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The Journal of Immunology, 1998, 161: 1320-1326.
Copyright © 1998 by The American Association of Immunologists

Pig MHC Mediates Positive Selection of Mouse CD4+ T Cells with a Mouse MHC-Restricted TCR in Pig Thymus Grafts1

Yong Zhao, Kirsten Swenson, Justin J. Sergio and Megan Sykes2

Bone Marrow Transplantation Section, Transplantation Biology Research Center, Surgical Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Remarkably normal immune function and specific T cell tolerance to discordant xenogeneic donors can be achieved by grafting fetal pig thymus and liver (FP THY/LIV) tissue to T cell and NK cell-depleted, thymectomized (ATX) mice. To determine whether or not host class II MHC molecules participate in the positive selection of mouse CD4+ T cells in FP THY/LIV grafts, we compared their development in ATX "AND" TCR-transgenic mice with positive selecting or nonselecting host MHC genotypes. Mouse TCR-transgenic CD4 single positive T cells repopulated the periphery significantly and to a similar extent in both T/NK cell-depleted, ATX AND mice with positive-selecting or nonselecting MHC backgrounds after grafting with FP THY/LIV. Therefore, MHC molecules from a widely disparate xenogeneic species can positively select T cells bearing a host class II MHC-restricted TCR without a contribution from the host MHC. These results, in combination with previous studies performed in this model, suggest that the T cell repertoire that is generated by the combination of positive selection on xenogeneic MHC and negative selection on both recipient and xenogeneic porcine MHC is tolerant of both donor and recipient and has sufficient cross-reactivity with host MHC/foreign peptide complexes to confer a high level of immunocompetence. The results have implications for the potential clinical applicability of xenogeneic thymic transplantation and also suggest a predominant role for the TCR recognition of species-conserved MHC residues in positive selection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
We have recently observed that efficient mouse thymopoiesis and mouse CD4+ T cell repopulation occurs in xenogeneic fetal pig thymus and liver (FP THY/LIV)3-grafted, thymectomized (ATX), T/NK cell-depleted B10 mice (1-3). These mouse CD4+ T cells that develop in FP THY/LIV grafts are phenotypically and functionally mature (4). Furthermore, the reconstituted T cells are tolerant to pig donor and mouse host Ags as indicated by nonresponsiveness to donor and host Ags in MLR assays and by long-term acceptance of donor swine leukocyte Ag (SLA)-matched pig skin grafts (1-3, 5). Evidence suggests that intrathymic clonal deletion is one of the mechanisms for the discordant xenogeneic tolerance induced with this approach (3, 5).

Since it is generally accepted that the MHC restriction of T cells is determined by thymic epithelial cells (6, 7), we expected these mice to show poor host MHC-restricted T cell responses and consequently to be immunoincompetent. Surprisingly, however, mouse CD4+ T cells maturing in FP THY/LIV grafts show remarkably normal immune function, including host MHC-restricted responses to keyhole limpet hemocyanin and the ability to clear opportunistic infection with Pneumocystis carinii (4). Using ATX class II MHC-deficient and H-2b wild-type mice as FP THY/LIV recipients, we have demonstrated that porcine MHC molecules participate in both the positive and negative selection of mouse T cells maturing in FP THY/LIV grafts, and that host class II MHC molecules regulate the terminal maturation of CD4 single positive (SP) thymocytes (8). However, these results did not rule out the possibility that host mouse class II MHC participates in positive selection in FP THY/LIV grafts in class II+ mouse recipients. To determine whether host mouse class II+ cells participate in positive selection in FP THY/LIV grafts, we compared the maturation of mouse T cells with a transgenic TCR in FP THY/LIV grafts in ATX, TCR-transgenic "AND" mice with positive selecting or nonselecting host MHC backgrounds. Mouse CD4+ T cells bearing a transgenic TCR were positively selected by pig MHC in FP THY/LIV grafts regardless of the MHC genotype of the recipients. This is the first direct evidence that MHC molecules from a widely disparate xenogeneic species can positively select T cells bearing host class II MHC-restricted TCRs; these results also suggest a predominant role for the TCR recognition of species-conserved MHC residues in positive selection. The results have implications for the potential clinical applicability of xenogeneic thymic transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

AND TCR-transgenic mice (TgN(TcrAND)53Hed) bearing a transgenic TCR (V{alpha}11, Vß3) from an I-Ek-restricted pigeon cytochrome c peptide 88-104-specific CD4 clone. This transgenic clone is positively selected by I-Ab (9, 10). The mice (on an H-2b background) were purchased from The Jackson Laboratory (Bar Harbor, ME). These mice were bred to B10.A(4R) mice (KkI-AkI-EbDb), and the F1 generation was subsequently intercrossed to generate transgenic mice on a homozygous, nonselecting MHC background (I-Ak/k). The H-2 genotype of mice in the F2 generation was determined by staining PBLs with anti-I-Ab (25-9-17) and anti-I-Ak (590H-4-2) mAbs (11) followed by flow cytometry (FCM) analysis. All mice were maintained in a specific pathogen-free facility and were housed in microisolator cages containing autoclaved feed, bedding, and acidified water.

Transplantation procedures

Thymectomy was performed as described previously (12, 13). Intraperitoneal injections of mAbs GK1.5 (rat anti-mouse CD4), 2.43 (rat anti-mouse CD8), 30-H12 (rat anti-mouse Thy1.2), and PK136 (mouse anti-mouse NK1.1) were administered to 8- to 12-wk-old euthymic or ATX AND mice in depleting doses, as described previously (14), on days -6 and -1 before FP THY grafting. On day 0, 3 Gy of whole body irradiation was administered to recipients, and one fresh, second trimester (gestational day 48-75, estimated by observed estrus or mating and confirmed by ultrasound examination of the fetuses), miniature swine FP THY/LIV fragment (~1 mm3 in size) was transplanted under the kidney capsule via a midline laparotomy incision. After the abdomen was closed in two layers, 1 x 108 fetal pig liver cells were injected i.p. On days 7 and 14, repeat injections of all four of the mAbs described above were administered i.p. Simultaneously prepared control groups included similarly T/NK cell-depleted ATX mice receiving FP LIV tissue without thymic grafts (immunodeficient controls) and non-ATX mice receiving similar T/NK cell depletion and FP LIV tissue (euthymic controls). Animal care was in accordance with both the American Association for the Accreditation of Laboratory Animal Care and institutional guidelines. Operations were performed under metofane inhalational anesthesia.

mAbs and FCM

The following mAbs were used: phycoerythrin (PE)-conjugated rat anti-mouse CD4 (RM 4-5), PE-conjugated rat anti-mouse CD8 (53-6.7), PE-conjugated anti-Vß3 (KJ25), FITC-labeled rat anti-mouse CD4, FITC-labeled rat anti-mouse CD8, FITC-labeled anti-V{alpha}11 (RR8-1), FITC-labeled mouse anti-mouse Qa-2 (1-9-9), FITC-labeled rat anti-mouse CD24 (heat stable Ag (HSA)) (J11d), biotin-conjugated anti-CD4, and biotin-conjugated anti-CD8. PE-conjugated rat IgG2a, biotinylated HOPC-1, and FITC-labeled HOPC-1 were used as nonstaining control mAbs. All mAbs were purchased from PharMingen (San Diego, CA), with the exception of HOPC-1, which was purified from ascites in our laboratory on protein A-Sepharose and conjugated by FITC or biotin according to standard procedures.

PBLs were prepared as described previously (5). Mouse spleen cell suspensions were prepared, and RBCs were lysed with ammonium chloride potassium as described previously (3). Cells were stained with PE-labeled anti-CD4 or CD8 mAbs vs FITC-labeled anti-V{alpha}11 mAb. Cells were analyzed by two-color FCM using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Nonviable cells were excluded using the vital nucleic acid stain propidium iodide. The percentage of cells that stained with a particular reagent or reagents was determined by subtracting the percentage of cells staining nonspecifically with the negative control mAb from those cells staining with the specific anti-mouse mAbs in the same dot-plot region.

For the three-color analysis of thymocytes, thymocyte suspensions were stained with biotin-labeled anti-CD8 mAb plus CyChrome-streptavidin (PharMingen), PE-labeled anti-CD4 mAb, and either FITC-labeled anti-V{alpha}11 mAb, FITC-labeled anti-CD24 mAb, FITC-labeled anti-Qa-2 mAb, or nonstaining HOPC-1 control mAb. To assay Vß3 usage among SP thymocytes, thymocytes were stained with biotin-labeled anti-CD4 mAb plus CyChrome-streptavidin, PE-labeled anti-Vß3 mAb, and FITC-labeled anti-CD8 mAb. A total of 5,000 to 10,000 gated CD4+CD8+ (double positive (DP)) or CD4+CD8- (CD4 SP) thymocytes were analyzed. The net percentages of cells that stained with a particular reagent were calculated by subtracting the percentage of the same gated cell population which stained with HOPC-1-FITC or rat IgG2a-PE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maturation of mouse thymocytes with a transgenic TCR in FP THY/LIV grafts

In TCR-transgenic AND mice, CD4+ T cells expressing a transgenic TCR (V{alpha}11Vß3) recognize pigeon cytochrome c peptide 88-104 in association with I-Ek- or I-Ab-encoded class II MHC molecules (9, 10). Thymocytes expressing this transgenic TCR are positively selected and become CD4 SP cells in mouse strains expressing MHC I-Ab or I-Ek. Transgenic T cells are not selected by I-Ak and are negatively selected by H-2s MHC molecules, to which the TCR cross-reacts (10, 15, 16).

We grafted SLAdd or SLAhh (both pig SLA types have the same class II MHC) FP THY/LIV tissue to ATX, T cell-depleted, TCR-transgenic AND mice with a positive-selecting MHC (H-2b) to determine whether TCR-transgenic mouse thymocytes could mature in FP THY/LIV grafts. As shown in Figure 1Go, A and B, ~10% of CD4 SP cells were detected in the FP THY/LIV grafts in ATX H-2b AND mice. This number was lower than that found in thymi of homozygous H-2b euthymic control transgenic mice, which contained ~30% transgenic CD4 SP cells (Fig. 1Go, A and B). The mean total number of thymocytes in FP THY/LIV grafts and normal AND thymi was 69.2 x 106 and 77.8 x 106, respectively. These results suggest that the maturation of mouse thymocytes with a transgenic TCR occurs in FP THY/LIV grafts, albeit somewhat less efficiently than is observed in the host thymi of I-Ab (positive-selecting) AND mice.



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  		FIGURE 1. Mouse thymopoiesis in FP THY grafts and the peripheral repopulation of mouse CD4+ cells bearing a transgenic TCR in FP THY/LIV grafted, T/NK cell-depleted, ATX AND mice. A, FCM analysis of a representative FP THY graft in an ATX, T/NK cell-depleted AND mouse and a euthymic control AND mouse thymus, showing subsets of mouse thymocytes. B, Results obtained from groups of six mice each are summarized. The mean ± SD of the total cell number of the indicated thymocyte subsets is shown for each group. Grafts were removed at 13 wk postimplantation. Cells were stained with PE-conjugated anti-mouse CD4 mAb and biotin-labeled anti-CD8 mAb plus CyChrome-streptavidin. C, Peripheral repopulation of CD4+ cells with transgenic TCR in ATX AND mice at 13 wk after grafting with FP THY/LIV (ATX AND FP THY/LIV), immunodeficient controls (ATX AND FP LIV), and in euthymic controls (EUTHYMIC AND CONTROL). Cells were stained with PE-conjugated anti-mouse CD4 mAb and FITC-conjugated anti-V{alpha}11 mAb. A total of 10,000 cells were analyzed from each sample. The number of animals included in the analysis for each group is shown in brackets. *p < 0.05 and **p < 0.01, compared among the indicated groups.

 
The maturation of CD4 SP cells in FP THY/LIV grafts resulted in the repopulation of TCR-transgenic mouse V{alpha}11+CD4+ cells in the peripheral blood, spleen, and lymph nodes of FP THY/LIV-grafted H-2b mice (Fig. 1GoC) at significantly higher levels than those observed in similarly treated ATX AND mice that received FP LIV without a thymic graft. These mouse CD4+ cells did not express CD8 (data not shown), excluding the possibility that immature DP cells repopulated the periphery. Very few CD8+ cells expressing the transgenic TCR were detected in the periphery (data not shown). Therefore, these results demonstrate that mouse class II MHC-restricted TCR-transgenic CD4+ T cells can mature in FP THY/LIV grafts and repopulate peripheral lymphoid tissues.

Pig MHC, but not mouse MHC, mediates positive selection of mouse thymocytes with a transgenic TCR in FP THY/LIV grafts

To investigate whether host mouse MHC molecules participated in the positive selection of transgenic AND CD4+ T cells in FP THY/LIV grafts, we compared their maturation in FP THY/LIV grafts in AND mice with positive selecting and nonselecting MHC backgrounds and in heterozygotes between the two. As shown for a representative animal in Figure 2Go, an average of 6.5% CD4 SP thymocytes were detected in the host thymi of I-Ab/k heterozygous mice, whereas ~30% of CD4 SP thymocytes were detected in the host thymi of I-Ab homozygous mice (Fig. 1Go). The decreased levels of CD4 SP cells in the thymi of heterozygous mice may be due to their lower levels of I-Ab expression, which may result in less efficient positive selection of the transgenic TCR. The host thymi of AND mice with a nonselecting MHC background (I-Ak, I-Eb) contained much lower percentages (mean ± SD: 3.3 ± 0.5%, n = 4) of CD4 SP cells than did the thymi of mice with the positive-selecting I-Ab or I-Ab/k MHC, consistent with previous results (10). However, a significantly greater percentage of CD4 SP thymocytes (mean ± SD: 9.0 ± 2.9%, n = 4; p < 0.05) was detected in FP THY/LIV grafts in ATX mice with nonselecting MHC compared with the host thymi of the euthymic nonselecting controls (Fig. 2Go). These percentages of CD4 SP cells obtained in the FP THY/LIV grafts of nonselecting AND mice were similar to those seen in the host thymi of heterozygous positive-selecting (I-Ab/k) mice (Fig. 2Go). The similar levels of CD4 SP cells in FP THY/LIV grafts placed in positive-selecting (I-Ab/k or I-Ab/b) and nonselecting (I-Ak/k) mice, which were significantly higher than those seen in the host thymi of euthymic nonselecting I-Ak/k mice (Fig. 2Go), indicate that positive selection was mediated entirely by pig MHC.



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  		FIGURE 2. Mouse thymopoiesis in FP THY grafts of positive-selecting or nonselecting MHC recipients. A FCM analysis of representative thymi in euthymic controls (top panels) and FP THY grafts (bottom panels) in ATX, T/NK cell-depleted AND mice with hemizygous-selecting (left panels) and nonselecting (right panels) MHC backgrounds is shown. The grafts were removed at 13 wk postimplantation. Cells were stained with PE-conjugated anti-mouse CD4 mAb and biotin-labeled anti-CD8 mAb plus CyChrome-streptavidin. Representative profiles from two separate experiments are shown.

 
The maturation of mouse T cells with transgenic TCR in FP THY/LIV grafts was further indicated by the efficient repopulation of mouse V{alpha}11+CD4+ cells in peripheral lymphoid tissues. As shown in Figure 3Go, there were much higher levels of mouse V{alpha}11+CD4+ cells in the PBLs and spleens of FP THY/LIV-grafted, T/NK cell-depleted, ATX mice with any of the three (I-Ab/b, I-Ab/k, and I-Ak/k) MHC backgrounds compared with FP LIV-grafted ATX mice, which had very low levels of V{alpha}11+CD4+ cells in the periphery. As expected, these peripheral CD4+ cells also expressed the transgenic Vß3, and there were very few CD8+ cells with transgenic TCR in the periphery (data not shown). Most importantly, the percentages of TCR-transgenic CD4+ cells in the periphery of I-Ab/b, I-Ab/k, and I-Ak/k mice receiving porcine thymic grafts were all similar to those in euthymic I-Ab/b and I-Ab/k mice and were significantly greater than those in euthymic I-Ak/k mice. Thus, the porcine thymus was solely responsible for the repopulation of these transgenic CD4+ cells in all strains of mice; no demonstrable contribution could be attributed to the presence of a selecting host mouse MHC.



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  		FIGURE 3. Repopulation of mouse CD4+ cells with transgenic TCR (V{alpha}11+) in the PBLs and spleens of FP THY/LIV-grafted ATX AND mice with a homozygous-selecting, heterozygous-selecting, or homozygous nonselecting MHC background. Tissues were harvested at 13 wk after grafting with FP THY. Cells were stained with PE-conjugated anti-mouse CD4 mAb and FITC-conjugated anti-V{alpha}11 mAb. A total of 10,000 cells were analyzed for each sample. A summary of two separate experiments is shown (three to eight mice per group). *p < 0.05, compared with both FP LIV-grafted ATX mice and euthymic nonselecting control mice.

 
Phenotypic maturation of mouse T cells bearing a transgenic TCR in FP THY/LIV grafts

To determine whether the presence of a selecting host MHC influences the pattern of transgenic T cell maturation in porcine thymus grafts, we examined the levels of expression of HSA, Qa-2, and TCR on CD4 SP cells in FP THY/LIV grafts. During the development of DP thymocytes into functional CD4 SP cells, positive selection by class II MHC molecules is required; such selection is intimately connected to the terminal differentiation of SP cells and to the acquisition of function (6, 17). The terminal differentiation of SP cells is associated with down-regulation of HSA expression and up-regulation of TCR, class I MHC, and Qa-2 (18-21). As shown in Figure 4Go, CD4 SP cells developing in FP THY/LIV grafts in mice with selecting or nonselecting MHCs showed a similar pattern of expression of transgenic TCR (V{alpha}11), Qa-2, and HSA. The proportion of mouse CD4 SP cells expressing the most mature TCRhigh phenotype in FP THY/LIV grafts was higher than that seen in the host thymi of mice with a nonselecting H-2, confirming that pig MHC supports the positive selection of AND transgenic TCR-bearing T cells with greater efficiency than nonselecting I-Ak/k mice. However, there was a lower proportion of CD4 SP cells expressing the most mature Qa-2high and HSAlow phenotype in FP THY/LIV grafts in mice of either MHC genotype compared with the host thymi of mice with a homozygous positive-selecting H-2. Overall, these results support the possibility that porcine MHC supports the positive selection of AND transgenic T cells more efficiently than nonselecting mouse MHC, but with somewhat lesser efficiency than the homozygous-selecting I-Ab/b thymi. Since similar maturation patterns of mouse CD4 SP cells were detected in FP THY/LIV grafts in ATX AND mice with positive-selecting and nonselecting MHC backgrounds, these results confirm that host mouse MHC is not involved in the positive selection of AND mouse thymocytes in FP THY/LIV grafts.



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  		FIGURE 4. The expression of V{alpha}11, Qa-2, and HSA on mouse CD4 SP cells in the thymic grafts of FP THY/LIV-grafted mice (I-Ab/b and I-Ak/k) and in the thymi of euthymic control mice (I-Ab/b and I-Ak/k). The grafts were removed at 13 wk postimplantation. Cells were stained as described in Materials and Methods. A total of 5,000 to 10,000 gated CD4 SP thymocytes were analyzed. Representative profiles from two separate experiments (n = 4 per group) are shown. The open histogram indicates nonspecific control mAb (HOPC-1) staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The repertoire of mature T lymphocytes emerging from the thymus is shaped by both positive and negative selection, which is mediated by interactions between MHC molecules expressed on stromal and hematopoietic cells and Ag-specific receptors on T cells (6, 22-24). Thymocytes with receptors that presumably recognize self peptides in association with self MHC molecules in the thymus with low affinity are rescued from programmed cell death and permitted to differentiate further (i.e., they are positively selected), whereas others with too low an affinity for self MHC/peptide complexes fail to be positively selected and undergo death by neglect. Other thymocytes, apparently those with receptors that recognize host MHC/peptide ligands with high affinity, undergo programmed cell death or negative selection, resulting in self tolerance (25-27). Accumulating studies suggest that class II MHC molecules plus peptides expressed on epithelial cells in the thymic cortex are responsible for the positive selection of CD4 cells and imprint the MHC restriction of mature T cells, while MHC molecules on bone marrow-derived dendritic cells and other cell types mediate negative selection and deletional tolerance (28-32). In addition, thymic epithelial cells are also capable of inducing tolerance by deletional and nondeletional mechanisms (33-39).

In our FP THY/LIV-grafted, T/NK cell-depleted, ATX mice (3), efficient cellular immunity is reconstituted; this reconstitution is indicated by allogeneic skin graft rejection and MLR responses (3, 5), by the ability to clear opportunistic infection, and by a host MHC-restricted responsiveness to peptide Ag (4). Two possibilities could explain this robust, host-restricted responsiveness: first, in contrast to results in homologous situations, host hemopoietic cells or other cell types might participate in positive selection within xenogeneic porcine thymic grafts and imprint T cells with host restriction specificity. Consistent with this possibility, we can routinely detect mouse class II+ cells in porcine THY/LIV tissue that has been grafted into ATX mice (5). Alternatively, pig MHC alone might mediate positive selection, but the cross-reaction between pig and mouse MHC may be sufficiently strong to allow effective mouse-restricted immune responses to occur. The studies reported herein were designed to distinguish between these two possibilities.

The results of our studies in FP THY/LIV grafted, TCR-transgenic AND mice indicate that mouse MHC does not play a role in the positive selection of CD4 T cells in porcine thymic grafts. Similar levels of mouse CD4 SP cells with similar TCR expression were detected in FP THY/LIV grafts in ATX AND mice with positive selecting and nonselecting MHC backgrounds (Fig. 2Go), and these levels were significantly higher in both groups compared with the host thymi of mice with nonselecting MHC backgrounds. Consistent with the thymocyte results, a similar repopulation of CD4+ cells expressing transgenic TCR was detected in the peripheral lymphoid tissues (PBLs and spleen) of T cell-depleted, ATX AND mice that received FP THY/LIV grafts, regardless of the H-2 type of the recipients. The levels of repopulating V{alpha}11+CD4+ cells in FP THY/LIV-grafted, ATX AND mice with either I-Ab/b, I-Ab/k, or I-Ak/k genotypes were markedly higher than those in ATX AND H-2b mice that received FP LIV only and were also higher than those in euthymic nonselecting AND I-Ak mice (p < 0.05). These results indicate that the peripheral CD4+ cells in FP THY/LIV-grafted ATX mice are not merely nondepleted, preexisting T cells, but are instead T cells that arise in the grafted porcine thymi, in which transgenic T cells at all stages of maturation can be detected (Figs. 1Go and 2Go). Together, our results demonstrate that donor pig MHC mediates the positive selection of mouse thymocytes in FP THY/LIV grafts with no demonstrable contribution from the host mouse MHC.

The similar levels of maturation of mouse CD4+ cells in FP THY/LIV grafts (i.e., up-regulation of transgenic TCR and Qa-2 and down-regulation of HSA expression) that were detected on subpopulations of CD4 SP thymocytes in FP THY/LIV grafts in mice of all MHC genotypes (Fig. 4Go) confirm that host MHC does not contribute to thymocyte selection in porcine THY/LIV grafts. In FP THY/LIV grafts in mice with all MHC genotypes, the level of terminal maturation of these cells was less than that observed in the host thymi of mice with a selecting MHC, perhaps reflecting a less avid interaction of the AND TCR with porcine MHC than with I-Ab. Consistent with this possibility, pig MHC positively selects the AND transgenic TCR with somewhat lesser efficiency than homozygous I-Ab thymi. While cells with this particular TCR may not mature efficiently in porcine grafts, our previous studies have shown that porcine MHC is capable of selecting a fully differentiated polyclonal mouse T cell repertoire that has excellent function (4).

Our previous studies comparing class II MHC-deficient and wild-type mice suggested that host mouse class II MHC molecules, when present, regulate the terminal maturation of CD4 SP cells in the porcine thymic microenvironment (8). The similarity of the maturation levels of mouse CD4 SP cells in FP THY/LIV grafts that was observed in recipients with positive- selecting or nonselecting MHC in the present studies (Fig. 4Go) suggests that this role for mouse class II MHC may not require a specific recognition of that MHC molecule by the TCR but may instead be dependent upon homologous CD4/class II MHC interactions.

In our view, it is highly unlikely that the apparent positive selection of TCR-transgenic CD4 SP cells in FP THY/LIV grafts merely reflects a failure of negative selection in these grafts. We have obtained several lines of clear evidence that both pig and mouse MHC are capable of efficient negative selection of mouse thymocytes in FP THY/LIV grafts. Evidence that pig MHC mediates negative selection includes the demonstration of intrathymic deletion of mouse CD4 SP cells that use Vß in response to the presentation of mouse endogenous superantigens by porcine MHC (3, 5, 8). The demonstration of this phenomenon in class II-deficient mouse recipients of FP THY/LIV grafts (8) is irrefutable proof that pig MHC can mediate such negative selection.

Our more recent studies have demonstrated that the grafting of FP THY alone to T/NK cell-depleted ATX B6 mice is sufficient to support mouse thymopoiesis and the peripheral repopulation of mouse CD4+ cells; the coimplantation of porcine fetal liver tissue is not required (Y.Z. and M.S., unpublished observations). Since porcine hematopoietic cells have been detected in long-term pig thymus tissue grafted without an additional source of pig hematopoietic cells, these cells may induce deletional tolerance, as has been observed for recipients of pig THY/LIV implants (5).

Evidence that the mouse class II+ cells detected in FP THY/LIV grafts participate in negative selection includes the more complete deletion of these Vß that is detected when the recipient mouse expresses the I-E molecules which can present endogenous superantigens in comparison with the deletion observed when these Ags can only be picked up and presented by porcine MHC (3, 5). Furthermore, we have recently demonstrated that AND TCR-bearing cells are efficiently deleted at the DP stage when they develop in FP THY/LIV grafts in AND transgenic mice with negatively selecting H-2s MHC (Y.Z. and M.S., manuscript in preparation). Finally, the observed tolerance to donor and host Ags (3-5) in mice in which CD4 cells have matured in FP THY/LIV grafts is further evidence that negative selection occurs efficiently.

The present studies were performed to address the question of why mouse T cells maturing in FP THY/LIV grafts show excellent host mouse MHC-restricted responsiveness in wild-type mice (4). Based on the above results showing the ability of porcine MHC to positively select mouse transgenic T cells with a known mouse MHC-restricting element, we conclude that the mouse T cell repertoire is positively selected exclusively by porcine MHC elements in FP THY/LIV grafts, but that this repertoire has sufficient cross-reactivity with host MHC/foreign peptide complexes to confer a high level of host MHC-restricted immunocompetence. It has recently been reported that large numbers of polyclonal thymocytes can be positively selected by a single MHC/peptide ligand, and that many of these maturing T cells react with the selecting MHC bound to other peptides and also react frequently with allogeneic MHC molecules (40). Furthermore, one recent study suggested that MHC reactivity is inherent in randomly rearranged TCR, independent of positive and negative thymic selection (41). Our results are consistent with both of these studies, and further suggest that the germline-encoded TCR components and MHC ligands with which these components interact are well-conserved between species.

Our observation that the MHC of the donor thymus is entirely responsible for positive selection is consistent with previous studies (29-32), which have led to the conclusion that the T cell repertoire is "restricted" to recognize peptides presented by MHC molecules shared by the thymic epithelium. However, while these studies showed a skewing of the repertoire toward a recognition of peptides in the context of host MHC (42-44), our studies (4) and those involving allogeneic thymic grafting in humans with congenital thymic aplasia (45) suggest that restriction is an overly exclusive term that might be better replaced by the term "skewing" of the repertoire. While our studies demonstrate the exclusive role of the thymic epithelium in positive selection in a highly disparate xenogeneic species combination, they show that a T cell repertoire generated by an MHC of one species is sufficiently cross-reactive on the MHC of another disparate species to confer excellent immunocompetence (4). The observed role of both donor and host MHC in the negative selection of this repertoire (3, 5, 8) may be most critical for the development of a competent T cell repertoire that is tolerant of both donor and host. The ability to develop such a repertoire suggests a novel approach to achieving donor-specific discordant xenograft tolerance.

Consistent with our results, a patient with complete DiGeorge syndrome receiving an HLA-mismatched allogeneic thymus graft showed recovery of CD4+ cells and normal Ab responses to tetanus toxoid and pneumococcal vaccine immunization, suggesting successful immune restoration (45). This restoration occurred despite the MHC incompatibility between the donor thymus and recipient APCs, similar to our results in pig THY/LIV grafted mice. These results support the possibility of using xenogeneic thymic grafts to restore a functional T cell repertoire that is tolerant of both the discordant xenogeneic donor and the host in patients who suffer from congenital or acquired immunodeficiencies associated with thymic dysfunction. Since a direct attack of HIV on the human thymus may result in the degeneration of the thymic epithelium (46-48), thymic allografting has recently attracted attention as a possible component of strategies to achieve immune restoration in HIV-infected persons (49). However, if this approach is successful, it is unlikely that the available human thymic tissue would be sufficient to meet the need of the HIV-infected population. Therefore, xenogeneic thymic replacement, which has the potential advantages of being available in unlimited quantities from donors of an optimal gestational age and of being potentially resistant to HIV-infection, might provide an important element that is needed for the achievement of immune restoration in patients receiving virus-suppressive therapies.


   Acknowledgments
 
We thank Drs. Boris Nikolic, John Iacomini, and Henry Winn for critically reviewing the manuscript. We also thank Vantran Tru for outstanding animal husbandry and Diane Plemenos for expert assistance in preparing the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant PO1-AI39755 and by a sponsored research agreement between Massachusetts General Hospital and BioTransplant, Inc. Y.Z. was supported in part by the Massachusetts General Hospital Fund for Medical Discovery. M.S. is a consultant to Biotransplant Inc. Back

2 Address correspondence and reprint requests to Dr. Megan Sykes, Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Building 149-5102, 13th Street, Boston, MA 02129. Back

3 Abbreviations used in this paper: FP THY/LIV, fetal pig thymus and liver; ATX, thymectomized; DP, double positive; FCM, flow cytometry; PE, phycoerythrin; SLA, swine leukocyte Ag; SP, single positive; HSA, heat stable Ag. Back

Received for publication January 21, 1998. Accepted for publication April 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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