (nea he immuniza ion si es) in he immunized coho s, which was mo e e iden o males (Figu e 2G). Rema kably, his obse a ion was in e ed o he axilla y lymph nodes, which we e mo e p ominen in emales. On he o he hand, he a e age equency o de ec able iliac nodes was lowe in he immunized coho (Figu e2G). Small MLNs de eloped in mos mice, ega dless o gende o immuniza ions (da a no shown), indica ing he possibili y ha he de elopmen o MLNs may be induced di e en ly. idCpp65-Immunized Mice demons a ed Func ional pp65-speci ic Memo y Cell Responses Lymph nodes o immunized mice we e a aluable compa - men o he de ec ion o high equencies o human T cells. Lymphocy es eco e ed om lymph nodes o h ee immu- nized mice we e pooled and main ained in i o o 48h o homeos a ic ac i a ion by beads and cy okines. Lymphocy es om non-immunized mice we e no used since, om p e ious expe ience, we we e no able o expand hem success ully in i o (19, 20). Mic ocul u es o cell suspensions we e incuba ed wi h au ologous “emp y” iDCs o wi h iDCpp65 a 10:1 T o DC a io o 1week o p omo e u he expansion o Tcells. The expan- sion was mo e p onounced o Tcells cul u ed in he p esence o iDCpp65 s imula ion han “emp y” iDCs ( old expansion ela i e he popula ion be o e s imula ion): 10.9 (iDCpp65) and 9.1 (iDCs) (Figu es3A,B). Ac i a ed CD8+ and CD4+ Tcells we e analyzed by low cy ome y o de ec ion o in acellula IFN-γ. Tcells expanded in he p esence o emp y iDC and e- s imula ed wi h WT1 o pp65-pep ide pools showed simila baseline equencies o CD8+ IFN-γ+ and CD4+ IFN-γ+ Tcells. In con as , Tcells expanded in he p esence o iDCpp65 and hen e-s imula ed wi h he pp65-pep ide pool showed a iable bu in a e age much highe ela i e equencies o CD8+ IFN-γ+ FIGURe 4 | Rela i e and absolu e quan i ica ion o human hema opoie ic lineages in lympha ic issues. Mean ela i e equency o human CD19+ (whi e), CD4+ (ligh g ay), CD8+ (da k g ay), and o he CD45+ cells (black) de ec ed by low cy ome y analyses o (A) bone ma ow, (B) SPL, (C) combined pe iphe al lymph nodes and MLN, and (d) MLN. F equencies o emale (F) and male (M) mice a e shown sepa a ely. (e) Absolu e cell coun s o bone ma ow and (F) spleen we e ob ained. Plo s ep esen a e age numbe o cells de e mined o con ol (g ay ba s) and induced dend i ic cells exp essing pp65 (iDCpp65)-immunized (black ba s) spli be ween emale and male mice. The numbe o mice analyzed pe g oup (n) and *p<0.05 is indica ed on he g aph. Fo (A,B,e,F) F/con ol n=5, F/iDCpp65 n=9, M/con ol n=6, M/iDCpp65 n=8. Fo (C) F/con ol n=3, F/iDCpp65 n=6, M/con ol n=4, M/iDCpp65 n=6. Fo (d) F/con ol n=4, F/iDCpp65 n=5, M/con ol n=6, M/iDCpp65 n=6. 9 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 and CD4+ IFN-γ+ Tcells (Figu es3C,D). The mean an i-pp65 esponse o CD8+ was 6.84 imes highe in he iDCpp65 han in iDC e-s imula ion g oup and o CD4+ Tcells in 6.27 imes, espec i ely. F- es compa ing a iances be ween iDC and iDCpp65 showed a s ong e idence o a iance di e ence be ween g oups in case o e-s imula ion wi h pp65-pep ide pool (p=0.025, CD8+; p=0.072, CD4+) bu no when g oups we e e-s imula ed wi h WT1 o no s imula ed a all (p>0.05 o all cases) (Figu es3C,D). As a complemen a y app oach, he abili y o Tcells o ec- ognize and be ac i a ed by pp65 epi opes p esen ed by an aAPC posi i e o HLA-A*02.01 was es ed by an IFN-γ-ELISPOT assay as p e iously desc ibed (32). Tcells expanded a e cocul- u e wi h iDCs o iDCpp65 we e exposed o e nigh o di e - en ypes o aAPCs, and he numbe s o eac i e Tcells we e quan i ied. Tcells expanded wi h iDCpp65 and cocul u ed wi h ei he aAPC loaded wi h pp65-pep ides o ansduced o pp65 exp ession showed on a e age signi ican ly highe equencies o ac i a ed T cells han when cocul u ed wi h aAPC loaded wi h con ol WT1 pep ides (p=0.016, p=0.026, espec i ely). No signi ican ampli ica ion o Tcell ac i a ion was obse ed when Tcells we e expanded in he p esence o “emp y” iDCs (p>0.05 o bo h cases) (Figu e3E). These da a con i med ha immuniza ions wi h iDCpp65 p omo ed a speci ic immune compe ence agains pp65 in humanized mice which was medi- a ed by human Tcells. he e ogeneous Pa e ns o human Lymphocy es in Lympha ic issues a e idCpp65 Immuniza ion Isola ed issues [BM, SPL, lymph nodes (PLN+MLN and MLN)] we e p ocessed o low cy ome y analyses and quan i ica ion o human lymphocy e equencies and absolu e numbe s. Fo BM, CD3+ T cells ep esen ed only a mino i y o huCD45+ cells, whe eas Bcells (CD19+) and o he CD45+ cells p e ailed. In e ms o ela i e equencies, a end o inc eased equencies o CD19+ cells was obse ed upon iDCpp65 immuniza ion, 16 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 The same analysis in MLN e ealed impo an di e ences in he a iance dis ibu ion in con ol and iDCpp65 mice (Figu e8E) and conside able co ela ion changes in he hea - map analysis (Figu e9E). In e se co ela ed signa u es could be seen be ween CD4% and CD8CM% (posi i e co ela ion in con ol mice, equal o 0.5969 and nega i e co ela ion in iDCpp65 ones, equal o −0.618), CD4% and CD8CM% (posi- i e co ela ion in con ol mice, equal o 0.8102, and a weakly nega i e co ela ion in iDCpp65 ones, equal o −0.384). The same in e sely co ela ed signa u e could be seen be ween CD4% and CD8N% (nega i e co ela ion in con ol mice, equal o −0.6346 and posi i e co ela ion in iDCpp65 ones, equal o 0.7513). Finally, in PB, he same analysis e ealed a s iking di e - ence in he a iance dis ibu ion (Figu e8F) and conside able di e ences in he hea -map analysis (Figu e 9F). In e sely co ela ed signa u es could be seen be ween CD4CM% and CD4EM% (posi i e co ela ion in con ol mice, equal o 0.6414 and nega i e co ela ion in iDCpp65 ones, equal o −0.598) as well as be ween CD4EM% and CD8CM% (posi i e co ela ion in con ol mice, equal o 0.6567 and weakly nega i e co ela- ion in iDCpp65 ones, equal o −0.3047). The posi i e s ong co ela ion be ween CD4EM% and CD8EM% in con ol mice (equal o 0.94) became neu al in iDCpp65 (−0.09983) and he nega i e s ong co ela ion be ween CD19% and CD4EM% in con ol mice (equal o −0.8088) becomes neu al in iDCpp65 (−0.06359). The abo e esul s showed how we could exploi he in insic complexi y and he e ogenei y o he inpu ma ke s o gain addi ional knowledge on he cha ac e is ic o an indi idual immunized mouse. Mo e speci ically, by checking he co ela ion pa e ns among speci ically selec ed ma ke s in an indi idual mouse, we could conclude abou hei immuniza ion s a us. Toge he wi h he classi ica ion pe o mance o ANN in he di - e en issues, hese esul s p edic ed mo e p onounced e ec s o immuniza ion in SPL and PLN combined wi h MLN compa ed wi h o he issues. dIsCUssIoN The use o immune de icien mice humanized wi h human HSCs o s udy and cha ac e ize he ma u a ion o human Tcells a e immuniza ions in di e en lympha ic issues gene a e la ge da a se s and highly complex esul s. Long- e m s udies (20 o mo e weeks a e ansplan a ion) and la ge mouse coho s (15 mice o mo e) ha e been commonly used (6). The ini ial HSC eng a men in BM, ea ly Tcell de elopmen in hymus and he eg ess o naï e Tcells o he pe iphe y ecapi ula e he gene al pa e ns ound in immune compe en mice and in humans (5, 33, 34) in he i s 10–15 weeks a e ansplan a ion. Howe e , analyses o he T cell ma u a ion in seconda y lymphoid o gans ha e shown o be mo e he e ogeneous and p edic i e o he quali y o he immune econs i u ion and de elopmen o ma u e T cells. This e lec s he “pe sonal- ized” condi ion o di e en CB dono s wi h he e ogeneous gene ic backg ounds, which is ampli ied in a xenog a sys em. In addi ion, al hough he eng a men o human HSCs (35) and he highe hymic ou pu in humanized emale mice (25) had been p e iously epo ed, he ele ance o he mouse sex in de e mining he impac o immuniza ions and he p edic - abili y o human Tcell esponses had no been p esen ed. All hese ac o s we e aken in accoun when explo ing humanized mice o es ing a new accine ype. In he p esen wo k, we sough o e alua e he mul idimen- sional spa ial e ec s o a po en cellula accine agains HCMV ma ched o he HSC dono and p o iding he h ee main ele an signals o bo h an igenic and homeos a ic ac i a ion o Tcells: an immune-dominan an igen p esen ed ia HLA class I and II, co-s imula o y ligands, and in lamma o y cy okines. Thus, iDCpp65 which a e iable o 2–3weeks in i o and e ec i ely mig a e o lymph node s uc u es (19) we e used o accele a e and po en ly boos he human Tcell de elopmen and unc ional esponses in humanized mice. As an icipa ed, imp o ed human Tcell de elopmen and ma u a ion we e longi udinally obse ed in PB and e minally in se e al lympha ic issues in iDCpp65- immunized mice a 20-weeks a e HSCT. These esul s comple- men ed p e ious indings ob ained in sho e (16weeks) and in longe (up o 36weeks) iDCpp65 immuniza ion models (20, 24). Ad anced s a is ical analyses showed ha iDCpp65 immuniza- ions p omo ed a ypical memo y Tcell signa u e (abo e all o CD8+ Tcells) which was mos p ominen o Tcells homing lymph nodes and spleen. As mos o he s udies using human- ized mice ha e ocused on analyses o human cells in blood and spleen (5, 8, 18) i is impo an o emphasize ha , as seen om he ANN and PCA analyses, he quan i y and quali y o human Tcell eac i i y in pe iphe al and MLNs (e en i hey a e small and di icul o be sampled) ha e o be aken in accoun , as lymph nodes ep esen he p ime issue o in e ac ions be ween an igen-p esen ing cells wi h naï e CD8+ and CD4+ T cells. Fu he , he le els o human IFN-γ in plasma inc eased upon immuniza ion. In gene al, bo h cellula and cy okine immune e ec s we e mo e accen ua ed o emale mice. This con i med and expanded ou p e iously epo ed obse a ion ha humanized emale mice ha e highe ou pu o naï e Tcells han humanized male mice un il 12weeks a e HSCT (25). A ound 16weeks a e HSCT, male mice showed highe de elopmen o ma u e Tcells and by 20weeks a e HSCT, he equencies o human CD45+ cells, naï e and memo y Tcells in PB equalized be ween he sexes. No a e al. showed ha be ween 10 and 12weeks a e HSCT, emales ansplan ed wi h limi ing amoun s o HSCs ob ained om se e al CB uni s gene ally exhibi ed a highe equency o huCD45+ cells han male mice (35). The e o e, CB-HSCT in humanized mice could po en ially mi o he e ec o sex s e oids on human immune econs i u ion since empo a ily blocking sex s e oids be o e HSCT in pa ien s, inc eased hymus unc ion and enhanced he a e o T-cell egene a ion (36). Responses o a ious ypes o accina ion a e o en highe among women [ o a e iew see Re . (37)], who a e able o moun s onge humo al esponses han men. A possible explana ion o his phenomenon is based again on majo sex s e oid ho mones such as he ypical “ emale” ho mone es adiol ha enhances he adap i e and inna e immune sys ems, and he “male” ho mone es os e one conside ed immune supp essi e. 17 Volk e al. Lea ning Machine o Humanized Mice F on ie s in Immunology | www. on ie sin.o g Decembe 2017 | Volume 8 | A icle 1709 No ewo hy, women display highe T helpe ype 2 (Th2) esponses, whe eas males a o Th1 esponses (37). Al hough sex-speci ic esponses o dis inc accines a e no usually con- side ed and ha e been epo ed in a ew clinical ials, his is an impo an ac o also o be conside ed in p eclinical esea ch, when es ing new accine ypes, including when humanized mice a e used as a po ency model. In he cu en model, we ob ained no only highe esponses, bu explo ing he ANN, also a be e p edic ion o esponse. Thus, as a logical app oach o educe he numbe s o humanized mice when es ing a accine is ini ially a o ing he use o emale mice. In addi ion, om now on, s udies on humanized mice should conside male and emale esponses as dis inc esponses, and should be analyzed sepa a ely and compa ed. We also showed ha he s a is ical me hods can be com- plemen ed wi h an ANN algo i hm in o de o pin down he complexi y o a mul idimensional da a se s including usual immune ma ke s such as equencies o he human cell pheno- ypes among lympha ic issues and conside ing mouse sexes. As gene ally p oposed o ANNs (38), we we e able o demon- s a e he e ha he ANN based on he humanized mouse da a could “lea n” o ecognize he immune p ope ies o immunized e sus con ol mice. Fo s udies in humans, ANNs we e buil wi h independen immunologic a iables such as cell p oli - e a ion, pheno ypic ma ke s, and cy okine exp ession in he con ex o p os a e cance and in HSCT pa ien s (39–41). To ou knowledge, he applica ion o ANNs o humanized mouse mod- els o p edic ing he accu acy o immune esponse o de ining signa u es o Tcell esponses was no p e iously pe o med. The iden i ica ion o lymph node and spleen as he mos p edic i e o gans o he immune s a e o con ol e sus immunized mice migh be u he imp o ed by eleasing he assump ion o s a is- ically independen issues. Along wi h local immune popula ion dynamics, i would be o in e es o in es iga e he immune cell a icking dynamics be ween di e en issues (42, 43). In his way, we could elax he assump ion o issue independence. Howe e , he immune cell a icking is an open challenge ye o be sol ed in u u e esea ch. Al oge he , he cu en app oach, modali ies o analyses and obse a ions gi e aluable in o ma ion o u he planning o in i o es ing o accines and immune modula o s in humanized mice. The 3R p inciple (Replace animal es ing, Reduce he num- be o animals, and Re ine he analyses) can hus be ad anced o Reduce and Re ine: (i) by using (a leas ini ially) emale mice and (ii) explo ing bio-in o ma ics me hods such as ANN o comple- men adi ional s a is ical analyses in o de o de ine he mos impo an issues (such as spleen and lymph nodes) and he PCA ha e eal signa u es and co ela ions o immune esponses o di e en lympha ic issues. e hICs s A eMeN All subjec s dona ing co d blood p o ided w i en in o med consen . This s udy was app o ed by he E hics Commi ee o Hanno e Medical School. AU hoR CoN RIBU IoNs RS planed he p ojec , designed expe imen s, ob ained unding and egula o y app o als, en olled collabo a o s, in e p e ed he da a, and w o e and edi ed he manusc ip . VV conduc ed expe i- men s, analyzed da a, and w o e he i s manusc ip d a . BS, ST, AS, LG, and CR assis ed in p epa a ion and analyses o human- ized mice. CF pe o med he human cy okine a ay analyses. CK assis ed in he p ocu emen and collec ion o HSC o he s udies. LS pe o med he s a is ical analyses. AR, PR, HH, and MM-H pe o med he ANN and PCA analyses, in e p e ed he da a, and w o e and edi ed he manusc ip . SK and UK assis ed in he execu ion o he iDCpp65 quali y con ol analyses, and e ised he manusc ip . ACKNoWLedGMeN s The au ho s hank all o he cu en and pas membe s o he Regene a i e Immune The apies Applied Labo a o y o hei aluable con ibu ions. The au ho s hank Sebas ian Binde o e ising he manusc ip . FUNdING This wo k was suppo ed by g an s o he Ge man Resea ch Council (DFG/SFB738 P ojec A6 o RS; DFG/REBIRTH Uni 6.4 o RS, Uni 6.3 o CF) and he Ge man Cen e o In ec ions Resea ch (DZIF-TTU07.803 o RS). VV ecei ed a DAAD/ZIB Ph.D. ellowship, ST ecei ed a RegSci Ph.D. ellowship and CDR ecei ed a CNPq “Sciences wi hou Bo de s” pos -doc o al el- lowship. HH and AR would like o acknowledge he SYSMIFTA ERACoSysMed g an (031L0085B) o he inancial suppo o his wo k. 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