Lack of acute xenogeneic graft-versus-host disease, but ... · KEY WORDS: humanized † HU-PBL-SCID...

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Lack of acute xenogeneic graft-versus-host disease, butretention of T-cell function following engraftment ofhuman peripheral blood mononuclear cells in NSG micedeficient in MHC class I and II expressionMichael A. Brehm,*,1 Laurie L. Kenney,* Michael V. Wiles,† Benjamin E. Low,† Roland M. Tisch,‡

Lisa Burzenski,† Christian Mueller,§ Dale L. Greiner,* and Leonard D. Shultz†

*Diabetes Center of Excellence and §Department of Pediatrics and Horae Gene Therapy Center, University of Massachusetts Medical School,Worcester, Massachusetts, USA; †The Jackson Laboratory, Bar Harbor, Maine, USA; and ‡Department of Immunology and Microbiology,University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

ABSTRACT: Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) supportpreclinical studiesofhumanpathogens, allograft rejection, andhumanT-cell function.However, amajor limitationofPBMC engraftment is development of acute xenogeneic graft-versus-host disease (GVHD) due to human T-cellrecognitionofmurinemajorhistocompatibility complex (MHC).Toaddress this,we created2NOD-scid IL-2 receptorsubunit g (IL2rg)null (NSG) strains that lackmurineMHC class I and II [NSG–b-2-microglobulin (B2M)null (IA IE)null

and NSG-(Kb Db)null (IAnull)]. We observed rapid human IgG clearance in NSG-B2Mnull (IA IE)null mice whereasclearance inNSG-(KbDb)null (IAnull)mice andNSGmicewas comparable. InjectionofhumanPBMCs intoboth strainsenabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cellfunction was documented by rejection of human islet allografts. Administration of human IL-2 to NSG-(Kb Db)null

(IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase inhuman regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data documentthat NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD andenable evaluation of human antibody therapeutics targeting human T cells.—Brehm, M. A., Kenney, L. L., Wiles,M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft-versus-host disease, but retention of T-cell function following engraftment of human peripheral bloodmononuclearcells in NSGmice deficient in MHC class I and II expression. FASEB J. 33, 000–000 (2019). www.fasebj.org

KEY WORDS: humanized • HU-PBL-SCID • GVHD • immunodeficient • humanized

Immunodeficient mice engrafted with human immunesystems are valuable preclinical tools for studying humanimmunity in a small animal model. In the early 2000s, anumber of laboratories developed immunodeficient micewith genetic mutations in the IL-2 receptor common g

chain (for reviews, see refs 1–6). These mice support en-graftment with high levels of human immune and hema-topoietic cells. Although a number of mouse strainsbearing the IL-2 receptor subunit g (IL2rg)null (NSG) mu-tation have been previously described (4), the NOD-scidIL2rgnull [NSG orNOD/Shi-scid/IL-2Rgnull (NOG)] strainsare the most widely used as recipients of human cells andtissues (7, 8). These mice lack T, B, and NK cells, and havedefects in innate immunity. In addition, theNSGandNOGstrains have a humanlike polymorphism in the Sirpa gene,whichcontrolsmacrophage recognitionand the removalofforeign cells via the Sirp-a/CD47 axis. The Sirpa allele inNSG and NOG mice supports enhanced engraftment ofhuman cells and tissues (9, 10).

A number of human tissues and cell populations havebeen engrafted into immunodeficient mice to model hu-man biology and immunity (2, 6). One approach is theengraftment of humanperipheral bloodmononuclear cells,or PBMCs [termed the Hu–peripheral blood leukocyte(PBL)–SCID model], first described in 1988 (11). Human

ABBREVIATIONS: AAV, adeno-associated virus; Ab, antibody; B6, C57BL/6;ds, double-stranded; DT, diphtheria toxin; DTR, diphtheria toxin receptor;FcRn, neonatal FcR; GVHD, graft-versus-host disease; IEQ, islet equivalent;IL2rg, IL-2 receptor subunit g; MHC, major histocompatibility complex;NOD, NOD/ShiLtJ; NOG, NOD/Shi-scid/IL-2Rgnull; NSG, NOD-scidIL2rgnull; PBL, peripheral blood lymphocyte; PBMC, peripheral bloodmononuclear cell; PD-1, programmed death-1; RIP, rat insulin promoter;TALEN, transcription activator–like effector nuclease; TEMRA, termi-nally differentiated effector memory CD45RA+ T cell; Treg, regulatory T1 Correspondence: Diabetes Center of Excellence, Program in MolecularMedicine, University of Massachusetts Medical School, 368 PlantationSt., AS7-2053, Worcester, MA 01605, USA. E-mail: [email protected]

doi: 10.1096/fj.201800636RThis article includes supplemental data. Please visit http://www.fasebj.org toobtain this information.

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T cells are the predominant cell type that engrafts in thismodel, whereas engraftment of other cell populations—such as B, myeloid, or NK cells—is relatively low. TheHu-PBL-SCIDmodel has been used to study human infectiousagents, tissue transplantation, and human T-cell immunefunction (2, 12–14). One of the primary uses of this modelis the study of acute graft-versus-host disease (GVHD)(15–25), amajor problem in clinical hematopoietic stem celltransplantation (26). NSG mice engrafted with humanPBMCs develop an acute xenogeneic GVHD-like diseaseupon recognition of themurine cells and tissues bymaturehuman T cells (23).

Although useful for the study of human GVHD, stud-ies of other human immune functions using the Hu-PBL-SCIDmodelhavebeen limitedby thebriefwindowoftime available to conduct experiments before the PBMCrecipients succumb to GVHD (23). We have reported thatmurine major histocompatibility complex (MHC) class Iand II antigens are the major components that are recog-nized by mature human T cells during development ofacute xenogeneic GVHD inNSGmice (23). These findingswere based on earlier observations following injection ofPBMCs into NSG mice lacking murine MHC class I orMHCclass II antigens.HumanCD4+Tcells predominatedinMHCclass I–deficientNSGmice,whereas humanCD8+

T cells predominated inMHC class II–deficientNSGmice,demonstrating that robust expansion of the respective cellsubsets depends on the type of murine MHC expressed(24). Moreover, NSG MHC class I–deficient mice engraf-ted with PBMCs exhibited delayed development ofGVHD when compared with NSG or NSG MHC classII–deficient mice, suggesting that human CD8+ T cells arepotent effectors in the GVHD disease (24).

To address the development of GVHD following hu-man PBMC injection, a strain of NOG mice lacking bothMHC class I and II has recently been described (25). It wasreported that the PBMC-engrafted MHC-deficient NOGmice had longer lifespans than those of the parental NOGstrain, and that the engrafted cells were able to generatecytotoxic T-cell activity against human tumor cells as wellas NK cell–mediated cytotoxicity against NK-sensitivetargets following treatmentwith anti–programmed death-1 (PD-1) antibody (Ab). To extend these observations, wenow describe the generation of 2 models of NSGmice thatlack bothmurineMHCclass I and II. In the firstmodel, IgGclearance is extremely rapid, whereas in the secondmodel,IgG clearance is much slower and comparable to that ob-served in NSGmice. Injection of human PBMCs into bothstrains led to long-term engraftment of functional humanT cells and lack of acute GVHD. Our data suggest NSGmice deficient in murineMHC class I and II can be used tostudy human immunity and the therapeutic effects of hu-manAb–baseddrugs on the human immune system in theabsence of acute GVHD.

MATERIALS AND METHODS

Mice

Allmiceused in these studieswere raised in thebreedingcoloniesofL.D.S. at The JacksonLaboratory (BarHarbor,ME,USA).NSG

mice have been previously described (8). NSG mice were main-tained through sib matings. NOD.Cg-Prkdcscid H2-K1tm1Bpe

H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl/SzJ [NSG-(Kb Db)null (IAnull)]mice were developed using transcription activator–like effectornucleases (TALENs). Exon 2 of the H2-Ab1 gene was targeted inNOD.Cg-Prkdcscid H2-K1tm1Bpe H2-D1tm1Bpe Il2rgtm1Wjl/SzJ [NSG-(Kb Db)null] (27) embryos. The cytoplasmic microinjection wasperformed in homozygous NSG-(Kb Db)null fertilized oocytes, de-livering 50 ng/ml of each TALEN mRNA (left TALEN targeting:TGGGGCGGCCAGACGCCG; right TALEN targeting: TCGCT-CCAGGATCTCCGG), prepared in RNase-Free TE supplementedwithRNasin (Promega,Madison,WI,USA)at a final concentrationof0.2U/ml.Toscreen forpotential candidates,PCRwasperformedusing the following primers: (forward, 59-TTCGTGTACCA-GTTCATGGGCG-39; reverse, 59-GATGCCTAACCGACCAC-TTT-39), which produces an 853 bp wild-type amplicon. Themutant allele that was fixed in this line yields a 292 bp productusing these primers. Sanger sequencing was used to characterizethis allele as an INDEL (net +7/2568), though it has 2 distinctdeletions (61 bp, 507 bp) separated by a 39 bp “island” of intactsequence as well as a 7 bp insertion (59-CCGTCAC-39). Themutant PCR product is shown below; bracketed bases indicatethe deletion sites and the insertion is underlined: 59-TTCGTG-TACCAGTTCATGGGCGAGTGCTACTTCACCAACGGGAC-GCAGCGCATACGATATGTGACCAGATACATCTACAAC-CGGGAGGAGTACGTGCGCTACGACAGCGACGTGGGCG-AGCACCGCGCGGTGACCGAGCTGGGGCGGCCAGACGC-CGAGTA[CA]CAACTACGAGGGGCCGGAGACCCACACC-TCCCTGCGG[CCCGTCACA]ACTCATTTCCGTTTCCAGCA-CACTCCCTGATACCCCCAGAGCCTCTCACCCGTGATGCC-AATTAAAGTGGTCGGTTAGGCATC-39. The offspring carryingthe null IAb allele (H2-Ab1em1Mvw) were identified by PCR and thenull IAb allele was fixed to homozygosity. NSG-(Kb Db)null (IAnull)mice are maintained through homozygous sib mating. MHCclass II molecules are heterodimers comprised of both a and bchains (28). Mice on the NOD background (H2g7) express boththe IAa andb chains and express a functional IAg7 protein.H2g7

mice also express an IE b chain but have a deletion mutationwithin the IEa chain and thereforedonot express a functional IEprotein (29). Hence disruption of the IA-b chain eliminates allexpression of MHC-class II in NSG mice.

NOD.Cg–b-2-microglobulin (B2m)tm1Unc Prkdcscid H2dlAb1-Ea

Il2rgtm1Wjl/SzJ [NSG-B2Mnull (IA IE)null] were made by intercross-ingNOD.Cg-B2mtm1Unc Prkdcscid Il2rgtm1Wjl/SzJ (NSG-B2Mnull) mice(23) with NOD.Cg-PrkdcscidH2dlAb1-Ea1Il2rg tm1Wjl/SzJ [NSG-(IAIE)null] (28) and intercrossing the F1 progeny followed byselecting the NSG mice doubly homozygous for the B2mtm1Unc

and H2dlAb1-Ea alleles. The NSG-B2Mnull (IA IE)null mice weremaintained through sib mating. MHC class I is a heterodimercomprised of a heavy chain and a B2M chain which are non-covalently linked, and both are required for cell surface expres-sion of the class I complex. Mutations that disrupt expression ofB2M abrogate the cell surface expression of MHC class I (30).

To create the NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-Ab1em1Mvw

H2-D1tm1Bpe Il2rgtm1Wjl Tg(Ins2-HBEGF)6832Ugfm/Sz transgene[NSG–rat insulin promoter (RIP)–diphtheria toxin receptor(DTR) (Kb Db)null (IAnull) strain], we backcrossed the Tg(Ins2-HBEGF)6832Ugfm, RIP-DTR transgene, onto the NSG strain (31,32), andthencrossed theNSG-DTRstrainwith theNSG-(KbDb)null

(IAnull) strain to create the NSG-RIP-DTR (Kb Db)null (IAnull) strain.Thesemice aremaintainedby sibmating ofmice homozygous forthe disrupted alleles and for the transgene.

All animals were housed in a specific pathogen–free facility inmicroisolator cages and given autoclaved food and acidifiedautoclaved water at the Jackson Laboratory or alternated weeklybetween acidified autoclaved water and sulfamethoxazole-trimethoprim–medicated water (Goldline Laboratories, FortLauderdale, FL, USA) at theUniversity ofMassachusettsMedicalSchool. All animal procedures were done in accordance with the

2 Vol. 33 March 2019 BREHM ET AL.The FASEB Journal x www.fasebj.org



guidelines of the Animal Care and Use Committee of the JacksonLaboratory and the University of Massachusetts Medical Schooland conformed to the recommendations in the Guide for the Careand Use of Laboratory Animals (National Institutes of Health,Bethesda, MD, USA). Supplemental Figure S1 and SupplementalTable S1 provide a direct comparison of the relevant strains usedfor experiments (28, 29, 33–36).

Abs and flow cytometry

The phenotypes ofmurine cells in theNSGMHCknockoutmicewere determined as described (8). Anti-murine mAbs werepurchasedasFITC,phycoerythrin, allophycocyanin, orperidininchlorophyll protein conjugates to accommodate 4-color flowcytometric analysis. Immune-competent NOD/ShiLtJ (NOD)and C57BL/6 (B6) mice (data not shown) were run with eachexperiment to ensure correct MHC staining. The B6 mice wereincluded to control for carryover of the linked MHC II gene re-gion adjacent to the classically knocked-out Ea genes, whichwasmade in 129 embryonic stem cells and backcrossed to NSG tomake NSG-B2Mnull (IA IE)null mice. Spleens were snipped intosmall pieces in 1mlof 200U/ml collagenaseD inDMEMwithoutserumon ice. Two additionalmilliliters of collagenaseD solutionwere added and the splenocytes were vortexed. Cells were in-cubated ina37°Cwaterbath for 30minwithoccasionalvortexingandmixing. The cellswerewashed and suspended inGey’s RBClysing buffer (8.3 g/L NH4Cl, 1 g/liter KHCO3, pH 7.2; all re-agents fromMilliporeSigma, Burlington, MA, USA), mixed andincubated 1 min on ice. Cells were then washed with flowcytometry (FACS) buffer and stained for 30 min at 4°C, washedtwicewith FACS buffer, suspended in 250ml of FACS buffer andstainedwithpropidiumiodide, and100,000 events analyzedonaBDBiosciencesLSR II FlowCytometer (San Jose,CA,USA).Anti-mouse Abs used were anti-H2Kb (clone AF6-885), H2Kd (SF1-1.1), CD11b (M1/70), CD11c (N418), I-Ab,d IEk,d (M5/114),Ly6G (1A8), Ly6c (HK1.4), and I-Ag7 (10-2.16).

Human immune cell populations were monitored in PBMC-engrafted mice using mAbs specific to the following humanantigens: CD45 (clone HI30), CD3 (clone UCHT1), CD4 (cloneRPA-T4), CD8 (cloneRPA-T8), CD20 (clone 2H7)CD45RA (cloneHI100), CCR7 (clone G043H7), PD-1 (clone EH12.2H7), andgranzyme B (clone GB11), purchased from either eBioscience(SanDiego,CA,USA), BDBiosciences, orBioLegend (SanDiego,CA, USA). Murine cells were identified and excluded fromanalysis by staining with a mAb specific to murine CD45 (clone30-F11; BD Biosciences).

Single-cell splenic suspensions were prepared from engraftedmice, and whole blood was collected in heparin. Single cell sus-pensionsof13106 cellsor100ml ofwholebloodwerewashedwithFACS buffer (PBS supplemented with 2% fetal bovine serum and0.02% sodium azide) and then preincubated with rat anti-mouseFcR11b mAb (clone 2.4G2; BD Biosciences) to block binding tomurine FcRs. Specific mAbs were then added to the samples andincubated for 30 min at 4°C. Stained samples were washed andfixed with 2% paraformaldehyde for cell suspensions or treatedwith BD FACS lysing solution for whole blood. At least 50,000events were acquired on LSRII or FACSCalibur instruments (BDBiosciences). For human cell phenotyping, murine cells were iden-tifiedandexcluded fromanalysisbystainingwithamAbspecific tomurine CD45 (clone 30-F11; BD Biosciences). Data analysis wasperformed with FlowJo (Treestar, Ashland, OR, USA) software.

Collection of human PBMCs

Human PBMCs were obtained from healthy volunteers undersigned informed consent in accordance with the Declarationof Helsinki and approval from the Institutional Review Board ofthe University of Massachusetts Medical School. PBMCs were

collected in heparin, purified by Ficoll-Hypaque density centri-fugation, and suspended in Roswell Park Memorial Institutemediumfor injection intomice at the cell doses indicated. In someexperiments, pheresis leukopaks were obtained as anonymousdiscarded units from the blood bank at the University of Mas-sachusetts Medical Center.

GVHD protocol

Mice were injected intraperitoneally with various doses ofPBMCs.Micewereweighed2–3 times/wkandtheappearanceofGVHD-like symptoms, including weight loss (.20%), hunchedposture, ruffled fur, reduced mobility, and tachypnea, was usedto determine when mice would be euthanized. This is indicatedas time of survival.

Human islet transplantation

The procurement and use of human islets were performed underprotocols approved by the institutional review board of the Uni-versity of Massachusetts Medical School. Human islets desig-nated for research were obtained from Prodo Laboratories (AlisoViejo, CA, USA). Then, 4000 human islet equivalents (IEQs) weretransplanted into the spleens of NSG-RIP-DTR (Kb Db)null (IAnull)mice. NSG-RIP-DTR (Kb Db)null (IAnull) mice were treated with40 ng diphtheria toxin 2–4 d prior to islet transplantation. Hy-perglycemia (.400 mg/dl) was confirmed using an Accu-ChekActive glucometer (Roche, Basel, Switzerland). Blood glucoselevels were checked at twice-weekly intervals following trans-plantation to monitor islet graft function. C-peptide levels weredetected in plasma using an ELISA kit specific to humanC-peptide (Alpco, Salem, NH, USA). Total insulin content withintransplanted spleenswasdeterminedaspreviouslydescribed (37)using an ELISA kit specific to human insulin (Alpco).

Double-stranded adeno-associated virus vectors

The double-stranded (ds) adeno-associated virus (AAV) vectorswere engineered and packaged as previously described (38).Briefly, full-length cDNA encoding human IL-2 or EGFP wassubcloned into a dsAAV plasmid (39) containing the murinepreproinsulin II promoter. dsAAV vector packaging with sero-type8 capsidproteinwas carried out as previouslydescribed (40,41) or produced by the Viral Vector Core at the University ofMassachusetts Medical School Horae Gene Therapy Center(Worcester, MA, USA). Recipient mice were intraperitoneallyinjected with 2.5 3 1011 particles of the purified AAV8-huIL-2(AAV-IL-2).

Statistical analyses

To compare individual pairwise groupings, we used 1-wayANOVA or 2-way ANOVA with Bonferroni posttests andKruskal–Wallis test with Dunn’s posttest for parametric and non-parametricdata, respectively.Significantdifferenceswereassumedfor values of P , 0.05. Statistical analyses were performed usingGraphPad Prism software v.4.0c (La Jolla, CA, USA).

RESULTS

Phenotypic characterization of NSG miceand 2 strains of NSG MHC class I/IIdouble-knockout mice

We created 2 NSG mouse strains that are doubly de-ficient inMHC class I and II, theNSG-(KbDb)null (IAnull)

NSG MICE LACKING MHC CLASS I AND II 3


and NSG-B2Mnull (IA IE)null knockout strains. The ab-sence of MHC class I and II in both strains was con-firmed by flow cytometry (Fig. 1). To compensate forthe lack of immune cells expressing readily detectablelevels of mouse MHC II, we enzymatically disaggregatedspleens and gated onCD11c1 cells to analyze the dendriticcell population. Figure 1Ademonstrates thegating strategyof excluding doublets and dead cells and proceeds to gateon monocyte-derived dendritic cells (CD11b+ Ly6cdim

CD11c+). The NSG mouse demonstrates the expectedstaining pattern ofH2Kd positive, H2Kb negative forMHCclass I (Fig. 1B), and I-Ag7 positive, I-Ab negative for MHCclass II (Fig. 1C). Both the NSG-(Kb Db)null (IAnull) and theNSG-B2Mnull (IA IE)null knockout mice strains lack MHC

class I and II molecules normally expressed by NOD andC57BL/6 mice.

Due to the requirement of B2M for appropriate ex-pression of murine neonatal FcR (42), the receptor re-sponsible for prolonging the half-life of IgG in thecirculation (43), we compared the clearance of humanIgG in both stocks of mice. Mice received an injection(200 mg, i.v.) of human IgG and were bled at intervalsfor ELISA analysis of circulating human IgG. The firstbleed at 2 min postinjection was considered 100% se-rum IgG. We observed rapid clearance of human IgGin NSG-B2Mnull (IA IE)null mice whereas IgG clearancein NSG-(Kb Db)null (IAnull) mice was much slower andsimilar to that observed in NSG mice (Fig. 2).

Figure 1. Representative flow cytometry of MHC class I and II expression in NSG, NSG-(Kb Db)null (IAnull), and NSG-B2Mnull (IAIE)null mice. Splenic monocyte–derived dendritic cells from NSG, NSG-(Kb Db)null (IAnull), and NSG-B2Mnull (IA IE)null knockoutmice were analyzed by flow cytometry. A) Monocyte-derived dendritic cells were identified in viable cells as CD11b+, Ly6Gdim,CD11c+, and Ly6C2. B, C) Monocyte derived dendritic cells recovered from each strain were evaluated for expression of murineH2Kd and H2Kb (B), and murine H2 IAg7 and H2 IAb (C). Representative staining is shown for all stains (n = 2).




Survival of PBMC-engrafted NSG mice andNSG-MHC class I knockout, NSG-MHC class IIknockout, and NSG-MHC I/II knockout mice

NSG-(Kb Db)null (IAnull)

We first determined whether the absence of mouse MHCclass I and II altered the incidence and kinetics of xeno-geneic GVHD following human PBMC engraftment intoNSG MHC I/II knockout mice. NSG strains deficient inMHC class I,MHC class II, or the 2NSGdouble-knockoutstrains were engrafted with 10 3 106 PBMCs, and theirsurvival was compared with that of the NSG mice. Aspreviously reported (23), bothNSG andNSG-(IAnull) miceshowed relatively short survival times, similar to thoseobserved inNSGmice (23). By contrast, as expected,NSG-(KbDb)nullmicehada longerperiodof survival thandid theNSG mice (23). When both MHC class I and II wereknocked out in NSG-(Kb Db)null (IAnull) mice, however,survivalwas.100d,with 13/15MHCI/II knockoutmiceexhibitingnosymptomsofGVHDforup to125d (Fig. 3A).

NSG-B2Mnull (IA IE)null

Similar extended survival resultswere observed inPBMC-engrafted NSG-B2Mnull (IA IE)nullmice. For this MHC I/IIknockout strain, we used the NSG-B2Mnull strain as thecontrol rather than the NSG-(Kb Db)null strain. Again, asexpected (23), NSG and NSG-(IAnull) knockout miceengrafted with human PBMCs demonstrated relativelyshort survival times. Survival of NSG-B2Mnull mice wassignificantly higher.As observed inNSG-(KbDb)null (IAnull)mice, long-termsurvivalof PBMC-engraftedNSG-B2Mnull

(IA IE)nullmice was achieved, with 15/18 surviving to thetermination of the experiment (125 d) with no symptomsof GVHD (Fig. 3B).

Human cell chimerism in PBMC-engrafted NSGmice and NSG-MHC class I knockout, NSG-MHCclass II knockout, and NSG-MHC I/IIknockout mice

The long-term survival of PBMC-engrafted NSGMHC I/II knockout mice could be the result of either a lack ofhuman cell engraftment or a lack of GVHD due to theabsence of MHC class I and II. To distinguish betweenthese 2 possibilities, we injected 10 3 106 PBMC IP intoboth NSG MHC I/II knockout strains and compared thelevels ofCD45+ cells in the circulationover timewithNSG,NSG class I knockout, and MHC class II knockout mice.

NSG-(Kb Db)null (IAnull) mice

As expected (23), we observed that human CD45 cell en-graftment increased rapidly in NSG mice and NSG-(IAnull)mice (Fig. 4A). Thepercentages of circulatinghumanCD45+

cells over time were lower in NSG-(Kb Db)null and NSG-(KbDb)null (IAnull)mice than inNSGandNSG-(IAnull)mice.

Figure 2. Human IgG half-life in the serum of NSG, NSG-(Kb Db)null

(IAnull), and NSG-B2Mnull (IA IE)null mice. Mice received aninjection (200 mg, i.v.) of human IgG and were bled at theindicated time points to recover serum. Serum was used forELISA analysis of circulating human IgG. The first bleed at 2 minpostinjection was considered to be 100% serum IgG. Each pointrepresents the mean 6 SE of IgG in 5 males, 2–3 mo of age.

Figure 3. Survival of NSG mice lacking the expression of mouseMHC class I and II following injection of PBMCs. Recipient micewere intravenously injected with 10 3 106 PBMCs and weremonitored for overall health and survival. A) NSG, NSG-(IAnull),NSG-(Kb Db)null, and NSG-(Kb Db)null (IAnull) mice received PBMCs.The data are representative of 3 independent experiments. B)NSG, NSG-(IA IE)null, NSG-B2Mnull, and NSG- B2Mnull (IA IE)null

mice received PBMCs. The data are representative of 3 inde-pendent experiments. Survival distribution between groups wastested using the logrank test.



In the spleen, the percentages of human CD45+ cells inNSG-(IAnull) and NSG-(Kb Db)nullmice were comparable tothat observed in NSGmice, but the percentages of humanCD45+ cells in the spleens of NSG-(Kb Db)null (IAnull) micewere significantly lower than in the other 3 strains (Fig. 4B).

NSG-B2Mnull (IA IE)null mice

In these experiments, we used the NSG-B2Mnull strain astheNSGMHCclass I knockout control.As observed in theNSG,NSG-(IAnull)mice,NSG-(KbDb)null andNSG-(KbDb)null

(IAnull) mice, the percentages of circulating humanCD45+ cells were higher in the NSG and NSG-(IAnull)mice than in NSG-B2Mnull and NSG-B2Mnull (IA IE)null

mice (Fig. 4C). The percentages of human CD45+ cells in

the spleen of NSG-B2Mnull (IA IE)null mice were signifi-cantly decreased (Fig. 4D).

Engraftment of human T and B cells in PBMC-engrafted NSG, NSG-MHC class I knockout,NSG-MHC class II knockout, and NSG-MHC I/IIknockout mice

NSG-(Kb Db)null (IAnull)

As expected (23), we observed that circulating humanCD45+ cells were predominately CD3+ T cells in NSG,NSG-(IAnull), andNSG-(KbDb)nullmice (Fig. 5A). Similarly,the majority of CD45+ cells in NSG-(Kb Db)null (IAnull) micewere also CD3+ T cells. In the NSG andNSG-(IAnull) mice,there were readily detectable numbers of CD20+ B cells

Figure 4. Human CD45+ cellchimerism levels in PBMC-engrafted NSG mice lackingthe expression of both mouseMHC class I and II. Recipientmice were intravenously in-jected with 10 3 106 PBMCsand were assessed for levels ofhuman cell chimerism by de-termining the proportion ofhuman CD45+ cells in theperipheral blood (A, C) andspleens (B, D). A) Human cellchimerism levels were moni-tored in the blood of NSG,NSG-(IAnull), NSG-(Kb Db)null,and NSG-(Kb Db)null (IAnull)mice injected with PBMCover a 10-wk period. The dataare representative of 3 in-dependent experiments. A2-way ANOVA was used todetermine significant differ-ences between groups at eachtime point. Week 6: NSGvs. NSG-(Kb Db)null, P , 0.01;NSG vs. NSG-(Kb Db)null

(IAnull), P , 0.001; NSG-(IAnull) vs. NSG-(Kb Db)null,P , 0.01; and NSG -(IAnull)vs. NSG-(Kb Db)null (IAnull),P , 0.001. B) Human cellchimerism levels were moni-tored in the spleens of NSG,NSG-(IAnull), NSG-(Kb Db)null,and NSG-(Kb Db)null (IAnull)mice injected with PBMCswhen mice were euthanizedafter developing GVHD or at 10 wk post–PBMC injection. A 1-way ANOVA was used to determine significant differences betweengroups. *P , 0.05, **P , 0.01. C) Human cell chimerism levels were monitored in the blood of NSG, NSG-(IA IE)null, NSG-B2Mnull,and NSG-B2Mnull (IA IE)null mice injected with PBMCs over a 10-wk period. The data are representative of 3 independent experiments.A 2-way ANOVA was used to determine significant differences between groups at each time point. Week 4: NSG vs. NSG-B2Mnull (IAIE)null; P , 0.01; NSG-(IA IE)null vs. NSG-B2Mnull (IA IE)null, P , 0.01; and NSG-B2Mnull vs. NSG-B2Mnull (IA IE)null, P , 0.05. Week 6:NSG vs. NSG-B2Mnull (IA IE)null, P , 0.05; NSG-(IA IE)null vs. NSG-B2Mnull, P , 0.05; and NSG-(IA IE)null vs. NSG-B2Mnull (IA IE)null,P, 0.01. Week 8: NSG vs.NSG-B2Mnull, P, 0.001; NSG vs.NSG-B2Mnull (IA IE)null, P, 0.01; NSG-(IA IE)null vs.NSG-B2Mnull, P, 0.001;and NSG-(IA IE)null vs. NSG-B2Mnull (IA IE)null, P, 0.01. Week 10: NSG vs. NSG-B2Mnull, P, 0.01; NSG vs. NSG-B2Mnull (IA IE)null, P,0.01; NSG-(IA IE)null vs.NSG-B2Mnull, P, 0.01; and NSG-(IA IE)null vs.NSG-B2Mnull (IA IE)null, P, 0.01. D) Human cell chimerism levelswere monitored in the spleens of NSG, NSG-(IA IE)null, NSG-B2Mnull, and NSG-B2Mnull (IA IE)null mice injected with PBMCs when micewere euthanized after developing GVHD or at 10 wk post–PBMC injection. A 1-way ANOVA was used to determine significantdifferences between groups. **P , 0.01.




2 wk post-engraftment, but these were essentially un-detectable 4 wk post-engraftment (Fig. 5B).

NSG-B2Mnull (IA IE)null

For this comparison we again used NSG-B2Mnull mice asMHCclass I knockout control. PBMCengraftment inNSG-B2Mnull (IA IE)null mice consisted of predominately CD3+

Tcells, similar toNSG,NSG-(IAnull), andNSG-B2Mnullmice(Fig. 5C). Although human CD20+ B cells were readilyapparent in theNSGandNSG-IAnullmiceat 2wk in the firstexperiments (Fig. 5B), levelswere significantly lower inall 4strains examined in Fig. 5D, likely reflecting variability indonor PBMCs. The variability between PBMC donors ismostly likely attributed todifferences inT- andB-cell levels,and activation status of the cells (unpublished results).

Phenotypic analysis of human T cellsengrafted in NSG, NSG-(IAnull), NSG-(Kb Db)null,and NSG-(Kb Db)null (IAnull) mice injectedwith PBMCs

The CD4:CD8 ratio in NSG mice at 4 wk post PBMC-engraftment, as expected (44), was ;4:1 (Fig. 6A). Bycontrast, very few CD4+ T cells were detected in NSG-(IAnull) mice, whereas relatively high levels of CD4+ T cellsengrafted in NSG-(Kb Db)null mice, resulting in very lowandveryhighCD4:CD8ratios, respectively.TheCD4:CD8ratio of CD3+ T cells in NSG-(Kb Db)null (IAnull) mice wassimilar to that observed inNSGmice (Fig. 6A), suggesting

that neither humanT-cell subset had a selective advantagefor engraftment in mice lacking both MHC class I andMHC class II. Themajority of CD4+ andCD8+ T cells in all4 strains expressed the activationmarker PD-1 (Fig. 6B, C).A representative histogram of CD4+CD3+ andCD8+CD3+

T cells (Fig. 6D) and of PD-1 staining of CD4+ and CD8+

cells is shown (Fig. 6E, F). To determine the differentiationstate of the CD4+ andCD8+ T cells, we stained each subsetfor CD45RA and CCR7. CD45RA+CCR7+ cells representnaive T cells, CD45RA2CCR7+ cells represent centralmemoryTcells,CD45RA2CCR72cells representT-effector/effector memory T cells, and CD45RA+CCR72 cells repre-sent terminally differentiated effector memory CD45RA+

T cells (TEMRAs) (45, 46). In both the CD4+ (Fig. 6G) andCD8+Tcellpopulations (Fig.6H),very fewnaiveTcellswereobserved in blood at 4 wk post-PBMC injection. A few cen-tralmemoryCD4+ andCD8+T cellswere detected,whereasalmost no TEMRACD4+ or CD8+ T cells were present. Themajority of CD4+ and CD8+ T cells were effector/effectormemory CD45RA2CCR72 T cells (Fig. 6G, H).

Phenotypic analysis of human T cellsengrafting in NSG, NSG-(IA IE)null, NSG-B2Mnull,and NSG-B2Mnull (IA IE)null mice injectedwith PBMC

The CD4:CD8 T-cell ratios in NSG mice were again ;4:1(Fig. 7A). MHC class II (IA IE)null knockout and class IB2Mnull knockout mice similarly had CD4:CD8 low andhigh T-cell ratios, respectively, as observed in the NSG-(IAnull) and NSG-(Kb Db)null mice (Fig. 6A). NSG-B2Mnull

Figure 5. Engraftment of hu-man T and B cells in PBMC-engrafted NSG mice lacking theexpression of both mouse MHCclass I and II. Recipient micewere injected intravenously with10 3 106 PBMC, and mice weremonitored for levels of humanCD3+ T (A, C) and CD20+ B(B, D) cells in peripheral blood.A, B) NSG (N = 7), NSG-(IAnull)(n = 5), NSG-(Kb Db)null (n = 7),and NSG-(Kb Db)null (IAnull) (n =8) mice received PBMCs. C, D)NSG (6), NSG-(IA IE)null (n = 6),NSG-B2Mnull (n = 5), and NSG-B2Mnull (IA IE)null (n = 7) micereceived PBMCs. The data arerepresentative of 3 independentexperiments. A 2-way ANOVAwas used to determine significantdifferences between groups ateach time point. *P , 0.05.



Figure 6. Phenotypic analysis of human T cells engrafting in PBMC-injected NSG, NSG-(IAnull), NSG-(Kb Db)null, and NSG-(Kb Db)null

(IAnull) mice. Recipient mice were intravenously injected with 10 3 106 PBMCs, and at 4 wk postinjection mice were monitoredfor levels of human CD3+/CD4+ and CD3+/CD8+ T cells (A, D) and T cell phenotype (B, C, E–H) in peripheral blood. A) Levelsof CD4+ and CD8+ T cells were determined by flow cytometry and expressed as a ratio of CD4+ to CD8+ T cells. B, C) PD-1expression by CD4+ and CD8+ T cells was determined by flow cytometry. D–F) Representative CD4, CD8, and PD-1 staining isshown. G, H) CD4+ and CD8+ T cells were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages ofT-cell subsets are shown with naive CD45RA+/CCR7+ cells, central memory CD45RA2/CCR7+ cells, effector/effector memoryCD45RA2/CCR72 cells, and TEMRA CD45RA+/CCR72 cells. The data are representative of 2 independent experiments. A 1-wayANOVA was used to determine significant differences between groups. *P , 0.05, **P , 0.01, ***P , 0.005, ****P , 0.001.




Figure 7. Phenotypic analysis of human T cells engrafting in PBMC-injected NSG, NSG-(IA IE)null, NSG-B2Mnull, and NSG-B2Mnull

(IA IE)null mice. Recipient mice were intravenously injected with 103 106 PBMCs, and at 4 wk postinjection mice were monitoredfor levels of human CD3+/CD4+ and CD3+/CD8+ T cells (A, D) and T-cell phenotype (B, C, E–H) in peripheral blood. A) Levelsof CD4+ and CD8+ T cell were determined by flow cytometry and expressed as a ratio of CD4+ to CD8+ T cells. B, C) PD-1expression by CD4+ and CD8+ T cells was determined by flow cytometry. D–F) Representative CD4, CD8, and PD-1 staining isshown. G, H) CD4+ and CD8+ T cells were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages ofT-cell subsets are shown with naive CD45RA+/CCR7+ cells, central memory CD45RA2/CCR7+ cells, effector/effector memoryCD45RA2/CCR72 cells, and TEMRA CD45RA+/CCR72 cells. The data are representative of 2 independent experiments. A 1-wayANOVA was used to determine significant differences between groups. *P , 0.05, **P , 0.01, ***P , 0.005, ****P , 0.001.



(IA IE)null mice (Fig. 7A) showed an ;4:1 CD4:CD8 ratiothat is similar to that observed in NSG and inNSG-(KbDb)null (IAnull)mice (Fig. 6A). Themajority of CD4(Fig. 7B) and CD8 (Fig. 7C) cells in all 4 MHC knockoutstrains expressed the activation marker PD-1. Represen-tativehistogramsofCD4andCD8staining (Fig. 7D) andofCD4(Fig. 7E) andCD8 (Fig. 7F) stainingwithanti-PD-1areshown. In all 4 strains, although there were few CD4 (Fig.7G) orCD8 (Fig. 7H) naiveorTEMRAcells observed, somecentral memory cells were present. The majority of T cellsexhibited the CD45RA2CCR7+ effector/effector memoryphenotype (Fig. 7G, H).

Engrafted human T cells in NSG-(Kb Db)null

(IAnull) mice are functional

We have previously reported that injection of humanPBMCs into NSG mice engrafted with human allogeneicislets leads to islet allograft rejection (47). To determine ifthe human immune cells engrafted in NSG MHC I/IIknockout mice were functional, we created a new mousestrain, NSG-RIP-DTR (Kb Db)null (IAnull). Injection of diph-theria toxin (DT) into mice expressing the DTR under thecontrol of the RIP led to murine b cell death and hyper-glycemia (48). Injection ofNSG-RIP-DTR (KbDb)null (IAnull)mice with DT led to the rapid development of diabetes(Fig. 8A). Intrasplenic transplantation of 4000human IEQsrestored normoglycemia in the mice within 1–2 d. Thesemice were then divided into 2 groups. To confirm thefunction of the human islets in the absence of an allogeneicimmune system, one islet-transplanted group was in-traperitoneally injected with 50 3 106 allogeneic PBMCswhereas theothergroupreceivednoPBMCs.Controlmicethat received only human islets remained normoglycemicthroughout the experimental period (n = 3). By contrast, 3of the 4 mice that received allogeneic human PBMCreverted to hyperglycemia after 3–4 wk (Fig. 8A).

The engraftment levels of human CD45+ cells in PBMC-injected, islet-transplanted mice trended toward higherpercentages in the bloodover time, andup to;70%humanCD45+ cellswere detected in the spleen at 7wkpost–PBMCinjection. This level of human CD45+ cell engraftment inNSG-RIP-DTR (Kb Db)null (IAnull) mice was higher than inPBMC-engrafted NSG-(Kb Db)null (IAnull) mice (Fig. 8B) andwasconsistentwith thenumberofhumanPBMCs(503106)that were injected into the NSG-RIP-DTR (Kb Db)null (IAnull)mice, a 5-fold increase over the 10 3 106 cells injected intoNSG-(Kb Db)null (IAnull) mice. The CD4:CD8 T-cell ratiochanged significantly in the blood over the course of theexperiment as thepercentageofCD4+Tcells decreased (Fig.8C). At the termination of the experiment, the ratios of CD4:CD8 T cells in the spleen also showed a significant increaseof CD8+ T cells (Fig. 8D). The levels of human C-peptide inthe blood at 6wkwas decreased in 3 of the 4 islet-engraftedmice that received human PBMCs; the 1mouse that did notrevert to hyperglycemia had levels of C-peptide similar tothat observed in islet recipients that were not injected withallogeneic PBMCs (Fig. 8E). In all 4 mice that were givenallogeneic PBMCs, however, the level of human insulinobserved in the islet grafts was significantly lower than thatfound in control islet transplant recipients (Fig. 8F).

Modulation of engrafted human T cells bytreatment with dsAAV-IL-2 in NSG and NSG-(KbDb)null (IAnull) mice transplanted with PBMC

Having shown that the engrafted human T cells in theNSG-(KbDb)null (IAnull)mice are functional (Fig. 8A) but donot mediate acute GVHD (Fig. 3), we next determinedwhether administration of human recombinant IL-2 couldmodulate the T cell populations. We have previouslyshown that administration of a dsAAV8 vector encodinghuman IL-2 (dsAAV8-huIL-2) increased human regula-tory T (Treg) cells in NSGmice humanized by engraftmentof human fetal liver and thymus [i.e., the BLTmodel (49)].In the current study, injection of dsAAV8-huIL-2 led to atransient expansion of human CD45+ cells in the blood ofNSG andNSG-(KbDb)null (IAnull) mice engraftedwith 103106 PBMC for 2wk (Fig. 9A). dsAAV8-huIL-2 did not alterthe proportion of humanCD45+ cells thatwere CD3+ over8wk (Fig. 9B).However, therewas a significant increase inthe proportion of CD4+ T cells that expressed a Treg phe-notype (CD4+CD25+CD1272FOXP3+) at 2, 4, and 6 wk inNSG mice, and at 2 and 4 wk in NSG-(Kb Db)null (IAnull)mice post–PBMC injection (Fig. 9C). Representativestaining of CD4+ T cells with CD25 and CD127 Abs isshown in the top row and the expression of FOXP3 inCD4+CD25+CD1272 T cells in NSG and NSG-(Kb Db)null

(IAnull) mice with or without administration of dsAAV8-huIL-2 is shown in the bottom row (Fig. 9D). The relativepercentage of Treg cells declined steadily from 2 to 8 wk inmice treated with AAV-IL-2 and were present at levels sim-ilar tountreatedmiceby8wkpost PBMCengraftment.NSGandNSG-(KbDb)null (IAnull)mice injectedwithdsAAV8-huIL-2 have detectable human IL-2 in blood as early as 2 wkpostinjection (2196 48 and 2626 40 pg/ml, respectively).

However, the administration of dsAAV8-huIL-2shortened the survival of NSG-(Kb Db)null (IAnull) mice tothat observed in NSG mice and NSG mice treated withdsAAV8-huIL-2 (Fig. 9E). The injection of dsAAV8-huIL-2also altered the CD4:CD8 T-cell ratio to that of pre-dominately CD8+ T cells in both NSG andNSG-(Kb Db)null

(IAnull) mice when compared with controls (Fig. 9F). Inaddition, treatment with dsAAV8-huIL-2 increased thelevel of effector/effector memory CD8+ T cells and de-creased the level of central memory CD8+ T cells as com-pared to untreated NSG-(Kb Db)null (IAnull) mice (Fig. 9G).No changes were observed in CD4+ T-cell subsets fol-lowing treatment with dsAAV8-huIL-2 (data not shown).Correlated to the increase in percentages ofCD8+ effector/effector memory T cells in dsAAV8-huIL-2 treated NSG-(KbDb)null (IAnull)micewas an increase in the percentage ofgranzyme B–expressing CD8+ T cells (Fig. 9H).

DISCUSSION

Humanizedmice have beenwidely used tomodel humanimmune cell function in vivo (1–6). In the Hu-PBL-SCIDmodel, a major limitation of studying human T-cell func-tion is the rapid development of fatal xenogeneic GVHDthat not only shortens the experimental time window butalso confounds theanalysisofhumanTcell functiondue tothe underlying ongoing acute GVHD that eventually kills




the mice (15–25). In the present study, we have overcomethis limitation by eliminating expression of murine MHCclass I and II in NSGmice. Using two different NSGMHCclass I/II knockout mouse models, we engrafted human

PBMCs inmice lackingmurineMHC, but thesemice failedto develop acute GVHD-like disease for #125 d afterPBMCengraftment. The engraftedhumanTcells remainedfunctional, as demonstrated by their ability to reject human

Figure 8. Rejection of human islet allografts in PBMC-engrafted NSG-RIP-DTR (Kb Db)null (IAnull) mice. Recipient NSG-RIP-DTR(Kb Db)null (IAnull) mice were generated as described in Materials and Methods. A) NSG-RIP-DTR (Kb Db)null (IAnull) mice weretreated with 40 ng of DT 6 d before PBMC injection, and then implanted with human islets (4000 IEQs) by intrasplenic injection.On d 0, one group of mice was intraperitoneally injected with 50 3 106 human PBMCs, and one group was left untreated. Bloodglucose levels were monitored; mice with blood glucose levels over 300 mg/dl for 2 consecutive tests were considered diabetic. B)Mice were monitored for levels of human cell chimerism by determining the proportion of CD45+ cells in the peripheral bloodover 6 wk and in spleen at 7 wk. C, D) Levels of CD3+/CD4+ and CD3+/CD8+ T cells were evaluated in peripheral blood andspleen. E) Levels of circulating human C-peptide in plasma was determined by ELISA at wk 6. F) Total insulin content fromspleens of islet-engrafted mice was determined at wk 7 by ELISA. The data are representative of 2 independent experiments.Student’s t test was used to determine significant differences between groups. *P , 0.05, **P , 0.01, ***P , 0.005.



Figure 9. Expression of human IL-2 in PBMC-engrafted NSG mice and NSG-(Kb Db)null (IAnull) mice enhances survival of human CD4+

Treg. Recipient NSG and NSG-(Kb Db)null (IAnull) mice were intraperitoneally injected with 2.53 1011 particles of dsAAV8-huIL-2 or PBS.Two weeks later mice were intraperitoneally injected with 13 106 PBMCs. A–C) Levels of human CD45+ cells (A), CD3+ T cells (B) andCD4+/CD25+/CD1272/FOXP3+ Treg cells (C) were determined by flow cytometry. A 2-way ANOVA was used to determine significantdifferences between groups. ***P , 0.005, ****P , 0.001. D) Representative staining of CD4+ T cells for CD25, CD127 and FOXP3 isshown for all groups. E) Survival of recipient mice was monitored, and survival distribution between groups was determined using thelogrank test. F, G) For the graphs shown, closed black triangles represent NSG mice, open black triangles represent NSG mice injectedwith AAV-IL-2, closed red circles represent NSG-(Kb Db)null (IAnull) mice and open red circles represent NSG-(Kb Db)null (IAnull) miceinjected with dsAAV8-huIL-2. F) Levels of CD4+ and CD8+ T cells were determined by flow cytometry and expressed as a ratio of CD4+ to

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islet allografts. Moreover, the human T cells could bemodulated in vivo, as seen after dsAAV8-huIL-2 injection.Administration of dsAAV8-huIL-2, however, resulted inthe restoration of a wasting GVHD. Importantly, in theNSG-(Kb Db)null (IAnull) strain, human IgG clearance wascomparable to that observed in NSG mice whereas IgGclearance in the NSG-B2Mnull (IA IE)null strain was ex-tremely rapid.

We have previously shown that the primary xenoge-neic targets of engraftedhumanPBMCTcells inNSGmiceare the murine MHC class I and II molecules (23). In thatstudy, we knocked out expression of the B2M molecule,which is required for MHC class I expression (50), therbyextending the survival time of PBMC-injected NSG mice.NOD and NSG mice do not express the IE molecule (34),and by knocking out the IAb gene, we eliminated the ex-pression ofMHC class II inNSGmice (23). The survival ofNSG-MHC class II IAb knockoutmicewas onlyminimallyextended over NSG mice, and in the present study,we failed to see a significant increase in survival of NSG(IA IE)null mice over that of NSG mice. In vitro analyses inour previous report documented that knocking out eitherMHC class I or II reduced the in vitro mixed-lymphocytexenoreactivity of the human CD8+ and CD4+ T cells, re-spectively, suggesting that the majority of xenoreactivitywas mainly directed against the murine MHC molecules.This was confirmed by the near absence of T-cell pro-liferation in response to antigen-resenting cells from NSGmice lacking both MHC class I and II expression (23).

NOGmice lacking both murineMHC class I (b2m) andII (IAb) have recently been described (25). The strategy tocreate the NOG-MHC I/II knockout was to cross theb2mnull allele from NOD-scid b2mnull mice (51) and theIabnull allele from a MHC class II–deficient B6 mouse (52)onto the NOG background. NOG-MHC I/II knockoutmice survived for#70 d following the injection of 13 107

human PBMCs. The engrafted T cells inhibited humantumor cell line growth following the injection of anti-PD-1,due to induction of cytotoxic T cell andNK cell killing (25).Inour studies,wegenerated2 strainsofNSG-MHCclass I/II–deficient mice: one based on NSG-b2mnull mice crossedwithNSG (IA IE)nullmice (23), and one by knocking out theIa gene in NSG-(KbDb)null mice (27). b2m is required forFcRn expression (42), and controls the half-life of IgG in thecirculation (43). Accordingly, we observed rapid clearanceof human IgG in NSG-B2Mnull (IA IE)null knockout mice,that lack a functional FcRn,whereas IgG clearance inNSG-(Kb Db)null (IAnull) mice was prolonged and similar to thatobserved inNSGmice, as these strains express a functionalFcRn. It would be expected that the NOG MHC I/IIknockout mice that are based on a B2M deficiency forknocking out expression of MHC class I would also showrapid IgG clearance (25). This is important because manyof the new biologic therapeutics entering the market are

Ab-based drugs, and preclinical studies using these drugswill be best performed in NSG-(Kb Db)null (IAnull) micerather than NSG-B2Mnull (IA IE)null mice.

Engraftment and function of human PBMCs in bothNSGstrainsofMHCI/IIknockoutmiceandthe respectivecontrol single MHC knockout mice were assessed by in-jection of 10 3 106 PBMC and by monitoring mice forengraftment, weight loss and survival. The survival ki-netics of NSG, NSG MHC class I and II knockout micewere similar towhat has been previously reported (23). Bycontrast, we observed that a majority of both strains ofNSG MHC I/II knockout mice survived to the end of theobservation period, ;125 d. NOG MHC I/II knockoutmice were observed for 70 d after PBMC engraftment forsurvival (25). Prolonged survival of NSG-B2Mnull andNSG-(Kb Db)null mice correlated with lower levels ofengrafted human CD45+ cells relative to NSGmice and inNSG class II knockout mice, peaking at;20%.

The predominant CD45+ cell subset in engrafted NSG,NSG class I or II knockout, and NSG MHC I/II knockoutmice was CD3+ T cells, with few other cell populationsengrafting beyond 2 wk. Interestingly, in NSG mice de-ficient in MHC class II, the CD4:CD8 T-cell ratio was low,indicating thatmurineMHC class II was amajor driver forhuman CD4+ T cell expansion. By contrast, in NSG micedeficient inMHCclass I, theCD4:CD8T-cell ratiowashigh,indicating the murine MHC class I was a major driver forhuman CD8 T cell expansion. The CD4:CD8 T-cell ratio inNSG MHC I/II knockout mice was ;4:1, similar to thatfound in NSGmice, where there was no selective pressurefor expansion of either T-cell subset. In all strains of NSGmice tested, effector/effector memory CD45RA2CCR72

CD3+ T cells were the predominant CD4+ and CD8+ T-cellpopulation. This observation suggests that even though awasting-like syndromewas not observed, engrafted CD4+

and CD8+ T cells nevertheless became activated.We have previously reported that human PBMC-

engrafted NSG mice reject human islet allografts (47). Totest the ability of human PBMC to reject islet allografts inhyperglycemic NSG MHC I/II knockout mice, we de-veloped the NSG-RIP-DTR (Kb Db)null (IAnull) strain. Thisnew model permits the complete, specific, and permanentablation of murine pancreatic b cells, avoiding the broadlytoxic effects of diabetogenic drugs such as streptozotocin(31, 32). Human PBMC readily engrafted in NSG-RIP-DTR(Kb Db)null (IAnull) mice that had been rendered hyperglyce-mic and restored to normoglycemia by engraftment of hu-man islets. The human islet allografts were rejected asevidenced by recurrent hyperglycemia, marked by reducedcirculating C-peptide and decreased insulin content of theislet grafts. Interestingly, the islet allograft recipients had anincreased frequencyofCD8+Tcells inboth thebloodand thespleen over time. This suggests that the presence of islet al-lograftspreferentially stimulatedandexpandedthecytotoxic

CD8+ T cells. G) CD8+ T cells were evaluated for expression of CD45RA and CCR7 by flow cytometry. Percentages of T-cellsubsets are shown for naive CD45RA+/CCR7+ cells, central memory CD45RA2/CCR7+ cells, effector/effector memoryCD45RA2/CCR72 cells, and TEMRA CD45RA+/CCR72 cells. H) Granzyme B expression by CD8+ T cells was determined by flowcytometry and representative staining is shown. Student’s t test was used to determine significant differences between micetreated with AAV-IL-2 and controls. The data are representative of 3 independent experiments. ***P , 0.005, ****P , 0.001.



CD8+ T-cell population. These data indicate that humanPBMC function can be evaluated in NSGMHC I/II knock-out mice in the absence of an ongoing GVHD response.

Many of the drugs being advanced to the clinic are im-munemodulators, andoneof these entering clinical trials isthe administration of recombinant IL-2.Highdose IL-2 hasbeenused for cancer therapy (53, 54)whereas lowdose IL-2has been applied toward the treatment of autoimmunediseases (55, 56). To determine if IL-2 canmodulate humanT cells in NSG-(Kb Db)null (IAnull) mice, we administered aninjection of dsAAV8-huIL-2 (41). High doses of thedsAAV8-huIL-2 vector led to elevatedhuman IL-2 levels inthe circulation and increased levels of Treg cells; however,the elevated levels of IL-2 also led to the development ofGVHD in NSG-(Kb Db)null (IAnull) mice. This suggests thatthe engrafted human T cells inNSG-(KbDb)null (IAnull) miceare responsive to immunomodulatory drugs, and that theinability to mediate GVHD in these NSG MHC I/IIknockoutmice can be overcome by strong T cell activationand expansion. Although the major targets of the xenor-esponse, murine MHC class I and II molecules, have beeneliminated there are multiple other xenoantigens that maystimulate mature human T cells, including murine minorMHC antigens andmurine antigens cross-presented to thehuman CD8+ T cells. High levels of IL-2 appear to lead toactivationof the engraftedhumanTcells that is sufficient toinitiate andmediate a lethalGVHD, even in thepresence ofincreased proportions of human Treg cells.

In summary, we have described 2 newmodels of NSGmice that are deficient in MHC class I and II and havedemonstrated that PBMC engraftment does not lead tolethal GVHD. We have also shown that the engraftedT cells are functional, reject islet allografts, and are re-sponsive to immunomodulators such as IL-2. Finally, wehaveovercomethemajor limitationof IgGclearance rate inNSG-B2Mnull (IA IE)null mice through the development ofthe NSG-(Kb Db)null (IAnull) strain, which has IgG clearancerates similar to that of NSG mice. These new models ofNSG MHC I/II knockout mice will facilitate the study ofhuman immunity in the absence of GVHD and permitevaluation of clinical uses of Ab-based therapeutics.

ACKNOWLEDGMENTS

This work was supported, in part, by U.S. National Institutes ofHealth (NIH) Office of the Director Grant 1R24 OD018259 andthe NIH National Institute of Diabetes and Digestive and KidneyDiseases–supported Human Islet Research Network (https://hirnet-work.org) Grants UC4 DK104218 (to M.A.B., D.L.G., and L.D.S.),CA034196 (to L.D.S.), 1R01 AI132963 (to M.A.B. and L.D.S.),HL131471 (to C.M.), 1DP3DK111898 (to M.A.B.), DK098252(to C.M.), and 1R01 DK1035486 (R.M.T. and M.A.B.). Thecontents of this publication are solely the responsibility ofthe authors and do not necessarily represent the official views ofthe NIH. M.A.B. and D.L.C. are consultants for The JacksonLaboratory. The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

M. A. Brehm designed and performed research, analyzeddata, and wrote the manuscript; L. L. Kenney performedresearch and analyzed data; M. V. Wiles, B.E. Low and

C. Mueller contributed new reagents; R. M. Tischcontributed new reagents and reviewed the manuscript;L. Burzenski performed research and analyzed data; D. L.Greiner analyzed data and wrote the manuscript; andL. D. Shultz designed and performed research, analyzeddata, and wrote the manuscript.

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Received for publication April 2, 2018.Accepted for publication October 1, 2018.



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