ABNORMAL REGULATORY AND EFFECTOR T CELL FUNCTION PREDISPOSE TO AUTOIMMUNITY FOLLOWING XENOGENEIC THYMIC TRANSPLANTATION

Abstract

Porcine thymus grafts support robust murine and human thymopoiesis, generating a diverse T cell repertoire that is deleted of donor and host-reactive cells, achieving specific xenograft tolerance. Positive selection is mediated exclusively by the xenogeneic thymic MHC. While thymectomized, T cell-depleted normal mice usually remain healthy following xenogeneic thymic transplantation, thymus-grafted congenitally athymic mice frequently develop multiorgan autoimmunity. We investigated the etiology of this syndrome by adoptively transferring lymphocyte populations from fetal pig (FP) thymus (THY)-grafted (FPG) BALB/c nude mice to secondary BALB/c nude recipients. FPG nude mice generated normal numbers of CD25⁺Foxp3⁺CD4 T cells, but these cells lacked the capacity to block autoimmunity. Moreover, thymocytes and peripheral CD4⁺CD25⁻ cells from FPG nude mice, but not those from normal mice, induced autoimmunity in nude recipients. Injection of thymic epithelial cells from normal BALB/c mice into FP THY grafts reduced autoimmunity and enhanced regulatory function of splenocytes. Our data implicate abnormalities in post-thymic maturation, expansion and/or survival of T cells positively selected by a xenogeneic MHC, as well as incomplete intrathymic deletion of thymocytes recognizing host tissue-specific antigens, in autoimmune pathogenesis. Regulatory cell function is enhanced and negative selection of host-specific thymocytes may potentially also be improved by co-implantation of recipient thymicepithelial cells in the thymus xenograft.

Introduction

We previously demonstrated that donor-specific xenograft tolerance can be achieved in thymectomized (ATX), T cell-depleted mice by grafting fetal pig thymus and liver tissue (FP THY/LIV) under the kidney capsule (1,2). In this xenogeneic pig-to-mouse model, mouse CD4⁺ T cells repopulated the periphery of T cell-depleted ATX mice after grafting with FP THY/LIV. These repopulated mouse CD4⁺ cells were tolerant to xenogeneic donor antigens, as indicated by specific nonresponsiveness to donor xenoantigens in mixed lymphocyte reactions and long-term acceptance of donor MHC-matched xenogeneic pig skin grafts, with the ability to reject third-party skin grafts (1,2). In addition, these repopulating mouse CD4⁺ T cells were immunologically functional (3). Therefore, xenogeneic thymic transplantation provides a promising approach to achieving xenograft tolerance.

Our previous studies have demonstrated that intrathymic clonal deletion is one of the major mechanisms of tolerance to host and donor antigens in thymic xenografts (1,2,4). The negative selection of host-reactive thymocytes correlates with the presence of host MHC class II^high cells with dendritic cell morphology in the porcine thymic grafts (2). While both donor pig and host mouse MHC molecules participate in the negative selection of mouse thymocytes in FP THY-grafted ATX mice, positive selection appears to be mediated only by pig MHC, with no demonstrable contribution from the host mouse MHC (5,6).

Despite clear evidence, by analysis both of superantigen-reactive Vβ and of a transgenic TCR with known host reactivity, that host APC participate in negative selection in FP thymic grafts, we previously reported that a small percentage of FP THY/LIV-grafted ATX B6 mice (approximately 10%) and a markedly higher percentage of FP THY/LIV-grafted (FPG) nude mice (approximately 60%) develop an autoimmune disease. The disease manifests clinically as a wasting syndrome with multi-organ infiltration by recipient CD4 cells, and can be induced by adoptive transfer into syngeneic nude mice of CD4⁺ T cells, which are essential for disease development (4). Co-transfer of normal syngeneic splenocytes prevented the occurrence of autoimmune disease in secondary BALB/c nude recipients of splenocytes from FPG nude mice (4), suggesting a possible failure of regulatory function in FPG nude mice. We have now employed an adoptive transfer approach to address the roles of regulatory cells and effector cell selection in mediating this phenomenon.

Materials and Methods

Animals

Female BALB/c (H2^d) and BALB/c nude mice were purchased from Charles River Laboratories (Wilmington, MA). All mice were maintained in a specific pathogen-free facility, and were housed in microisolator cages containing autoclaved feed, bedding, and acidified water. Second trimester (gestational age 60–75 days, estimated by observed estrus or mating and confirmed by ultrasound examination of the fetuses) partially inbred swine leukocyte antigen Massachusetts General Hospital (MGH) miniature swine fetuses were used as donors of porcine thymic and liver tissue. MGH miniature swine have been bred to homozygosity for the swine leukocyte antigen complex (7). Animal handling and care were in accordance with the American Association for the Accreditation of Laboratory Animal Care and institutional guidelines.

Transplantation Procedures

Eight to 12-week old BALB/c nude mice were transplanted with a miniature swine fetal pig thymic and liver fragment (FP THY/LIV), each about 2mm³ in size, under the kidney capsule (8). All surgical interventions were performed with i.p. injection of Ketamine (0.08 mg/g) and Xylazine (0.012 mg/g) in combination with inhaled methoxyflurane (Pitman-Moore, Mundelein, IL, USA) to maintain stage III anesthesia.

Adoptive Transfer

Eleven to 12 weeks after transplantation of FP THY/LIV, splenocytes were harvested from FP THY/LIV-grafted BALB/c (FPG) nude mice or control normal BALB/c mice. BALB/c nude mice that served as adoptive recipients received 3 Gy total body irradiation and I.V. injection of 2 × 10⁷ splenocytes from FPG mice (GSPL) or normal BALB/c mice (nSPL). In co-transfer experiments, the indicated cell populations were transferred to BALB/c nude mice together with GSPL. The body weights of BALB/c nude recipients were followed weekly.

Cell purification

CD4⁺ or CD8⁺ T cells were purified from splenocytes by positive selection using mouse CD4 or CD8 Microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The purity of the resulting CD4⁺ and CD8⁺ T cells were > 90% and > 95%, respectively. To purify CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells, CD4⁺ T cells were isolated from spleen by negative selection using the mouse CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. CD4⁺-enriched cells were then stained with FITC-labeled anti-CD4 mAb (RM4-5) and PE-labeled anti-CD25 mAb (PC61), and sorted on a MoFlo (Cytomation). The purities of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were > 90% and >99%, respectively.

Cell staining, monoclonal antibodies (mAbs) and flow cytometry (FCM)

Mice were tail bled at regular intervals post-transplant to obtain peripheral blood lymphocytes, which were prepared with Histopaque gradient 1077^® (Sigma, St.Louis, MO). Splenocyte and lymph node (LN) cell suspensions were prepared by standard techniques. Levels of T cell-reconstitution in the FPG mice that underwent transplantation and the phenotype of cells were determined by multicolor flow cytometric (FCM) analysis using various combinations of the following mAbs: anti–mouse CD4 (RM4–5), CD8 (53–6.7), CD25 (PC61), CD69 (H1.2F3), CD44 (IM7), CD62L (MEL-14), CD45RB (16A), CD45 (30–F11) and isotype control mAbs (all purchased from BD Pharmingen, San Diego, CA). Non-viable cells were excluded from analysis by gating out lower forward scatter and high propidium iodide–retaining cells. For intracellular staining, cells were washed twice with wash buffer and surface antigens were stained with Abs. The cells were then fixed and permeabilized with the Cytofix/Perm kit (BD Pharmingen), followed by incubation with anti-mouse CTLA-4 (UC10-4F10-11, BD PharMingen, San Diego, CA) or Foxp3 (FJK-16s, eBioscience, San Diego, CA) for 30 mins at 4°C. Cells were washed twice in Perm/Wash Buffer (BD Pharmingen, San Diego, CA) and resuspended in PBS before analysis. All samples were acquired using a FACScan, FACSCalibur or LSR-II cytometers (BD Biosciences, Mountain View, CA) and data analyses were performed using WinList (Verity Software House, Topsham, ME) and Flowjo (TreeStar, Ashland, OR). The percentage of cells staining with a particular reagent or reagents was determined by subtracting the percentage of cells staining nonspecifically with the isotype control mAb from those staining in the same dot-plot region with the anti-mouse mAbs.

Isolation of thymic epithelial cells and implantation into FP THY grafts

Thymi of 7-week old BALB/c mice were finely minced and rinsed with RPMI medium. The tissue fragments were gently stirred in RPMI medium for 30 min at room temperature to release the bulk of free thymocytes. The remaining tissue fragments were digested by collagenase/dispase/deoxyribonuclease in four incubations of 30 min each and pooled. To remove haematopoietic cells from the digested cell suspension, CD45⁺ cells were depleted using anti-mouse CD45 Microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Less than 5% of these cells were positive for CD45. Freshly isolated thymic epithelial cells were injected into fetal pig thymus grafts at the time of initial transplantation and/or 5 weeks later.

Histology and immunofluorescence staining

Various organs were fixed with 10% formalin, processed for hematoxylin and eosin staining. FP Thy/Liv grafts and normal BALB/c murine and porcine thymic tissues were cryosectioned and fixed with cold acetone for 10 minutes. After blocking with 1% bovine serum albumin (BSA) for 20 minutes, tissue sections were incubated with pre-titrated primary rat anti-mouse I-A/I-E Ab (2G9, BD) and rabbit anti–cytokeratin Ab (Z622, Dako, Carpinteria, CA) for 45 minutes at room temperature, washed, and incubated with FITC–conjugated goat anti-rat IgG and Texas red-conjugated goat anti-rabbit IgG (Vector, Burlingame, CA). After washing with PBS, the coverslips were mounted onto slides using Dako fluorescent mounting medium (Dako, Carpinteria, CA).

Mixed lymphocyte reaction and suppression assay

To compare the suppressive capacity of CD4⁺CD25⁺ T cells from FPG nude mice and normal BALB/c mice, we performed mixed lymphocyte reaction (MLR) coculture assays. CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells were isolated from splenocytes by MACS using the mouse CD4⁺ T Cell Isolation Kit and CD25 microbeads (Miltenyi) according to the manufacturer’s instructions. Pooled CD4⁺CD25⁻ T cells from normal BALB/c mice (8 × 10⁴ per well) were incubated with irradiated (30 Gy) C57BL/6 splenocytes (8 ×10⁴ per well) and CD4⁺CD25⁺ T cells isolated from individual normal Balb/c or FPG nude mice at various ratios in U-bottomed 96-well plates in RPMI 1640 medium supplemented with 15% CPSR-3 (Sigma) with 2 mM L-glutamine, 0.1 mM nonessential amino acids (Life Technologies, Grand Island, NY), 1 mM sodium pyruvate, 10 U/ml penicillin, 10 µg/ml streptomycin, 1% HEPES buffer, and 1×10⁻⁵ M 2-mercaptoethanol (Sigma) at 37°C in 5% CO₂ for 5days. Cells were harvested after 16 h of incubation with 1µCi of [³H]thymidine. [³H]thymidine incorporation was measured by a β-counter. Data are expressed as stimulation index (cpm of stimulated culture/cpm of unstimulated culture). Unstimulated control cultures were the same responder cells incubated in medium alone.

Statistical analysis

All data are presented as the mean ± SEM. Statistical analyses were performed using Student’s t-test with Welch’s correction, one way ANOVA and two way repeated measure ANOVA (for analyses of weight change) were used to compare groups with GraphPad Prism software. A p value less than 0.05 was considered to be statistically significant.

Results

Reconstitution of mouse T cells and phenotypic characteristics of CD4⁺CD25⁺ T cells in FPG BALB/c nude mice

To confirm T cell reconstitution and assess the level of Treg development in BALB/c nude mice grafted with FP THY/LIV (FPG nude mice), we evaluated peripheral reconstitution of mouse T cells by FCM. Increased numbers of CD4⁺ cells and of CD4⁺CD25⁺ cells were detected in spleens of FPG nude mice compared to those from normal BALB/c and BALB/c nude controls 11 weeks after FP THY/LIV transplantation (Figure 1A). The percentages of CD25⁺ cells among CD4 cells were similar between the two groups (data not shown). Thus, FP THY/LIV transplantation allows excellent peripheral reconstitution of mouse CD4⁺ and CD4⁺CD25⁺ T cells in BALB/c nude mice.

Figure 1. Reconstitution of peripheral murine CD4⁺ cells and CD25⁺CD4⁺ cells inBALB/c nude mice receiving FP THY/LIV grafts.

(A) Numbers of murine CD4⁺ cells in spleens of normal BALB/c mice, FP THY/LIV grafted BALB/c (FPG) nude mice and age-matched BALB/c nude mice at 11 weeks after FP THY/LIV transplantation (left). Total numbers of CD4⁺CD25⁺ cells in spleens of normal BALB/c mice and FPG nude mice (right). All data are expressed as mean +/− SEM (n = 4 per group). *P<0.05, **P<0.01, ***P<0.001; compared with indicated group.

(B) CD25 expression on CD4⁺ splenocytes from normal BALB/c mouse (upper left) and a representative FPG nude mouse (upper right) and intracellular expression of Foxp3 in gated CD4⁺CD25⁺ cells of both mice (lower right and lower left, respectively) 11 weeks after FP THY/LIV transplantation. (C) Proportions of Foxp3⁺ cells among CD4⁺CD25⁺ cell in spleens of normal BALB/c mice and FPG nude mice. All data are expressed as mean +/− SEM (n = 4 per group). There was no significant difference between the two groups.

Although constitutive CD25 expression is a marker for thymus-derived regulatory CD4⁺ T cells (Treg), CD25 is also expressed on activated T cells. To distinguish whether CD4⁺CD25⁺ T cells in FPG nude mice were activated T cells or Treg, the expression of Foxp3, a unique transcription factor in Treg, was analyzed in CD4⁺CD25⁺ cells of FPG nude mice by intracellular staining. As shown in Figure 1B, CD4⁺CD25⁺ cells in FPG nude mice were predominantly Foxp3⁺. Almost all CD4⁺CD25⁺ cells expressed Foxp3 (Figure 1C) and the intensities of Foxp3 in CD4⁺CD25⁺ cells of FPG nude mice were similar to those in normal BALB/c mice (Figure 1B). Significantly increased proportions of CD4⁺ cells expressing CTLA4, another characteristic marker for Treg, were detected among CD4CD25⁺ cells from FPG nude mice (71.8 ± 5.2 %) compared with normal BALB/c controls (63.3 ± 6.5 %, P<0.05). These data show that CD4⁺CD25⁺ T cells of FPG nude mice express typical Treg markers.

Co-transfer of normal CD4⁺ or CD8⁺ splenocytes can prevent autoimmune disease in adoptive recipients

We have previously reported that normal BALB/c splenocytes (nSPL) can suppress the ability of splenocytes from FPG nude mice (GSPL) to induce autoimmune disease in secondary BALB/c nude recipients (4). To identify splenocyte populations with this suppressive activity, we co-transferred various cell subsets of nSPL along with GSPL to lightly irradiated (3Gy) Bulb/c nude mice. Co-transfer of 10⁷ CD4⁺ nSPL completely suppressed the weight loss and tissue injury induced by 2×10⁷ GSPL in secondary BALB/c nude recipients. In contrast to recipients of GSPL alone, secondary recipients of a mixture of GSPL and CD4⁺ nSPL showed increasing weight and no clinical signs of autoimmune disease, similar to recipients of 2×10⁷ nSPL alone (data not shown). Co-transfer of 10⁷ CD8⁺ nSPL also protected secondary BALB/c nude recipients from any clinical or histologic evidence of autoimmune disease induced by 2×10⁷ GSPL (data not shown). These results are consistent with the ability of both subsets of syngeneic T cells to suppress autoimmunity induced by lymphocytes from nude rats receiving hamster thymus xenografts {9382}.

To quantitatively compare the suppressive activity of CD4⁺ and CD8⁺ nSPL, we co-transferred various doses of CD4⁺ or CD8⁺ nSPL. As shown in Figure 2, co-transfer of 10⁷ or 3×10⁶ CD4⁺ nSPL suppressed the autoimmunity induced by 2×10⁷ GSPL in secondary BALB/c nude recipients to a similar extent. Figure 2 also illustrates that the potency of CD8⁺ nSPL as suppressors of the disease was lower than that of CD4⁺ nSPL, since co-transfer of 3×10⁶ CD8⁺ nSPL in the same experiment failed to suppress autoimmunity, while 10⁷ CD8⁺ nSPL again completely suppressed the disease.

Figure 2. Co-transfer of CD4 or CD8 normal BALB/c splenocytes (nSPL) protects against autoimmune disease induced by adoptive transfer of splenocytes from FPG nude mice (GSPL).

Mean ±SEM body weight changes are shown as the ratio of body weight at the indicated time point compared to pre-infusion weight. Co-transfer of 1×10⁷ (◇) or 3×10⁶ (◆) CD4⁺ nSPL completely suppressed the autoimmunity induced by 2×10⁷ GSPL in secondary BALB/c nude recipients. However, co-transfer of 1×10⁷ (△) but not 3×10⁶ (▲) CD8⁺ nSPL completely suppressed the autoimmune disease. *P<0.05, **P<0.01, ***P<0.001; compared with the group receiving no splenocytes (▽)

The phenotype of CD4⁺ cells in spleen and lymph nodes of nude mice 7 weeks after adoptive transfer of GSPL was consistent with a high level of activation and possibly lymphopenia-driven expansion. CD4⁺ cells in both spleen and lymph nodes of recipients of GSPL alone showed significantly increased levels of CD69 and CD44 and decreased CD62L and CD45RB expression compared to those of recipients of nSPL (data not shown). Co-transfer of CD4⁺ nSPL with GSPL led to significantly reduced levels of CD69 and CD44, with increased proportions of CD62L⁺ and reduced proportions of CD45RB^low cells compared to secondary recipients of GSPL alone (data not shown). These effects of nSPL CD4 cells may reflect reduced activation of GSPL-derived CD4 cells and/or dilution by non-activated, non-expanding nSPL-derived CD4 cells. However, co-transfer of CD8⁺ nSPL led to reduced CD69 expression on GSPL-derived CD4 cells, indicating that they suppressed GSPL CD4 cell activation. nSPL-derived CD8 cells did not, however, preserve CD62L or CD45RB expression on GSPL CD4⁺ cells (data not shown).

Cotransfer of normal CD4⁺CD25⁺ or CD4⁺CD25⁻ splenocytes can inhibit autoimmune disease in secondary recipients

To identify subpopulations of CD4⁺ nSPL with suppressive activity, we fractionated co-transferred CD4⁺ nSPL populations. As shown in Figure 3A, co-transfer of 9×10⁵ CD4⁺CD25⁺ nSPL completely suppressed the autoimmunity induced by 2×10⁷ GSPL in secondary BALB/c nude recipients. This suppression was dependent on the number of CD4⁺CD25⁺ nSPL co-transferred, as shown in Figure 3B.

Figure 3. Co-transfer of CD4⁺CD25⁺ or CD4⁺CD25⁻ splenocytes from normal BALB/c mice (nSPL) can prevent autoimmune disease in secondary recipients.

Mean ±SEM body weight changes are shown as the ratio of body weight at the indicated time point compared to pre-infusion weight. (A) Co-transfer of 9×10⁵ CD4⁺CD25⁺ or 5×10⁶ CD4⁺CD25⁻ nSPL initially suppressed the autoimmunity induced by 2×10⁷ GSPL in secondary BALB/c nude recipients. ** P<0.01, *** P<0.001; compared with the groups receiving no splenocytes (▽), receiving only nSPL (○) or receiving both GSPL and 9×10⁵ CD4⁺CD25⁺ nSPL (◇) or receiving both GSPL and 5×10⁶ CD4⁺CD25⁻ nSPL (▲). (B) The suppressive activity of CD4⁺CD25⁺ nSPL is dose-dependent. CD4⁺CD25⁻ cells were less potent suppressors than the CD25⁺ subset, as 4×10⁶ CD25⁻ cells (▲) delayed weight loss in adoptive recipients less markedly than approximately 1/10^th that number of CD4⁺CD25⁺ cells (□). *P<0.05, ***P<0.001; compared with the groups receiving GSPL (■). ^###P<0.001; compared with the groups receiving no splenocytes (▽). Statistical analyses were performed using data from 20 weeks after adoptive transfer.

Co-transfer of 5×10⁶ CD4⁺CD25⁻ nSPL also initially suppressed the autoimmunity induced by 2×10⁷ GSPL (Figure 3A). However, as shown in Figure 3B, CD4⁺CD25⁻ nSPL were less potent suppressors than the CD25⁺ subset, as 4×10⁶ CD25⁻nSPL reduced the late weight loss in adoptive recipients less markedly than approximately 1/10^th that number of CD4⁺CD25⁺nSPL.

Defective accumulation in lymphopenic hosts of T cells generated in FP THY/LIV grafts

The ability of CD4⁺ nSPL, including CD25⁻ cells, as well as CD8⁺ nSPL to suppress T cell activation and autoimmune disease induced by GSPL raised the possibility that their suppressive function might be due in part to their ability to respond to lymphopenia-driven stimuli and hence “compete for resources” (9). To address this possibility, we compared the accumulation of T cells derived from FPG nude mice to those from normal BALB/c mice following adoptive transfer into BALB/c nude mice. As shown in Figure 4A, transfer of 3×10⁵ CD4⁺CD25⁻ GSPL led to expansion in secondary recipients, but there was significantly reduced accumulation in peripheral blood at 3 and 6 weeks (p<0.05 and p<0.01, respectively) (Figure 4A) compared to recipients of similar cell populations from nSPL. No significant differences were detected at 6 weeks in splenic or LN T cell accumulation in recipients of 3×10⁵ CD4⁺CD25⁻ GSPL versus those receiving 3×10⁵ CD4⁺CD25⁻ nSPL. When 3×10⁶ CD4⁺CD25⁻ GSPL cells were transferred, significantly reduced CD4 T cell accumulation was detected in blood at 3 and 6 weeks (p<0.001), and in both spleen and LNs at 6 weeks (p<0.01) compared with recipients of 3×10⁶ CD4⁺CD25⁻ nSPL (Figure 4A). These data are consistent with a defect in lymphopenia-driven expansion or survival among CD4⁺CD25⁻ cells generated in a xenogeneic thymic graft.

Figure 4. Defective expansion and/or survival in T cell-deficient hosts of T cells generated in FP THY/LIV grafts.

(A) Transfer of 3×10⁵ CD4⁺CD25⁻ splenocytes from FPG nude mice (GSPL) led to significantly reduced accumulation in peripheral blood at 3 and 6 weeks compared to recipients of similar cell populations from normal BALB/c donors (nSPL) (left, *P<0.05, ***P<0.001). No significant differences were detected between the two groups at 6 weeks in splenic or LN T cell accumulation. When 3×10⁶ CD4⁺CD25⁻ cells were transferred, significantly reduced accumulation was detected in peripheral blood at 3 and 6 weeks (left) and in spleen (middle) and LNs (right) at 6 weeks in secondary nude recipients of CD4⁺CD25⁻ GSPL compared with those of CD4⁺CD25⁻ nSPL (**P<0.01, ***P<0.001). (B) 3×10⁵ CD4⁺CD25⁺ nSPL or GSPL were adoptively transferred to secondary BALB/c nude mice. The accumulation of these cells in blood (right), spleen (middle) and LNs (right) at 6 weeks was significantly reduced in recipients of CD4⁺CD25⁺ GSPL compared with those of CD4⁺CD25⁺ nSPL (*P<0.05, ***P<0.001, n=5 for each group).

Since CD4⁺CD25⁺ Treg have been shown to require self class II MHC for their maintenance in the periphery (10), we performed similar comparisons following adoptive transfer of 3×10⁵ CD4⁺CD25⁺ nSPL or GSPL. As shown in Figure 4B, while evidence for expansion of the injected cells was observed in both groups, the accumulation of these cells in blood, spleen and lymph nodes at 6 weeks was also significantly reduced in recipients of CD4⁺CD25⁺ GSPL versus those receiving CD4⁺CD25⁺ nSPL (p<0.001, p<0.05, p<0.05, respectively). These data are consistent with a defect in lymphopenia-driven expansion and/or survival among Tregs generated in a xenogeneic thymic graft.

Abnormalities in both CD4 effector cells and Treg developing in FP THY/LIV grafts

To further analyze the etiology of autoimmunity in secondary BALB/c nude mouse recipients of GSPL, we asked whether CD25⁺ cells or CD25⁻ cells from these animals are responsible for inducing autoimmunity in secondary recipients. As shown in Figure 5A, adoptive transfer of 3×10⁵ CD4⁺CD25⁺ GSPL or nSPL did not cause clinical evidence of autoimmune disease in secondary BALB/c nude recipients. In contrast, transfer of 3×10⁵ or 3×10⁶ CD4⁺CD25⁻ GSPL led to rapid weight loss (Figure 5A) and other signs of autoimmune disease (not shown) in secondary BALB/c nude mouse recipients. Transfer of up to 3×10⁶ CD4⁺CD25⁻ nSPL did not cause autoimmunity in secondary BALB/c nude recipients (Figure 5A). Thus, CD25⁻ and not CD25⁺CD4 T cells generated in porcine thymus xenografts have an increased tendency to induce autoimmunity compared to those that develop in a normal mouse thymus.

Figure 5. Abnormalities in both CD4 effector cells and Treg developing in FP THY/LIV grafts.

Mean ±SEM body weight changes are shown as the ratio of body weight at the indicated time point compared to pre-infusion weight. (A) Adoptive transfer of 3×10⁵ CD4CD25⁺ cells from spleens of FPG nude mice (GSPL) or from normal BALB/c mice (nSPL) did not cause clinical evidence of autoimmune disease in secondary BALB/c nude recipients. In contrast, transfer of 3×10⁵ or 3×10⁶ CD4⁺CD25⁻ GSPL led to rapid weight loss. Transfer of up to 3×10⁶ CD4CD25⁻ nSPL did not cause autoimmunity in secondary BALB/c nude recipients. (B) Co-transfer of 5×10⁵ CD4 CD25⁺ nSPL completely suppressed the weight loss in BALB/c nude mice receiving 2×10⁷ GSPL (◇). In contrast, 5×10⁵ CD4⁺CD25⁺ GSPL did not suppress autoimmunity (◆). ** P<0.01, *** P<0.001; compared with the group receiving no splenocytes (▽). No significant differences were detected between mice receiving GSPL with or without 5×10⁵ CD4⁺CD25⁺ GSPL (■ and ◆).

While co-transfer of 5×10⁵ CD4⁺CD25⁺ nSPL completely suppressed the weight loss (Figure 5B) and all clinical evidence of autoimmunity in BALB/c nude mice receiving 2×10⁷ GSPL (not shown), similar numbers of CD4⁺CD25⁺ GSPL did not suppress autoimmunity. Thus, Treg in spleens of FPG nude mice were defective in the ability to suppress autoimmune disease.

Phenotypic analyses and in vitro functional analyses of Tregs from GSPL were consistent with this result. As shown in Figure 6A, CD4⁺CD25⁺ T cells from FPG nude mice showed a marked defect, compared to those from normal BALB/c mice, in their ability to suppress alloresponses of syngeneic BALB/c CD4⁺CD25⁻ T cells. The phenotype of Tregs from FPG mice was also abnormal, with increased CD44, increased CD45RB and decreased CD62L expression compared to Treg from normal BALB/c mice (Figure 6B and C).

Figure 6. Abnormal phenotype and function of Treg from FPG mice.

(A) Impaired suppressive activity of CD4⁺CD25⁻ T cells of FPG nude mice. MLR was performed 16 weeks after transplantation using normal BALB/c or FPG nude mice with >10% CD3⁺ PBMC. Splenic CD4⁺CD25⁻ T cells (8 × 10⁴) isolated from normal BALB/c mice were pooled and incubated with irradiated (30Gy) splenocytes of C57BL/6 mice (8 × 10⁴) and CD4⁺CD25⁺ T cells isolated from individual normal BALB/c or FPG nude mice at the indicated ratio for 5 days. Cultures were pulsed with [³H] thymidine at day 4 and harvested 16 hr later. Data are expressed as mean +/− SEM (n = 3 mice per group). *P<0.05; compared with FPG nude mice (two way ANOVA and Bonferroni post tests).

(B) and (C), Phenotypic comparison of CD4⁺CD25⁺ T cells in normal Balb/c mice and FPG nude mice. Fourteen weeks after thymic transplantation, FPG nude and normal Balb/c mice were bled, PBMCs were obtained by denisty separation and PBMCs were stained with anti-CD4, -CD8 and -CD25 mAbs vs. anti-CD44, -CD45RB, -D62L, and isotype control mAbs. Gated CD4⁺CD8⁻CD25⁺ T cells were analyzed by FCM. (B) Shows a representative phenotypic profile of CD4CD25+ T cells from normal BALB/c (upper panels) and FPG nude (lower panels) mice. The shaded line represents the staining with isotype control mAb and the thick line represents the test staining. The frequencies of each portion among CD4⁺CD25⁺ T cells are shown. (C) Proportions of cells expressing the indicated marker among CD4⁺CD25⁺ T cells in normal BALB/c mice (n=5) and FPG nude mice (n=8). Data are expressed as mean +/− SEM. **P<0.01; compared with indicated group (two way ANOVA and Bonferroni post tests).

Since CD25⁺ “natural” Treg develop intrathymically, we next assessed the ability of thymocytes derived from FPG nude mice to suppress autoimmunity in the adoptive transfer model. As shown in Figure 7A, co-transfer of 2×10⁷ thymocytes containing 7.4×10⁴ or 10.2×10⁴ CD4⁺CD8⁻CD25⁺ cells from FP THY grafts harvested 5 or 11 weeks after implantation, respectively, not only failed to suppress autoimmunity induced by GSPL, but actually accelerated autoimmunity. Aoptive transfer of thymocytes (2×10⁷) from FPG nude mice alone also caused autoimmunity. In contrast, similar numbers of thymocytes from normal BALB/c mice (n-thymocytes) completely suppressed the autoimmunity induced by GSPL, and did not cause autoimmunity on their own (Figure 7B). These data demonstrate that selection in a porcine thymic xenograft results in an intrinsic tendency of T cells to cause autoimmune disease.

Figure 7. Failure of thymocytes from FP THY grafts to suppress and tendency to induce autoimmunity in BALB/c nude mice.

Mean ±SEM body weight changes are shown as the ratio of body weight at the indicated time point compared to pre-infusion weight. (A) Co-transfer of 2×10⁷ thymocytes containing 7.4×10⁴ or 10.2×10⁴ CD4⁺CD8⁻CD25⁺ thymocytes from FP THY grafts harvested 11 or 5 weeks after implantation (◇ and ◆), respectively, accelerates autoimmunity induced in BALB/c nude recipients by 2×10⁷ GSPL. Unfractionated thymocytes (2×10⁷) from FPG nude mice alone induce autoimmunity in adoptive recipients (△ and ▲). *P<0.05, **P<0.01; compared with the group receiving GSPL alone (■) (B) Similar numbers of thymocytes from normal BALB/c mice (n-thymocytes) completely suppressed the autoimmunity induced by 2×10⁷ GSPL (◇ and ◆), and did not cause autoimmunity on their own (○ and ●).

Correction of autoimmune defects by co-implantation of mouse thymic epithelial cells (TEC) with porcine THY grafts

We hypothesized that the addition of murine, host-type thymic epithelial cells (mTEC) to the porcine thymic graft might prevent the development of autoimmunity by improving the negative selection of host-specific T cells, and/or by promoting the positive selection of CD25⁺CD4⁺ regulatory cells. We evaluated the effect of injecting normal BALB/c mTEC obtained by collagenase/dispase/DNase digestion and negative selection (with anti-CD45-MACS beads) into porcine thymus grafts at the time of implantation into BALB/c nude mice and/or 5 weeks later (by laparotomy and injection into the graft, which had enlarged markedly by this time). As shown in Figure 8A, GSPL from FPG nude mice that received FP THY/LIV grafts into which normal BALB/c mTEC were injected on Day 0 and at 5 weeks induced significantly less wasting syndrome in BALB/c nude mouse adoptive recipients. Moreover, co-transfer of GSPL from recipients of FPG with mTEC partially suppressed the wasting syndrome induced by GSPL from mice grafted without normal BALB/c mTEC. The figure also shows that injection of normal BALB/c mTEC at 5 weeks, without the Day 0 injection, did not reduce the ability of GSPL to induce wasting syndrome. Two-color immunohistochemical staining of the thymic grafts that were injected with mTEC, using anti-mouse class II mAb and an antibody specific for cytokeratin (Figure S1), clearly showed the presence of mTEC in the grafts of animals in which these cells had been injected at Day 0 and 5 weeks (Figure 8B). However, the FP THY grafts injected with mTEC only at Week 5 contained very few mTEC (Figure 8B).

Figure 8. Correction of autoimmune defects by co-implantation of murine thymic epithelial cells (TEC) with porcine THY grafts.

(A) GSPL from nude mice that received FP THY/LIV grafts into which normal BALB/c mTEC were injected on Day 0 and at 5 weeks (◇) induced significantly less wasting syndrome in BALB/c nude mouse adoptive recipients than GSPL from control FPG nude mice grafted without normal BALB/c mTEC (■). Co-transfer of GSPL from nude mice that received FP THY/LIV grafts into which normal BALB/c mTEC were injected on Day 0 and at 5 weeks (△) partially suppressed the wasting syndrome induced by adoptive transfer of 2×10⁷ GSPL from control FPG nude mice grafted without normal BALB/c mTEC. Injection of BALB/c mTEC at 5 weeks, without the Day 0 injection, did not inhibit wasting syndrome. Mean ±SEM body weight changes are shown as the ratio of body weight at the indicated time point compared to pre-infusion weight. *P<0.05, **P<0.01, ***P<0.001; compared with the group receiving GSPL alone from FPG nude mice grafted without mTEC. (B) Two-color immunohistochemical staining of the thymic grafts that were injected with or without murine TEC (mTEC), Anti-mouse class II mAb and an antibody specific for cytokeratin (Figure S1) showed the presence of mTEC in the grafts in which these cells had been injected at Day 0 and 5 weeks (right). However, the FP THY grafts injected with mTEC only at Week 5 had very few double positive mTEC (middle). Green; mouse class II, red; cytokeratin

Discussion

Fetal porcine thymic grafts can efficiently support mouse and human thymopoiesis, resulting in the generation of a T cell repertoire that is specifically tolerant of the xenogeneic donor (1,2,11). Recent extension of this approach using vascularized porcine thymic grafts in a non-human primate model has allowed, for the first time, acceptance of life-supporting αGal knockout porcine kidney grafts for months with no evidence for rejection (12).

While normal mice that are thymectomized and T cell depleted usually display excellent health following T cell reconstitution from FP THY grafts, a high proportion of FP-grafted nude mice, which lack a native thymus, eventually develop a multiorgan autoimmune syndrome characterized by murine CD4 cell infiltration of lung, liver, ovary and intestine (4). A similar phenomenon has been reported in nude rats receiving fetal hamster thymic xenografts, in which disease transfer to secondary recipients could also be blocked by CD4⁺ or CD8⁺ T cells from euthymic syngeneic donors {9382}. Normal numbers of cells with phenotypic characteristics of Tregs were detected in the thymus-grafted animals {9382}. In our pig to mouse model, we used FoxP3 staining to demonstrate that increased numbers of Tregs repopulate the the periphery of thymus xenograft recipients compared to euthymic mice, and that sorted Tregs from these animals show defective suppressive activity in vitro and in vivo. Natural Treg are CD25⁺CD4⁺ FoxP3⁺ T cells that are generated intrathymically (13,14). However, abnormal natural Tregs are not the sole cause of autoimmunity, as we have demonstrated an abnormal propensity of sorted CD25⁻ T cells from xenogeneic thymus grafted mice to cause autoimmunity, as well as abnormalities in the homeostasis of both effector and regulatory CD4⁺ T cells originating in xenogeneic thymus grafts. Our data show that, while both CD4⁺ and CD8⁺ cells from euthymic syngeneic mice suppress the ability of adoptively transferred T cells from thymus xenograft recipients to cause autoimmunity, the potency of the CD4 subset is greater than that of CD8 cells. Moreover, both CD25⁺ and CD25⁻ CD4 cells could suppress disease, though the CD25⁺ subset showed more than 10-fold greater potency.

The increased susceptibility of FPG congenitally athymic nude mice to the autoimmune syndrome compared to immunocompetent mice may reflect the presence in the latter of Treg in the periphery prior to T cell depletion with mAbs. Some of these Treg may persist after conditioning, protecting the mice from autoimmunity following porcine thymic implantation, as BALB/c Tregs have been shown to be relatively resistant to in vivo CD4 depletion with GK1.5 (15), the mAb used in our studies.

The reduced capacity of both effector T cells and Tregs from FPG mice to accumulate in T cell-deficient hosts may contribute to the autoimmunity in nude mice by failing to compete with activated autoreactive T cells for cytokines and other “resources”. A defect in lymphopenia-driven expansion could explain the reduced accumulation of adoptively transferred T cells from FPG mice compared to those from normal mice in secondary BALB/c nude recipients. The same MHC/peptide complexes responsible for positive selection may be required in the periphery to allow optimal T cell survival and expansion in a lymphopenic environment (38), and such complexes are not found in the periphery by T cells positively selected by a xenogeneic thymic MHC. We observed a slight increase in the decay of memory-type cells from FPG compared to syngeneic murine thymic grafts following graftectomy (39), consistent with a defect in homeostatic expansion.

The inability of thymocytes and peripheral CD4⁺CD25⁺ cells from FPG nude mice to suppress the autoimmunity induced by CD25⁻CD4⁺ T cells from FPG nude mice most likely reflects a reduced ability of the porcine thymic epithelium to positively select Tregs that recognize murine MHC/peptide complexes in the periphery. The thymic epithelium plays an important role in the positive selection of Treg (16–19). We have demonstrated that conventional CD4 T cells in FPG mice are positively selected by porcine MHC, with no measurable contribution from the murine MHC (5,6), and the same is likely to apply to Treg. Implantation of normal BALB/c thymic epithelial cells in FP THY grafts enhanced the ability of GSPL to suppress autoimmunity, suggesting an approach to overcoming this defect.

Additionally, our large animal studies utilize primarily vascularized thymic tissue rather than thymic fragments, and studies in the rat to mouse model suggest that vascularization can prevent autoimmunity in thymus xenograft recipients, in association with enhanced Treg generation (20).

Treg require interactions with self MHC in the periphery in order to become fully functional (10) and the same peptide/MHC complex in the periphery as that which led to positive selection in the thymus is needed to promote lymphopenia-driven expansion (21). Consistent with a failure of Tregs developing in thymus xenografts to re-encounter the selecting ligands in the periphery, we observed less expansion of Tregs transferred from FPG mice than those from normal mice in T cell-deficient recipients. Thus, Treg positively selected on porcine MHC/peptide complexes in the thymus may fail to interact effectively with the murine MHC/peptide complexes in the periphery and hence fail to become fully functional and capable of suppressing autoimmunity. Moreover, reduced homeostatic expansion may result in failure of Tregs to compete with autoreactive T cells with higher affinity for self antigens transferred to nude mice, resulting in reduced suppression of autoimmunity (9).

Porcine thymus xenografts are nevertheless capable of generating functional mouse Treg that suppress anti-donor responses {Rodriguez-Barbosa, 2001 7215 /id} and specifically prolong donor skin graft survival in an adoptive transfer model (Y. Zhao and M. Sykes, unpublished data). Sun et al reported that nude mice receiving neonatal porcine thymic grafts have FoxP3⁺ Tregs in the periphery that suppress anti-pig responses but show defective suppression of alloresponses in vitro (23), consistent with our results. However, in contrast to our results, these cells suppressed autoimmunity following adoptive transfer to nude mice (23). This discrepancy most likely reflects the much greater ratio of Tregs to CD25⁻ CD4 cells transferred by Sun et al, and the fact that the CD25⁻ cells in their study were derived from normal BALB/c mice rather than FPG mice (see below). Together, these studies suggest that the defect in suppression of autoreactivity by Treg from FPG mice is relative and not absolute. Effective suppression of anti-pig responses with reduced suppression of anti-host responses is consistent with an exclusive role of the xenogeneic thymic epithelium in positively selecting Tregs. While further studies are needed to address this hypothesis, the reduced (compared to normal syngeneic mice) capacity of Tregs from FPG mice to suppress alloresponses may reflect the same phenomenon. Since the potency of alloresponses is thought to reflect the cross-reactivity with allogeneic MHC/peptides of T cells positively selected on self MHC/peptide complexes, the same phenomenon may result in the presence of a relatively high frequency in the normal Treg repertoire of cells that suppress alloresponses. This situation would not prevail among Tregs selected by a xenogeneic MHC. Additional effects of the xenogeneic disparities between pig and mouse may also contribute to the reduction in Treg function. The altered phenotype of Tregs in FPG mice resembles that observed in cyclosporine-treated mice, in which regulatory function was also reduced {12726}. The reduced CD62L and increased CD45RB expression we observed is consistent with the reduced suppressive function, as the most potent suppression has been attributed to CD62L⁺ and CD45RB^low subsets of CD25+ CD4 T cells {10983;14236;14237}.

However, a lack of natural Treg specific for recipient antigens cannot explain the increased autoimmunity induced by CD25⁻CD4⁺ T cells and thymocytes from FPG mice. These data suggest a possible defect in the negative selection of host-reactive thymocytes in FP THY grafts. While mouse MHC class II⁺ hematopoietic cells in porcine thymus grafts contribute effectively to negative selection of murine thymocytes (5,6), the thymic epithelium “ectopically” produces proteins that are otherwise produced only in specialized peripheral organs (24–26). FP THY grafts, whose epithelium only expresses porcine tissue-specific antigens, might therefore fail to delete mouse tissue-specific antigen-reactive T cells. Consistently, the successful engraftment of normal BALB/c mTEC in FP grafts reduced the capacity of thymocytes from FP grafts to induce autoimmunity.

In the fetal hamster to nude rat thymic transplantation model discussed above, autoimmunity could be prevented by co-implanting several types of syngeneic fetal epithelial tissues, including the thymus, but not heart tissue, into the graft (27,28). While these results suggested that the addition of recipient epithelial cell antigens might prevent the autoimmunity, our study is the first to demonstrate that purified host-type thymic epithelial cells can ameliorate disease, persist long-term within the thymic xenograft and allow the development of regulatory cells that can inhibit autoimmunity upon adoptive transfer.

Consistent with our observation that normal CD8 T cells suppressed autoimmune potential and CD4 cell activation from GSPL, a paucity of regulatory CD8 cells was implicated in autoimmunity in rat to nude mouse and hamster to nude rat thymic transplantation models (28–31). CD8⁺ Tregs have been implicated in a number of tolerance models (32–35). Phenotypically mature, normal and functional murine CD8 single positive T cells are effectively generated in porcine thymic grafts, but these cells fail to repopulate the periphery (36). Fortunately this defect is not observed for human T cells developing in porcine thymic grafts (11,37).

“Self’ MHC-peptide complexes downmodulate the function of autoreactive T cells (10). It is possible that the absence in the periphery of FPG mice of the thymic (porcine) positively selecting MHC/peptide complexes leads to a failure of this modulation. Consistent with this possibility, increased levels of the CD69 activation marker were detected on adoptively transferred peripheral T cells from FPG nude mice compared to normal mice. Since CD69 is not upregulated on T cells that expand due to lymphopenic stimuli (38,40–43), the upregulation of this marker strongly suggests T cell activation.

Despite all of the possible ramifications of positive selection of thymocytes on a xenogeneic epithelium discussed above, the T cell repertoire that is positively selected in a xenogeneic thymus is broad {4404;14153}, and this may allow sufficient cross-reactivity on host MHC to explain the robust responses to protein antigens and ability to clear opportunistic infection that is conferred by xenogeneic thymus grafting {4040}. Our data suggest that positive selection of both effector and regulatory T cells recognizing recipient MHC may be enhanced by adding recipient thymic epithelial cells to the xenografts.

In summary, our data implicate several mechanisms in the propensity of T cells from FPG mice to induce autoimmunity in nude mice, including defects in positive selection and peripheral activation of host-specific Treg, defects in negative selection and peripheral modulation of autoreactive T cells and possible defects in lymphopenia-driven expansion and survival. All of these defects can be potentially overcome by co-implantation of recipient-type thymic epithelial cells. Further exploration of this approach will be of considerable importance for the clinical applicability of this promising approach to inducing xenograft tolerance.

Non-standard abbreviations

FP

fetal pig

THY

thymus

FPG

fetal pig thymus grafted

ATX

thymectomized

FP THY/LIV

fetal pig thymus and liver tissue

MLR

mixed lymphocyte reactions

GSPL

spelenocytes from FPG mice

nSPL

normal splenocytes

LN

lymph node

FCM

flow cytometric

Treg

regulatory T cells

mTEC

thymic epithelial cells