Xenograft Tolerance and Immune Function of Human T Cells Developing in Pig Xenografts

This information is current as Hannes Kalscheuer, Takashi Onoe, Alexander Dahmani, of September 29, 2021. Hao-Wei Li, Markus Hölzl, Kazuhiko Yamada and Megan Sykes J Immunol 2014; 192:3442-3450; Prepublished online 3 March 2014;

doi: 10.4049/jimmunol.1302886 Downloaded from http://www.jimmunol.org/content/192/7/3442

Supplementary http://www.jimmunol.org/content/suppl/2014/03/01/jimmunol.130288 Material 6.DCSupplemental http://www.jimmunol.org/ References This article cites 49 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/192/7/3442.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Xenograft Tolerance and Immune Function of Human T Cells Developing in Pig Thymus Xenografts

Hannes Kalscheuer,*,†,1,2 Takashi Onoe,*,1,3 Alexander Dahmani,† Hao-Wei Li,*,† Markus Ho¨lzl,† Kazuhiko Yamada,* and Megan Sykes*,†

Transplantation of xenogeneic thymus tissue allows xenograft tolerance induction in the highly disparate pig-to-mouse model. Fetal swine thymus (SW THY) can support the generation of a diverse human repertoire that is tolerant of the pig in vitro. We demonstrate that SW THY generates all human T cell subsets, including regulatory T cells (Tregs), in similar numbers as fetal human thymus (HU THY) grafts in immunodeficient mice receiving the same human CD34+ cells. Peripheral T cells are specif- ically tolerant to the mouse and to the human and porcine donors, with robust responses to nondonor human and pig Ags. Specific tolerance is observed to pig skin grafts sharing the THY donor MHC. SW THY–generated peripheral Tregs show similar function, but include lower percentages of naive-type Tregs compared with HU THY–generated Tregs. Tregs contribute to Downloaded from donor-pig specific tolerance. Peripheral human T cells generated in SW THY exhibit reduced proportions of CD8+ T cells and reduced lymphopenia-driven proliferation and memory-type conversion, accelerated decay of memory-type cells, and reduced responses to protein Ags. Thus, SW thymus transplantation is a powerful xenotolerance approach for human T cells. However, immune function may be further enhanced by strategies to permit positive selection by autologous HLA molecules. The Journal of Immunology, 2014, 192: 3442–3450. http://www.jimmunol.org/ enotransplantation could resolve the supply-demand survival in nonhuman primates, but such treatments are highly discrepancy in human , and pigs toxic (9, 10, 13–15). Thus, induction of T cell tolerance is likely to X are considered the most suitable xenogeneic source an- be critical for clinical application of . imal (1–5). However, transplants from pigs are subject to vigorous Thymic xenotransplantation can induce robust tolerance across rejection in primates (5, 6). Although hyperacute rejection owing xenogeneic barriers (9, 16–18). In a pig-to-mouse model, murine to anti–Gala1-3Gal (Gal) natural Abs (7) is avoided by using Gal recipients of fetal swine thymus (SW THY) grafts showed donor- KO pigs (3, 4, 8–10), T cell–dependent Abs recognizing other pig specific unresponsiveness in vitro and specific skin acceptance specificities can cause acute humoral rejection (11, 12). T cell– with immunocompetence (19, 20). Porcine thymic grafts can gen- suppressive treatments significantly prolonged porcine xenograft erate diverse and functional human T cell repertoires (21) in mice that by guest on September 29, 2021 are specifically unresponsive to the donor pig in vitro (22, 23). Ex- tension of this approach from the pig to the baboon model has ach- *Transplantation Biology Research Center, Department of Surgery, Massachusetts ieved lasting pig kidney xenograft survival in nonhuman primates (9). General Hospital, Harvard Medical School, Boston, MA 02114; and †Columbia Center for Translational Immunology, Department of Medicine, Surgery and Micro- We have now used this pig-to–humanized mouse model to biology/Immunology, College of Physicians and Surgeons, Columbia University, demonstrate that SW THY–generated T cells are tolerant to the New York, NY 10032 human HSC donor and the murine recipient and show specific tol- 1 H.K. and T.O. are cofirst authors. erance toward swine leukocyte Ag (SLA)-matched porcine skin 2Current address: Department of Internal Medicine I and Clinical Chemistry, Uni- grafts. Regulatory T cells (Tregs) develop normally in SW THYand versity of Heidelberg, Heidelberg, Germany. contribute to the donor SLA-specific unresponsiveness. However, 3Current address: Institute for Clinical Research, National Hospital Organization, Kure Medical Center/Chugoku Cancer Center, Kure, Japan. human SW THY–generated T cells demonstrate functional alter- ations in the periphery that may be due to ineffective postthymic Received for publication October 25, 2013. Accepted for publication January 18, 2014. interactions with HLA molecules. These results suggest approaches This work was supported by National Institutes of Health Grants P01 AI045897 and to optimizing immune function when this powerful approach is used R01 AI084903. T.O. was supported in part by a Postdoctoral Fellowship for Research to induce xenograft tolerance in human T cells. Abroad of the Japan Society for the Promotion of Science and a Postdoctoral Fel- lowship for study abroad from the Uehara Memorial Foundation. H.K. and T.O. designed and performed experiments, analyzed and interpreted data, Materials and Methods and wrote the paper; A.D., H.-W.L., and M.H. performed experiments and analyzed Animals and tissues and interpreted the data; K.Y. provided porcine tissues; and M.S. designed experi- ments, interpreted the data, and wrote the paper. NOD-severe combined immunodeficient (NOD/SCID) mice from Jackson Laboratory (Bar Harbor, ME) were housed in a specific pathogen-free Address correspondence and reprint requests to: Dr. Megan Sykes, Columbia Center environment. Human fetal thymus (HU THY) and liver (gestational age, for Translational Immunology, 622 West 168th Street, PH 17-111, New York, NY 10032. E-mail address: [email protected] 17–20 wk) tissues were obtained from Advanced Biosciences Resource (Alameda, CA). SW THY was harvested from fetuses (60–90 gestational The online version of this article contains supplemental material. days) of Massachusetts General Hospital SLA-defined miniature swine Abbreviations used in this article: DC, dendritic cell; FCM, flow cytometry; FL, (provided by Dr. David H. Sachs). Studies were done with approval from human fetal liver; FLC, fetal liver cell; HU, human; HU THY, fetal human thymus; Institutional Review Boards and Animal Care and Use Committees. LN, ; rhSCF, recombinant stem cell factor; SLA, swine leukocyte Ag; SW THY, fetal swine thymus; TEC, thymic epithelial cell; THY, fetal human thymus; Human and porcine fetal tissue transplantation Treg, regulatory T cell; TT, tetanus toxoid. NOD/SCID mice (6–10 wk old) were conditioned with 2.5 Gy total Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 body irradiation. Human (HU) or SW THY plus human fetal liver (FL) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302886 The Journal of Immunology 3443 fragments were implanted under the kidney capsule as described pre- 10% human AB serum, 1% HEPES buffer, and 1 3 1025 M 2-ME (Sigma- viously (22). In some experiments, SW THY fragments were cryo- Aldrich, St. Louis, MO). Irradiated human allogeneic or swine xenogeneic preserved and thawed prior to transplantation as described (24). Fresh PBMCs (3000 cGy, 105 cells per well) stimulators were cocultured for 5 d + 5 or cryopreserved CD34 human fetal liver cells (FLCs; 1–5 3 10 ), at 37˚C in 5% CO2. Irradiated NOD/SCID mouse splenocytes or human FL isolated as described (24), were administered i.v. donor-derived dendritic cells (DCs) were also used as stimulators. Human FL donor-derived DCs were generated from CD34 FLCs by culture for 3 d Flow cytometry in rhGM-CSF (50 ng/ml) and recombinant human stem cell factor (recombinant human stem cell factor [rhSCF]; 50 ng/ml); followed by 3 d Human hematopoietic chimerism was assessed by flow cytometry (FCM) as a described (24). Fluorochrome-labeled mAbs purchased from BD Phar- in rhGM-CSF (100 ng/ml), rhSCF (50 ng/ml), and rhTNF- (1 ng/ml); then 6 d in rhGM-CSF (100 ng/ml), rhIL-4 (20 ng/ml), and rhTNF-a mingen (San Diego, CA) were used: anti-mouse CD45 and Ter119, anti- (0.2ng/ml); and an additional day in rhGM-CSF (100 ng/ml), rhIL-4 (20 human CD4, CD8, CD14, CD19, CD45, CD3, CD45RA, CD45RO, CD62L, a m CD127, HLA-DR, CCR7, FOXP3, anti-pig CD2, and isotype control mAbs. ng/ml), TNF- (20 ng/ml), and PGE2 (3 g/ml). Cytokines were purchased from R&D Systems (Minneapolis, MN). Cells were harvested after 16 h of FCM was performed with exclusion of dead cells and murine erythroid m 3 3 cellsasdescribed(24). incubation with 1 Ci of [ H]thymidine, and [ H]thymidine incorporation was measured as described previously (25). Data are presented as mean of For intracellular staining, cells were washed, surface Ag stained, and then 3 fixed and permeabilized with the FOXP3 Staining Buffer Set (eBioscience, [ H]thymidine incorporation in triplicate cultures or as stimulation index (mean cpm of the indicated coculture divided by that of responder cells San Diego, CA), followed by incubation with anti-human FOXP3. Cells alone). were washed in permeabilization buffer and resuspended in PBS. To determine the possible role of Tregs in donor SLA-specific unre- In vitro T cell responses sponsiveness, enriched human T cells from splenocytes and LNs were depleted of human CD25+ cells by MACS Microbeads (Miltenyi Biotec) Splenocytes and lymph node (LN) cells were harvested from humanized and cultured in U-bottom 96-well plates as described above. mice and mononuclear cells isolated by Ficoll separation. Human T cells Downloaded from were enriched (purity 90%) by depletion of anti-mouse CD45 and Ter- 119 microbeads (Miltenyi Biotec, Auburn, CA) followed by T cell puri- fication (Pan T cell isolation kit II; Miltenyi Biotec). Split thickness (2.3 mm) skin from donor SLA-matched and mismatched Responder T cells (105 per well) were cultured in U-bottom, 96-well Massachusetts General Hospital miniature swine was grafted on the lateral plates in AIM-V medium (Invitrogen, Carlsbad, MO) supplemented with thoracic wall 12 wk after THY transplantation. Skin grafts were evaluated http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Similar peripheral human immune cell reconstitution in NOD/SCID mice receiving SW versus HU THY grafts plus FL CD34 cells i.v. (A) NOD/SCID mice received 2.5 Gy , followed by transplantation of an HU fetal THY/liver graft plus human FL CD34 cells (white bars; n = 48) or SW THY/human liver plus human FL CD34 cells (black bars; n = 48). Human cell reconstitution in PBMCs was measured at the indicated time points. The figure shows combined results of five experiments. (B) Immune reconstitution in PBMCs of NOD/SCID mice after transplantation of fresh, untreated SW THY (black bars; n = 29) versus HU THY in combination with human FL CD34 cells (white bars; n = 31). The figure shows combined result of three experiments. (C) Immune reconstitution in PBMCs of NOD/SCID mice after transplantation of cryopreserved/thawed fetal SW THY (black bars; n = 19) versus HU THY with human FL CD34 cells (white bars; n = 17). The figure shows combined results of two experiments. (D) Peripheral pig CD2+ detection following transplantation in NOD/SCID mice with untreated (white bars; n = 35) and cryopreserved/thawed (black bars; n = 19) fetal porcine thymus/human liver grafts plus human CD34 cells. (E) Reduced CD8+:CD4+ T cell ratio detected among PBMCs of SW/HU (black bars; n = 19) compared with HU/HU mice (white bars; n = 19) 16 wk after THY/LIV/FL CD34 transplantation. The figure shows combined results of two experiments in which the SW thymus graft had been fresh or cryopreserved at the time of implantation. *p , 0.05, **p , 0.01, ***p , 0.001 two-way ANOVA Bonferroni test or Mann–Whitney test. 3444 HUMAN T CELL FUNCTION WITH XENOGENEIC THYMOPOIESIS daily from day 7 until 4 wk, then once every 3 d and considered rejected measured with a b-counter. Data are expressed as actual cpm. In self- when ,10% of the graft remained viable. stimulated control cultures, responder cells were incubated with irra- diated autologous PBMCs. Data are shown as mean of [3H]thymidine Adoptive transfer incorporation in triplicate cultures or as stimulation index that was cal- culated by dividing the stimulated response with the mean [3H]thymidine Eighteen to 20 wk after transplantation, human cells were prepared from and peripheral LNs of humanized mice. Naive T cells enriched using incorporation with autologous stimulators. 2 anti-CD45RO microbeads (.97% CD45RO ) and labeled with CFSE Statistical analysis (Molecular Probes, Eugene, OR), and 2 3 106 cells were transferred into secondary recipient mice that previously received fetal CD34 cells from Statistical analyses were performed with PRISM 4.0 (GraphPad, San Diego, the same donor without a THY graft and therefore contained autologous (to CA). Data in bar graphs are expressed as mean 6 SEM. Student t test, the T cells) APCs without T cells as described previously (26). Fourteen Mann–Whitney U test, or ANOVAwere used to compare groups. Pearson’s days later, LN and cells were harvested from secondary recipients, correlation coefficient test was used to analyze curve regression; p , 0.05 pooled from each individual mouse, and analyzed as described (26). was considered statistically significant. T cell decay study To analyze decay of HU or SW THY-generated T cells, unilateral ne- Results phrectomy of the kidney bearing the graft was performed. Multilineage human immune reconstitution in humanized mice with a porcine thymus Tetanus responses of humanized mice We compared the kinetics of human T cell reconstitution in sub- Sixteen weeks after transplantation, THY-grafted mice were immunized with 100 ml tetanus toxoid (TT; 1 flocculation unit per mouse; Sanofi lethally irradiated NOD/SCID mice given a SW or autologous HU Pasteur, Swiftwater, PA), or PBS in controls, emulsified in 100 mlCFA THY graft plus a HU FL graft along with i.v. injected CD34 cells Downloaded from (Sigma-Aldrich) s.c. on the back and boosted with 100 ml TT (or PBS) in isolated from the same human FL. These mice are termed SW/HU 100 ml IFA (Sigma-Aldrich) 2 and 5 wk later. Three weeks after reboost, and HU/HU mice, respectively. As shown in Fig. 1A, similar splenocytes and LNs were harvested and human cells were enriched as . human peripheral blood chimerism was detected, with similar described above, to purity 90%. Responder human cells were cocultured + + + with a neoantigen-, chicken Ig- (10 mg/ml, Sigma-Aldrich), a recall Ag, percentages of human CD45 , CD19 , and CD14 cells. However, + TT-pulsed human FL donor-derived DCs (1 mg/ml, EMD Millipore, CD3 cell recovery was slightly reduced in SW/HU compared

Billerica, MA), or medium alone for 5 d at 37˚C in 5% CO2. Cells were with HU/HU mice when all five experiments were combined http://www.jimmunol.org/ 3 3 harvested after 16 h of incubation with 1 mCi [ H]thymidine. [ H]Thy- (Fig. 1A). This difference was apparent, however, only in experi- midine incorporation was measured with a b-counter. All data are shown as mean stimulation index. ments involving fresh SW THY tissue (Fig. 1B) and disappeared when the graft was cryopreserved and thawed prior to implanta- In vitro suppression assay tion (Fig. 1C). In mice that received a fresh SW THY graft, a + Human T cells of HU or SW THY–grafted mice were enriched from significantly greater proportion of peripheral pig CD2 T cells was spleens and LNs, stained and sorted on a FACSAria (Becton Dickinson) detected (Fig. 1D). + 2 + + – to purify CD4 CD25 (responders) and CD4 CD25 CD127 (suppressor) Despite the overall similar peripheral human T cell reconsti- cells. Normal human control cells were selected from PBMCs using the . tution, the CD8:CD4 ratio was significantly reduced in SW/HU

same method. Purities were 97%. Cell culture medium was AIM-V by guest on September 29, 2021 medium (Invitrogen, Carlsbad, CA) supplemented with 10% human AB compared with HU/HU mice, regardless of whether the implan- serum, 1% HEPES buffer, and 1 3 1025 M 2-ME (Sigma-Aldrich). ted SW THY graft had been fresh or cryopreserved (Fig. 1E). In suppression assays, responders (2 3 104 cells per well) were cultured in a U-bottom, 96-well plate with suppressors at indicated ratios in the Donor-specific xenotolerance of human T cells in SW/HU mice presence of plate-bound anti-CD3 mAb (UCHT1, 1 mg/ml) and soluble in vitro and in vivo anti-CD28 mAb (CD28.2, 2.5mg/ml) for 4 d or in the presence of irradiated human allogeneic PBMCs (3000 cGy, 4 3 104 cells per well) for 5 d in Previously, we demonstrated donor-specific xenogeneic tolerance culture media at 37˚C in 5% CO2. Cells were harvested after 16 h of in- of SW THY–generated human (22). We assessed the cubation with 1 mCi of [3H]thymidine. [3H]Thymidine incorporation was tolerance of peripheral T cells in SW/HU mice. We compared the

FIGURE 2. Specific tolerance to human and porcine donors and mouse recipients in SW/HU mice. (A) To analyze func- tionality 20 wk after transplantation, human T cells were enriched from the spleen and peripheral LNs of NOD/SCID mice that received a HU or SW THY graft plus human CD34 cells. “Human” denotes normal human donor from whom responders were purified T cells from PBMCs. Human FL donor-derived DCs were generated from CD34 FLCs. Human T cells enriched from human (n = 2), HU/HU mice (n = 4), or SW/HU mice (n = 4) were cultured with indicated stimulators. Data are expressed as mean 6 SEM. The figure shows rep- resentative result of two experiments. (B) Twelve weeks after transplantation, SW/HU mice received SLA-matched and mismatched skin grafts. HU/HU mice and naive NOD/SCID mice served as controls. Grafts were defined as rejected when ,10% of the graft remained viable. The Journal of Immunology 3445 reactivity of splenic and LN T cells toward allogeneic human Comparable human subsets and Treg development PBMCs, DCs generated from the fetal liver of the original human in HU and SW grafts donor, donor SLA-matched and third party pig PBMCs, and the We compared thymocyte numbers and phenotypes in SW and HU recipient mouse strain (Fig. 2A). Although human T cells in SW/HU grafts generated with the same human FL donor. As shown in and HU/HU mice showed robust in vitro alloresponses, self- Fig. 3A, similar, large numbers of human thymocytes were gen- tolerance toward DCs from the human CD34 cell donor and to the erated in both sets of grafts with normal percentages of double- NOD/SCID recipient mouse was observed in both groups. Fur- positive and CD4 and CD8 single-positive thymocytes (Fig. 3A; thermore, T cells in the SW/HU but not HU/HU mice showed Supplemental Fig. 1A), as described (21). Thus, the reduced CD8: specific tolerance to the porcine thymus donor, demonstrating un- CD4 ratio in the periphery of SW/HU compared with HU/HU responsiveness to donor SLA-matched PBMCs, with strong re- mice cannot be explained by reduced CD8 cell development in sponses to unrelated pig PBMCs (Fig. 2A). In contrast, T cells the SW/HU mice. Human natural Tregs were present in both sets from the HU/HU mice showed similarly strong xenoresponses to of grafts, with similar proportions and numbers of FOXP3+ cells both SLA haplotypes. among CD4 SP thymocytes (Fig. 3A; Supplemental Fig. 1A). After T cell reconstitution, the animals received donor SLA- matchedand-mismatchedskingrafts.AsshowninFig.2B, Reduced proportions of naive Tregs in periphery of SW//HU four of five SW/HU mice accepted their thymus donor SLA- compared with HU/HU mice matched skin graft for .60 d, and three of these four rejected Significantly reduced percentages of human FOXP3+CD25+ 2 +

SLA-mismatched porcine grafts. All HU/HU control mice rejected CD127 Tregs were detected among CD4 T cells of SW/HU Downloaded from xenogeneic skin grafts. As expected, unreconstituted NOD/SCID compared with HU/HU mice in two of three experiments (Fig. 3B; mice failed to reject porcine skin. Supplemental Fig. 1B–D), whereas no difference was seen in a http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Comparison of thymocyte subsets and peripheral Treg phenotypes in SW/HU and HU/HU mice. (A) Similar cellularity and normal subset distribution as well as Treg percentages of single positive CD4 thymocytes were detected in porcine (black bars; n = 3) versus human fetal thymus grafts (white bars; n = 3) implanted with fetal liver 18 wk earlier in NOD/SCID mice that also received HU FL CD34 cells. (B) Reduced percentages of human FOXP3+CD25+CD1272 Tregs were detected among the CD4+ T cells in PBMCs of SW/HU compared with HU/HU mice in two of three experiments 16 wk after THY/LIV/FL CD34 transplantation. The indicated number of mice was analyzed in each experiment. ***p , 0.001 Mann–Whitney U test. (C) Sixteen weeks after THY/LIV/FL CD34 transplantation, HU/HU and SW/HU mice were bled, and the proportions of CD45RO+ and HLA-DR+ cells among human FOXP3+CD25+CD1272CD4+ cells in PBMCs were compared. The figures show combined results of three independent experiments. Data are expressed as means 6 SEM. *p , 0.05, **p , 0.05, ***p , 0.001, compared in the indicated combinations (two-way ANOVA, Bonferroni test.). 3446 HUMAN T CELL FUNCTION WITH XENOGENEIC THYMOPOIESIS Downloaded from

FIGURE 4. Treg suppression and participation in SLA-specific unresponsiveness. (A) To examine peripheral Treg participation in SLA-specific unre- + + sponsiveness, enriched human CD4 T cells from splenocytes and lymph nodes from two SW/HU and two HU/HU mice were depleted of human CD25 http://www.jimmunol.org/ cells by MACS microbeads and cultured with irradiated stimulator cells as indicated. Data are expressed as mean of triplicates 6 SEM. Titrated numbers of Tregs were added to assess suppression of alloresponses by CD25+CD1272CD4+ cells sorted and pooled from spleens and LNs of three HU/HU mice and four SW/HU mice. The average cpm values of CD252CD4+ cells from HU/HU mice in the presence of CD25+CD1272CD4+ cells from the indicated group (HU/HU mice; open circles, SW/HU mice; closed circles) in response to allogeneic human PBMC stimulators—(B) representative of two similar experiments—or upon stimulation with anti-CD3/-CD28 (C) is shown. Data are expressed as mean 6 SEM. *p , 0.05, Student t test, comparing cpm of CD25-depleted (closed bar) versus undepleted (open bar) CD4+ T cells to the indicated stimulators in each mouse. third experiment. In normal, adult human PBMCs, most FOXP3+ CD25+CD4+ cells were added back in various numbers to – + 2 + Tregs are characterized by the CD45RA CD45RO “memory” CD25 CD4 cells from the same spleens in allogeneic MLRs. Peri- by guest on September 29, 2021 phenotype and ∼40% express HLA-DR (Fig. 3C; Supplemental pheral Tregs isolated from SW/HU mice showed generally similar Fig. 1E). However, 16–18 wk after thymus grafting, the per- suppressive activity as Tregs isolated from HU/HU mice, but was centage of “memory”-type cells in HU/HU and SW/HU mice was significantly reduced compared with normal adult humans, consistent with the younger immune systems in the animals (Fig. 3C). The reduced Treg proportions in SW/HU mice, when present, reflected only reductions in naive-type Tregs, with no difference from HU/HU mice in the proportions of CD4+ cells that were memory-type Tregs (Fig. 3C; Supplemental Fig. 2).

SW THY–generated Tregs participate in donor-specific unresponsiveness To examine the role of peripheral Tregs in maintaining donor SLA-specific tolerance in SW/HU mice, we assessed responses of human splenic and LN T cells depleted of CD25+ Tregs (Fig. 4A). Alloreactivity and xenoreactivity of T cells from two of two HU/HU mice tested was significantly increased in vitro after Treg depletion. In contrast, in vitro T cell responses from two SW/HU mice toward allogeneic PBMC did not change significantly after Treg depletion (Fig. 4). However, depletion of Tregs revealed a modest proliferative response toward donor SLA-matched (SLAdd) FIGURE 5. Proliferation assay with Ag stimulation 1 wk after boost pig stimulators that was statistically significant in one animal, but with IFA plus TT. HU/HU and SW/HU mice immunized with CFA plus TT just missed significance in the other (p = 0.06), suggesting a role for were boosted with IFA plus TT twice and assessed for proliferative responses 3 wk later. Human T cells isolated from spleen and lymph nodes Tregs in the donor-specific unresponsiveness of peripheral T cells + + in SW/HU mice. by depletion of mouse CD45 and Ter119 cells and magnetic cell sorting for human T cells, served as responders. Responder cells were cultured Suppressive function of peripheral Tregs in SW/HU and with indicated stimulators. The mean stimulation index values are shown HU/HU mice for HU/HU and SW/HU mice immunized with PBS or tetanus toxoid (n =3, each). Data are expressed as mean 6 SEM. ***p , 0.001, comparing re- 2 + + We purified CD25 and CD25 human CD4 cells from the spleens sponse to chicken Ig-pulsed DC (two-way ANOVA, Bonferroni test). One and LNs of SW/HU and HU/HU mice to examine Treg function. representative result of two experiments is shown. The Journal of Immunology 3447 slightly lower for SW/HU compared with HU/HU Tregs at lower human T cells from spleens and LNs of HU/HU and SW/HU mice Treg dilutions (Fig. 4B). Upon stimulation with anti-CD3/CD28, were sorted and adoptively transferred into NOD/SCID mice the level of suppression of nonspecific T cell responses was similar that previously received FL CD34 cells from the same human for Tregs from SW/HU and HU/HU mice (Fig. 4C). donor but no thymus graft. Fourteen days later, T cell recovery in adoptive recipients was strongly correlated to the level of hu- Comparison of Ag-specific T cell responses in SW/HU and man chimerism at the time of transfer, as previously observed HU/HU mice (26) (Fig. 6A). However, recovery of human CD4 and CD8 T cells We immunized SW/HU and HU/HU mice with tetanus toxoid (TT) that were generated in SW THY was significantly reduced com- and measured proliferative responses. Human T cells from SW/HU pared with that of T cells transferred from HU/HU mice (Fig. mice demonstrated markedly weaker responses to TT compared 6A, 6B). with HU/HU mice (Fig. 5). No responses to a neoantigen (chicken Rapidly proliferating human T cells convert to the memory IgG) were detected in either group (Fig. 5). phenotype when transferred to T cell–deficient humanized mice (26). Similar numbers of slowly proliferating CD4 and CD8 cells Suboptimal homeostatic maintenance of human T cells were recovered from both groups, and these consisted largely of developing in porcine thymus naive-type cells (Fig. 6C, 6D). Among the rapidly proliferating In HU/HU mice, we previously demonstrated that naive human subset of CD4+ T cells recovered from adoptive recipients, the T cells undergo expansion and conversion to the effector/memory majority were central memory and effector memory cells. How- phenotype upon transfer to a T cell deficient environment con- ever, the numbers of these cells were significantly lower among taining autologous APCs (26). Using the same approach, we com- CD4 cells transferred from SW/HU compared with HU/HU mice Downloaded from pared T cells developing in SW versus HU THY. Naive CD45RO2 (Fig. 6D). Rapidly dividing CD8+ T cells generated in SW/HU http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Suboptimal maintenance and homeostasis of human T cells generated in a porcine thymus graft. Naive SW and HU THY–generated human T cells were labeled with CFSE and transferred into T cell–deficient secondary recipient mice that previously received fetal CD34 cells from the same donor. Adoptive recipient mice were sacrificed 14 d after transfer, and recovery of transferred cells was analyzed with FCM. Correlations between human chimerism levels in T cell–deficient adoptive recipients at the time of transfer and recovered total (left panel), CD4 (middle panel), and CD8 T cell numbers (right panel) are shown 14 d after transfer. (A) Recovered cell number was calculated from the total recovered cell number, and the proportion of each T cell subset in spleen and LNs. (B) The total number of each cell type was calculated from the total recovered cell number and the proportion of each human T cell subset among total recovered cells determined by FCM (n = 4 mice in each group). Data are expressed as mean 6 SEM. *p , 0.05, ***p , 0.001, comparing indicated combinations (two-way ANOVA, Bonferroni test). (C) Naive human T cells isolated from the peripheral lymphoid tissues of HU/HU (top panel) or SW/HU (bottom panel) mice were CFSE-labeled and transferred into T cell deficient recipient humanized mice. Pooled splenocytes and LN cells from each individual recipient mouse were analyzed phenotypically with FCM 14 d after transfer. FCM plots showing CFSE intensity and expression of indicated markers of recovered CD4+ and CD8+ T cells from one representative recipient mouse are shown. The rapidly and slowly proliferating populations are defined by the left and right bars, respectively, above the histograms showing CFSE intensity. (D) Phenotypic characteristics of rapidly or slowly proliferating population of CD4 (top panel) or CD8 (bottom panel) T cells recovered in T cell–deficient recipients of naive T cells from SW/HU (black bars, n = 4) or HU/HU (white bars, n = 4). Naive, central memory (CM), effector memory (EM), and EM cells that re-express CD45RA (EMRA) subpopulations were defined as CD45RA+CCR7+,CD45RA2CCR7+,CD45RA2CCR72, and CD45RA+CCR72, respectively. Data are expressed as mean 6 SEM. Results are shown from one representative experiment (n = 4 mice in each group) of two experiments. ***p , 0.001, comparing indicated combinations (two-way ANOVA, Bonferroni test). 3448 HUMAN T CELL FUNCTION WITH XENOGENEIC THYMOPOIESIS mice largely failed to convert to the memory phenotype upon to prevent the strong immune responses to xenoantigens. SW THY adoptive transfer to T cell–deficient recipients, in contrast to those can support human thymopoiesis, leading to donor-specific unre- generated in HU/HU mice (Fig. 6C, 6D). sponsiveness in vitro (22, 23) and normal TCR diversity (21). To our knowledge, we have demonstrated for the first time xenoge- Decay of T cells is accelerated in SW/HU mice after neic tolerance of a human by the stringent crite- graftectomy rion of long-term acceptance of SLA-matched xenogeneic skin Twenty-two weeks after transplantation, T cell decay in SW/HU grafts. Acceptance of xenogeneic porcine skin is restricted to skin and HU/HU mice was compared after thymic graftectomy. As sharing the SLA Ags of the thymus donor, because SLA-mis- + shown in Fig. 7A, CD4 T cell decay in SW/HU mice was sig- matched grafts were rejected. Thus, central tolerance induced nificantly greater after 2 wk compared with that in HU/HU mice in by thymic xenografting leads to xenograft tolerance. The donor- graftectomized but not control mice. This difference between matched skin graft rejection observed in one of five SW/HU mice SW/HU and HU/HU mice only achieved significance for memory- might reflect reactivity to minor histocompatibility Ags expressed + type and not for naive-type CD4 T cells (Fig. 7B, 7C). However, by the skin graft donor that were not shared by the fetal THY both naive and memory-type CD4 cells showed significantly donor, because these animals were inbred only at the SLA. greater decay in graftectomized compared with control SW/HU T cell tolerance induction to donor porcine Ags in the thymus mice, whereas neither subset showed a significant difference be- graft most likely reflects negative selection by porcine thymic tween graftectomized and control HU/HU mice in the 2-wk period epithelial cells and APCs that persist in the graft (22). The unre- of follow-up. These results demonstrate clearly that maintenance sponsiveness to recipient mouse Ags likely reflects central clonal of both naive and memory CD4 subsets was more dependent on deletion due to the presence of murine APCs, which we have Downloaded from continuous thymic graft output in SW/HU than in HU/HU mice. detected in SW THY (22). However, the unresponsiveness to CD8 T cells were barely detectable in either group 2 wk after donor porcine Ags may also have involved positive selection of . Tregs that suppress anti-donor responses, as depletion of Tregs revealed some anti-pig donor MLR responsiveness in peripheral Discussion T cells of SW/HU mice. Clonal deletion of donor-specific thy-

In this study, we report that SW THY supports the development of mocytes may be incomplete because of the expected eventual http://www.jimmunol.org/ functional human T cells and Tregs from human FL CD34 cells. replacement of SW hematopoietic APCs with human and mouse These T cells show robust alloresponses and xenoresponses with APCs in the graft, resulting in partial reliance on Tregs to maintain specific immunologic tolerance in vivo and in vitro toward the tolerance. discordant xenogeneic THY donor and to the murine recipient, with We show in this study that peripheral human T cell reconstitution self-tolerance to the human donor. However, human T cells gen- can occur just as efficiently from porcine as from human THY erated in SW THY exhibit an increased CD4:CD8 ratio, defects in grafts. Consistent with our previous observations for HU THY homeostasis, and reduced responses to protein Ags compared with (24), cryopreservation and thawing of SW THY reduced the level those generated in autologous human thymus grafts. of peripheral porcine T cell reconstitution, permitting faster Pig xenotransplantation could overcome the human organ T cell reconstitution from the i.v. injected human CD34 cells. by guest on September 29, 2021 shortage; however, tolerance approaches are most likely necessary Thus, creation of open niches by thymocyte depletion accelerates

FIGURE 7. Accelerated decay of CD4+ T cells in SW/HU mice after graftectomy. Twenty weeks after transplantation, the THY graft-bearing kidney was removed and (A) the percentage of CD4+ T cells in PBL 2 wk after graftectomy was compared with levels before graftectomy. The percentage of (B) naive and (C) memory-type CD4+ cells in PBL was compared with values before graftectomy. The Journal of Immunology 3449 thymopoiesis from peripheral stem cells. Collectively, our data limited by the absence of the same peripheral MHC/peptide com- argue against any major species barriers to human thymopoiesis in plexes as those encountered on thymic epithelial cells (TECs). The porcine thymic grafts. experiment-to-experiment variability in observing this difference By studying thymectomized, T cell–depleted wild-type, knock- might reflect different degrees of cross-reactivity between different out, and TCR transgenic mice receiving SW THY, we previously combinations of human and porcine MHC genotypes in each ex- showed that pig and mouse MHCs mediate negative selection of periment. Furthermore, human APCs migrating into the SW thy- mouse T cells developing in SW THY, but only pig MHC medi- mus may contribute to Treg-positive selection as described (45), ates positive selection (19, 27–29). Application of these principles resulting in Tregs that interact efficiently with human HLA/peptide to the SW/HU model would imply that human, pig, and mouse complexes in the periphery. APCs present in SW THY grafts (22) contribute to negative se- In conclusion, we demonstrate that human T cells generated in lection, explaining the highly specific, three-species tolerance porcine thymus grafts are functional, self-tolerant, and specifically observed in SW/HU mice, with tolerance to the mouse recipient tolerant toward the porcine donor. Functional Tregs develop nor- strain in vitro and in vivo (the animals did not develop GVHD) mally in the porcine thymus and contribute to tolerance. However, and specific unresponsiveness to the human and the porcine human T cells generated in porcine thymus grafts demonstrate donors. immune defects that might be attributable to suboptimal post- Significant T cell recall responses to tetanus immunization were thymic interactions with human APCs following positive selection demonstrated in SW/HU mice, although at reduced levels com- on porcine MHC. For clinical application of this tolerance ap- pared with HU/HU mice. The lack of positive selection on human proach, methods of optimizing T cell function following devel- thymic MHCs can render SW THY–generated T cells less efficient opment in SW THY are needed. These methods could include Downloaded from at recognizing Ag presented on autologous human HLA mole- cotransplantation of human TECs to add an element of positive cules. However, studies in the pig THY to immunocompetent selection on autologous TECs, which was effective in the pig mouse model showed that cross-reactivity of murine T cells thymus to nude mouse transplant model (28, 46–49), or the use of generated in SW THY allowed mouse class II–restricted respon- HLA transgenic porcine source animals. siveness and protective responses to opportunistic infection (18,

27). A normal, diverse human T cell repertoire is generated in SW Acknowledgments http://www.jimmunol.org/ THY grafts (21). Further study is needed to elucidate the effects We thank Drs. Scott Arn and David H. Sachs for providing pigs and of development in a pig thymus on protective human T cell reagents, Drs. Yong-Guang Yang and Donna Farber for critical review of responses. the manuscript, and Melicia Walker for assistance with the manuscript The exclusive positive selection by porcine thymic epithelium in submission. SW/HU mice could explain the reduced CD8 T cell percentages, accelerated memory cell decay, and reduced lymphopenia-driven Disclosures expansion and CD8 cell memory differentiation in the periphery The authors have no financial conflicts of interest. of these mice compared with HU/HU mice. 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