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Induction of Alloreactive CD4 T Cell Tolerance in Molecular Chimeras: A Possible Role for Regulatory T Cells¹

Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K^b were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K^b, albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K^b-transduced bone marrow permanently accepted K^b disparate skin grafts. CD4⁺CD25⁺ T cells from mice reconstituted with H-2K^b-transduced bone marrow prevented rejection of K^b disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.

	Introduction

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Induction of donor-specific transplantation tolerance has the potential to eliminate the requirement for life-long administration of immunosuppressive drugs and the serious side effects that are associated with their use (1, 2, 3). Historically, one proven way to induce donor-specific tolerance is the use of allogeneic bone marrow transplantation to establish a state of donor-recipient mixed hemopoietic chimerism (4). Unfortunately, this approach is associated with complications such as graft-versus-host disease (5) and the incomplete recovery of immunocompetence (6, 7), which severely limit the application of allogeneic bone marrow transplantation as an approach to induce donor-specific tolerance. Moreover, it has been shown that induction of mixed chimerism through allogeneic bone marrow transplantation may not reliably prevent chronic rejection (8).

We have previously shown that tolerance to a variety of Ags, including MHC Ags, can be achieved by reconstituting conditioned hosts with genetically modified autologous hemopoietic stem cells resulting in a state of molecular rather than mixed cellular chimerism (9, 10, 11, 12). In transplantation studies, reconstitution of lethally irradiated B10.AKM mice with syngeneic bone marrow cells infected with retroviruses carrying the allogeneic MHC class I gene H-2K^b resulted in stable and lifelong expression of K^b on bone marrow-derived cells (9). These K^b molecular chimeras were shown to be specifically tolerant to K^b and permanently accepted K^b disparate skin grafts (9). Importantly, expression of a retrovirally transduced MHC Ag in bone marrow-derived cells resulted in the same robust tolerance to allogeneic skin grafts that is achieved by inducing a state of mixed cellular chimerism, while completely eliminating the possibility of graft-versus-host disease (13). More recently, we have shown that molecular chimerism can be induced in hosts receiving nonmyeloablative conditioning and similarly results in tolerance to K^b disparate skin grafts (14).

Both CD8 and CD4 T cells are able to reject MHC class I disparate skin grafts (15, 16). Therefore, in order for molecular chimeras to accept K^b disparate skin grafts, both CD8 and CD4 T cells that recognize K^b through the direct and indirect pathways must be tolerized. Using TCR transgenic BM3.3 mice (17), we have previously shown that developing alloreactive CD8 T cells, which recognize K^b through the direct pathway, undergo negative selection in the thymus of molecular chimeras expressing K^b on bone marrow-derived cells (18). To examine mechanisms responsible for alloreactive CD4 T cell tolerance in K^b molecular chimeras, we used Tg361 TCR transgenic mice that express an alloreactive TCR on CD4 T cells specific for K^b-derived peptides presented through the indirect pathway (19). T cells expressing the Tg361 TCR were readily detected in mice expressing the retrovirally transduced K^b gene in bone marrow-derived cells, albeit at lower levels than observed in controls. Despite the presence of these potentially alloreactive CD4 T cells in the periphery, molecular chimeras permanently accepted K^b disparate skin grafts, suggesting that alloreactive CD4 T cells may be controlled by regulatory T (Treg)⁴ cells that develop in these mice. Using an adoptive cell transfer model, we observed that CD4⁺CD25⁺ T cells from K^b molecular chimeras were able to prevent rejection of K^b disparate skin grafts by naive effector T cells. These results suggest that induction of molecular chimerism leads to the generation of donor-specific Treg cells that may be involved in the maintenance of tolerance.

	Materials and Methods

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Mice

Female B10.AKM/SnJ (H-2K^k, I^k, D^q), B10.MBR (H-2K^b, I^k, D^q), B10.BR (H-2^k), BALB/c (H-2^d), CBA/CaJ (H-2^k), and Rag-1-deficient CBA/Ca mice were purchased from The Jackson Laboratory. Tg361 TCR transgenic mice (H-2^k, CBA/Ca background) (19) and CBK mice (20) were provided to us by Dr. A. L. Mellor (Medical College of Georgia, Augusta, GA) and bred in our facility. CBK MHC class I transgenic mice express K^b on the CBA/Ca background. Rag-1-deficient mice on the B10.AKM background were generated in our facility as described (21). Mice were housed using microisolator conditions in autoclaved cages and maintained on irradiated feed and autoclaved acidified drinking water. All sentinel mice housed in the same colony were viral Ab-free. Six- to 12-wk-old female mice were used in all experiments. These studies have been reviewed and approved by Harvard Medical School, Center For Animal Resources and Comparative Medicine (Boston, MA).

Retroviruses

Construction and production of the vesicular stomatitis virus (VSV) G protein enveloped retroviruses carrying the gene encoding H-2K^b (VSV-Kb) used in this study is previously described (9). All virus preparations were made in affiliation with the Harvard Institute for Human Genetics Gene Therapy Initiative (Boston, MA). The viral titer obtained for the preparation of VSV-Kb used in this report was 2 x 10⁶ infectious particles per milliliter.

Retroviral transduction of bone marrow cells

Bone marrow cells were harvested from B10.AKM mice and transduced as previously described (9, 10). Briefly, bone marrow cells from mice treated 7 days prior with 5-fluorouracil (150 mg/kg) were cultured in Retronectin (Takara Biomedicals)-coated 6-well nontissue culture plates in transduction medium (DMEM containing 15% lot-tested FCS and cytokines to achieve a final concentration of 100 ng/ml human IL-6 (R&D Systems), 100 ng/ml recombinant mouse stem cell factor (BioSource International), 50 ng/ml recombinant mouse thrombopoietin (R&D Systems), and 50 ng/ml recombinant mouse Flt-3 ligand (R&D Systems)). Retronectin-coated plates were prepared according to the manufacturer’s instructions. All transductions were performed at 37°C with 5% CO₂ for 96 h. Bone marrow cells were cultured at a density of 10⁷ cells/ml together with VSV-Kb to achieve a multiplicity of infection of 1. After 24 h, a second dose of viral supernatant was added to the cultured bone marrow cells. Viral supernatant and transduction media were replaced 72 h after the start of the transduction. Mock transductions were performed in the same manner, except transduction media were used instead of viral supernatant. Twenty-four hours later, the cells were harvested, washed twice in HBSS, and counted.

Bone marrow transplantation

Tg361 bone marrow was harvested from mice treated 7 days prior with 5-fluorouracil as described. All Tg361 bone marrow donors also received a depleting dose of anti-CD8 (116-13-1; 1.5 mg/mouse) and anti-CD4 (GK1.5; 0.2 mg/mouse) on days –4 and –1 relative to bone marrow harvest to deplete alloreactive T cells in vivo. One day before bone marrow transplantation, mice received 10.25 Gy of irradiation. On the day of reconstitution, recipients were injected i.v. with a 6:1 ratio of mock- or K^b-transduced B10.AKM bone marrow cells and freshly isolated Tg361 bone marrow cells along with a mix of mAbs (1.5 mg of 116-13-1 (anti-CD8), 2 mg of 2.43 (anti-CD8), 0.2 mg of GK1.5 (anti-CD4), and 0.2 mg of MR1 (anti-CD40L)) to eliminate host alloreactive T cells in vivo. We used two different anti-CD8 Abs because B10.AKM mice express the Ly2.2 allele of CD8, whereas CBA/Ca mice express the Ly-2.1 allele, thus allowing us to deplete mature CD8 T cells from both mouse strains. Finally, mice received additional injections of anti-CD40L (MR1; 0.2 mg/mouse) and anti-CD4 (GK1.5; 0.2 mg/mouse) on days +4 and +7 after reconstitution.

Abs and flow cytometry

All cell surface staining and flow cytometry were performed as previously described (15, 22). Abs specific for H-2K^b (AF6–88.5), H-2D^q (KH117), V10 TCR (B21.5), CD4 (L3T4), CD8 (52-6.7), CD11b (Mac-1, M1/70), Gr-1 (RB6-8C5), CD11c (HL3), CD25 (PC61), TCR -chain (H57-597), and B220 (RA3-6B2) were obtained from BD Pharmingen.

Adoptive cell transfers

B10.AKM mice were given 10.25 Gy of whole body irradiation and reconstituted 1 day later with 4 x 10⁶ VSV-Kb-transduced B10.AKM bone marrow cells. At 12–16 wk later, populations of CD25^–, CD4⁺, CD4⁺CD25^–, or CD4⁺CD25⁺ splenocytes were purified from these mice using MACS (Miltenyi Biotec). A total of 5 x 10⁵ cells from each of these populations were than adoptively transferred into RAG.B10.AKM mice (Rag-1 knockout mice on the B10.AKM background) along with 10⁷ B10.AKM splenocytes from untreated mice. One day after adoptive transfer of cells, mice received B10.MBR and third party B10.BR skin grafts. In some experiments, T cell populations described were isolated from mice reconstituted with either VSV-Kb- or mock-transduced bone marrow and adoptively transferred into Rag-1-deficient CBA/Ca mice along with CBA/CaJ splenic effectors. The following day, these mice received CBK and third party BALB/c skin grafts.

Skin grafting

Tail skin grafting was performed and evaluated as previously described (15).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Development of a TCR transgenic model to examine the fate of alloreactive CD4 T cells in molecular chimeras

Tg361 TCR transgenic mice express an alloreactive TCR on CD4 T cells specific for K^b-derived peptides that are presented through the indirect pathway of Ag presentation in the context of I-A^k (19). We therefore used Tg361 mice to study the fate of CD4 T cells that recognize alloantigen through the indirect pathway in mice reconstituted with H-2K^b-transduced bone marrow. Bone marrow was harvested from B10.AKM mice treated 7 days prior with 5-fluorouracil (150 mg/kg i.v.) and either transduced with VSV G protein enveloped retroviruses carrying the gene encoding H-2K^b (VSV-Kb) or mock-transduced as described (9). B10.AKM recipients were then conditioned with lethal irradiation and a depleting dose of anti-CD4 and anti-CD8 mAbs as described (18). Conditioned mice were then reconstituted the following day with a 6:1 ratio of either VSV-Kb- or mock-transduced B10.AKM (3.0 x 10⁶ cells) and bone marrow cells from Tg361 mice (0.5 x 10⁶ cells) treated with anti-CD8 (116-13-1) and anti-CD4 (GK1.5) mAbs before bone marrow harvest to deplete mature alloreactive T cells in vivo (>99% depletion of CD4 and CD8 T cells) (data not shown). Recipient mice were also treated with anti-CD40L (MR1; 0.2 mg/mouse) and anti-CD4 (GK1.5; 0.2 mg/mouse) Abs on days +4 and +7 after reconstitution to prevent rejection of K^b expressing cells.

Induction of molecular chimerism in mice reconstituted with a mixture of VSV-Kb-transduced B10.AKM and Tg361 bone marrow

Bone marrow-derived cells expressing K^b on their surface were detectable in the blood of mice receiving Tg361 and VSV-Kb-transduced B10.AKM bone marrow at all time points analyzed over a 23 wk follow-up period (Fig. 1A). The percentage of cells expressing K^b in the blood remained stable over time. Expression of K^b was detected on T cells (CD4⁺, CD8⁺), B cells (B220⁺), macrophages (CD11b⁺), granulocytes (Gr-1⁺), and dendritic cells (CD11c⁺) (Fig. 1B). Similar frequencies of K^b-expressing cells were also detected in peripheral lymphoid tissues (data not shown). As expected, cells expressing K^b were not detected in control mice receiving Tg361 and mock-transduced B10.AKM bone marrow (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Expression of Kb in mice reconstituted with VSV-Kb-transduced bone marrow. A, The frequency of lymphocytes expressing Kb in the blood of mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced () (n = 15) or mock-transduced () (n = 15) B10.AKM and Tg361 bone marrow are shown over a 24-wk follow-up period. Data shown are the cumulative mean values with SD obtained from three separate experiments. B, Expression of Kb on hemopoietic cell lineages in the blood of molecular chimeras. The frequency of CD11b+, CD8+, CD4+, CD11c+, B220+, and Gr1+ cells expressing Kb on their surface in mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced () (n = 9) or mock-transduced () (n = 8) B10.AKM and Tg361 bone marrow 10 wk after transplantation is shown. Data shown are the cumulative mean values with SD obtained from three separate experiments.

We next set out to examine the fate of CD4 T cells that recognize K^b through the indirect pathway in molecular chimeras. An anti-clonotypic Ab specific for the Tg361 TCR is not available. Therefore, to analyze development of Tg361 bone marrow-derived alloreactive CD4 T cells by flow cytometry in reconstituted mice, we took advantage of the MHC class I difference between B10.AKM and Tg361 mice. Tg361 mice are on the CBA/Ca background. Therefore, T cells expressing the Tg361 TCR are D^q–V10⁺CD4⁺, whereas B10.AKM T cells are D^q+.

Tg361-derived D^q–V10⁺CD4⁺ T cells were readily detectable in the peripheral blood of B10.AKM mice that received mock-transduced B10.AKM and Tg361 bone marrow (Fig. 2A). Approximately 40% of CD4 T cells in the blood of these mice were derived from the Tg361 bone marrow 4 wk after reconstitution (Fig. 2, A and B). The frequency of Tg361-derived CD4 T cells in the blood of these mice increased over the next 4 wk and then became stable (Fig. 2B). Interestingly, we were also able to detect D^q–V10⁺CD4⁺ T cells derived from Tg361 bone marrow in the blood of mice receiving VSV-Kb-transduced bone marrow (Fig. 2A). Approximately 30–40% of CD4 T cells in these mice were derived from Tg361 bone marrow in mice expressing K^b on bone marrow-derived cells (Fig. 2B). Tg361-derived CD4 T cells were detectable at all time points analyzed, albeit at lower levels than observed in mice receiving mock-transduced B10.AKM bone marrow (Fig. 2B, p < 0.05 at all time points after 4 wk). CD4 T cells expressing the Tg361 TCR were also present in the lymphoid tissues of mice receiving VSV-Kb-transduced bone marrow (Fig. 2C). As observed in the blood, although Tg361 bone marrow-derived D^q–V10⁺CD4⁺ T cells were readily detectable in these mice, the overall frequency was less than observed in controls receiving mock-transduced B10.AKM bone marrow (Fig. 2C). These results suggest that expression of the retrovirally transduced K^b gene on bone marrow-derived cells does not prevent the development of alloreactive CD4 T cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Development of T cells expressing the Tg361 TCR in molecular chimeras expressing Kb on bone marrow-derived cells. A, Analysis of Dq–V10+CD4+ T cells in the blood of Tg361 and B10.AKM controls (top) as well as mice reconstituted with either mock-transduced or VSV-Kb-transduced bone marrow (bottom). Expression of Dq and V10 on the cell surface after gating on CD4+ cells is shown. Dead cells were gated out of the analysis. Data are representative of three separate experiments for mice 8–10 wk after bone marrow transplantation. B, Long-term survival of CD4 T cells expressing the Tg361 TCR in blood. B10.AKM mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced () (n = 15) or mock-transduced () (n = 15) B10.AKM and Tg361 bone marrow were bled starting at 4 wk after bone marrow transplantation, and development of T cells expressing the Tg361 TCR was analyzed by cell surface staining and flow cytometry. The percentage of CD4 T cells expressing the Tg361 TCR (Dq–V10+) is shown. Data shown are the cumulative mean values and SD obtained from three separate experiments. C, Presence of Tg361 CD4 T cells in various tissues of mice receiving VSV-Kb-transduced bone marrow is shown. B10.AKM mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced () (n = 4) or mock-transduced () (n = 4) B10.AKM and Tg361 bone marrow were sacrificed 14 wk after bone marrow transplantation. Various tissues were examined for the presence of CD4 T cells expressing the Tg361 TCR by cell surface staining and flow cytometry. Statistical difference (*, p < 0.05) is indicated. Data are representative of three separate experiments. D, The majority of T cells expressing the Tg361 TCR in molecular chimeras are CD25–. The frequency of Tg361 T cells expressing CD25 in the blood of mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced (dashed lines) (n = 15) or mock-transduced (solid lines) (n = 15) B10.AKM and Tg361 bone marrow over a 24 wk follow-up period is shown.

Incomplete deletion of Tg361 CD4 T cells in the thymus of molecular chimeras

The presence of CD4 T cells expressing the Tg361 TCR in the periphery of molecular chimeras suggested that expression of K^b on bone marrow-derived cells failed to negatively select alloreactive T cell in the thymus. To further examine this possibility, mice reconstituted with a mixture of either VSV-Kb- or mock-transduced B10.AKM and Tg361 bone marrow were sacrificed 10 wk after bone marrow transplantation, and development of CD4 T cells expressing the Tg361 TCR in the thymus was examined by cell surface staining and flow cytometry. CD4⁺CD8⁺ double positive T cells and CD4⁺ single positive T cells expressing the Tg361 TCR were readily detected in the thymus of mice receiving a mixture of mock-transduced B10.AKM and Tg361 bone marrow cells (Fig. 3). Interestingly, we were also able to detect CD4⁺CD8⁺ double positive T cells and CD4⁺ single positive T cells expressing the Tg361 TCR in mice receiving a mixture of VSV-Kb-transduced B10.AKM and Tg361 bone marrow cells (Fig. 3), even though bone marrow-derived cells expressing K^b were present in the thymus (Fig. 1B). The absolute number of CD4⁺CD8⁺ double positive T cells expressing the Tg361 TCR in mice receiving VSV-Kb-transduced (1.9 x 10⁷± 5.6 x 10⁶, n = 7) and mock-transduced (2.3 x 10⁷± 6.6 x 10⁶, n = 5) bone marrow was similar (p = 0.21 between groups). However, the absolute number of single positive CD4⁺ T cells expressing the Tg361 TCR was reduced in mice receiving VSV-Kb-transduced bone marrow (3.5 x 10⁶± 1.2 x 10⁶, n = 7) when compared with controls (5.6 x 10⁶± 2.2 x 10⁶ n = 5) (p = 0.05 between groups). These data suggest that T cells expressing the Tg361 TCR are able to escape negative selection in the thymus of reconstituted mice.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Analysis of Tg361 CD4 T cell development in the thymus. Mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced (right) or mock-transduced (left) B10.AKM and Tg361 bone marrow were sacrificed 10 wk after bone marrow transplantation, and development of T cells expressing the Tg361 TCR in the thymus were analyzed by cell surface staining and flow cytometry. Expression of CD4 and CD8 on thymocytes of reconstituted mice after gating on Dq–V10+CD4+ cells is shown. The values indicated are the absolute number of each population in the thymi of representative mice.

To examine whether molecular chimeras containing CD4 T cells expressing the Tg361 TCR were tolerant to K^b, we analyzed the ability of these mice to accept H-2K disparate skin allografts. Groups of mice receiving a mixture of either VSV-Kb- or mock-transduced B10.AKM and Tg361 bone marrow received both K^b disparate B10.MBR and third party BALB/c (H-2^d) skin grafts 8–12 wk after reconstitution. Control mice receiving a mixture of mock-transduced B10.AKM and Tg361 bone marrow quickly rejected their B10.MBR skin grafts (median survival time (MST) = 13 days, n = 8) (Fig. 4). In contrast, mice receiving a mixture of VSV-Kb-transduced B10.AKM and Tg361 bone marrow permanently accepted their B10.MBR skin grafts (MST >141 days, n = 9). Mice in both groups rapidly rejected third party BALB/c skin grafts (MST <15 days for both groups, data not shown) demonstrating that the mice remained immunocompetent. These results suggest that molecular chimeras containing alloreactive Tg361 derived CD4 T cells are functionally tolerant to K^b.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Permanent survival of Kb disparate skin grafts on mice receiving VSV-Kb-transduced bone marrow. B10.AKM mice reconstituted with a 6:1 mixture of either VSV-Kb-transduced () (n = 9) or mock-transduced () (n = 8) B10.AKM and Tg361 bone marrow received B10.MBR and third party BALB/c skin grafts 8–12 wk after bone marrow transplantation. Third party skin grafts were rejected with a MST <15 days by mice in both groups (data not shown). Shown are the combined results of allograft survival from three independent experiments.

The presence of CD4 T cells expressing the Tg361 TCR in mice that are functionally tolerant to K^b led us to hypothesize that CD4 T cell tolerance following the induction of molecular chimerism may involve the generation of Treg cells. B10.AKM mice were lethally irradiated and reconstituted with VSV-Kb-transduced bone marrow to generate molecular chimeras as previously described (9). Twelve to sixteen weeks later, the mice were sacrificed and splenocytes harvested. We then purified CD25^–, CD4⁺, CD4⁺CD25^–, or CD4⁺CD25⁺ splenocytes and adoptively transferred 5 x 10⁵ cells from each population separately intoRAG-1-deficient B10.AKM mice along with 10⁷ B10.AKM splenocytes. The following day, mice received both a B10.MBR and third party B10.BR skin graft. Mice receiving B10.AKM splenocytes alone rejected their B10.MBR skin grafts (MST = 28 days, n = 10) (Fig. 5A). Adoptive transfer of CD25^– (n = 8) or CD4⁺CD25^– (n = 10) T cells along with B10.AKM splenocytes had no effect on rejection of B10.MBR skin grafts (MST = 26 and 33.5 days, respectively) (Fig. 5A). Adoptive transfer of purified CD4⁺ T cells along with B10.AKM splenocytes slightly prolonged survival of B10.MBR skin grafts (MST = 36 days, n = 9) (Fig. 5A). The prolonged survival observed, although modest, was statistically significant (p = 0.019) when compared with survival in mice receiving B10.AKM splenocytes alone. Mice receiving CD4⁺CD25⁺ T cells together with B10.AKM splenocytes accepted their B10.MBR skin grafts indefinitely (MST >110 days, n = 5). Mice receiving CD25^–, CD4⁺, or CD4⁺CD25^– rejected their third party B10.BR skin grafts with MST <30 days (data not shown). Mice that received CD4⁺CD25⁺ T cells along with B10.AKM splenocytes rejected their B10.BR skin grafts within 52 days.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. CD4+CD25+ T cells from mice reconstituted with VSV-Kb-transduced bone marrow prevent the rejection of Kb disparate skin grafts. A, RAG-1-deficient B10.AKM mice received 107 naive B10.AKM splenocytes either alone or together with 5 x 105 CD25–, CD4+, CD4+CD25–, or CD4+CD25+ splenocytes from B10.AKM mice reconstituted with 4 x 106 VSV-Kb-transduced bone marrow cells 12–16 wk prior. One day after adoptive transfer of cells, mice received B10.MBR and third party B10.BR skin grafts. Shown are the combined results of allograft survival from two independent experiments. B, RAG-1-deficient CBA/Ca mice received 107 naive CBA/Ca splenocytes either alone () or together with 5 x 105 CD4+CD25+ from B10.AKM mice reconstituted with either VSV-Kb-transduced () or mock-transduced (•) bone marrow cells 8–12 wk prior. One day after adoptive transfer of cells, mice received Kb disparate CBK skin grafts. Shown are the combined results of allograft survival from two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Induction of molecular chimerism following reconstitution of conditioned recipients with genetically modified autologous or syngeneic bone marrow has been show to result in robust tolerance toward a variety of Ags (13). Defining the mechanisms that lead to tolerance following the establishment of molecular chimerism is critical in terms of predicting the stability of the tolerance induced. The establishment of central tolerance via negative selection in the thymus is perhaps the most robust form of transplantation tolerance because Ag-reactive T cell clones are eliminated from the repertoire. In contrast, tolerance resulting from the induction of T cell anergy or the generation of Treg cells has the potential of being broken because alloreactive T cells are not eliminated, but rather controlled, in a way that results in long-term transplant survival. We have previously shown that developing alloreactive CD8 T cells, which recognize K^b via the direct pathway of Ag recognition, are negatively selected in the thymus of molecular chimeras (18). However, MHC class I disparate allografts can be rejected by both CD4 and CD8 T cells via the indirect and direct pathways, respectively (15, 16). Therefore, CD4 T cells that recognize alloantigen through the indirect pathway must also be tolerized to allow permanent allograft survival.

To address mechanisms leading to CD4 T cell tolerance, we made use of Tg361 TCR transgenic mice (19), which allowed us to specifically track the development of CD4 T cells that recognize K^b via the indirect pathway in molecular chimeras. Based on our previous results related to the fate of CD8 T cells that recognize K^b through the direct pathway (18), we anticipated that expression of K^b on bone marrow-derived cells would most likely result in negative selection of T cells expressing the Tg361 TCR in the thymus. However, CD4 T cells expressing the Tg361 TCR were observed in the periphery of mice reconstituted with a mixture of VSV-Kb-transduced B10.AKM and Tg361 bone marrow that stably expressed K^b on bone marrow-derived cells. CD4 T cells expressing the Tg361 TCR were detected in all lymphoid tissues analyzed at approximately one-half of the frequency observed in control mice. Analysis of T cell development in the thymus of mice receiving a mixture of VSV-Kb-transduced B10.AKM and Tg361 bone marrow demonstrated that both CD4⁺CD8⁺ double positive as well as CD4⁺ single positive T cells expressing the Tg361 TCR were present in the thymus of mice expressing K^b on their bone marrow-derived cells. The presence of double positive T cells expressing the Tg361 TCR in the thymus of these mice suggested that CD4 T cells expressing this receptor in the periphery of K^b molecular chimeras developed from bone marrow progenitors and were not simply mature T cells contaminating the Tg361 bone marrow. Although the absolute number of single positive T cells expressing the Tg361 TCR was reduced, the presence of these cells in the thymus and periphery demonstrates that expression of K^b on bone marrow-derived cells was not sufficient to mediate negative selection of CD4 T cells that recognize K^b through the indirect pathway. These data contrast sharply with our previous results demonstrating that developing alloreactive CD8 T cells that recognize K^b through the direct pathway undergo negative selection in the thymus of molecular chimeras that express K^b on bone marrow-derived cells (18).

Despite the presence of potentially alloreactive T cells expressing the Tg361 TCR, mice reconstituted with a mixture of VSV-Kb-transduced B10.AKM and Tg361 bone marrow accepted K^b disparate B10.MBR skin grafts long term. These data suggest that although expression of K^b on bone marrow-derived cells in molecular chimeras was not sufficient to negatively select all CD4 T cells expressing the Tg361 TCR in the thymus, the mice were nevertheless functionally tolerant to K^b. Because it has been known for some time that Treg cells can play a significant role in preventing allograft rejection (23), we hypothesized that alloreactive T cell responses in molecular chimeras may also be controlled by Treg cells. To address this issue, we made use of an adoptive cell transfer system in which it is possible to examine the ability of various CD4 T cell populations isolated from molecular chimeras to prevent rejection mediated by naive effector T cells. For these experiments, we chose to isolate T cells with putative regulatory function from molecular chimeras reconstituted with either VSV-Kb- or mock-transduced syngeneic bone marrow rather than a mixture of transduced B10.AKM and Tg361 bone marrow, to ensure that we were isolating a polyclonal population of Treg cells and to avoid transferring Tg361 TCR-expressing alloreactive T cells. Furthermore, if induction of molecular chimerism is to eventually be tested in preclinical models, it is most critical to examine mechanisms of tolerance in mice with a normal T cell repertoire.

Mice receiving CD4⁺CD25⁺ T cells isolated from K^b molecular chimeras along with effector splenocytes accepted their B10.MBR skin grafts long term. We also observed prolonged survival of K^b disparate B10.MBR skin grafts on mice that received CD4⁺ T cells. Together, these data suggest that Treg cells develop in molecular chimeras that are able to prevent allograft rejection and that regulator T cell functional activity resides in the CD4⁺CD25⁺ fraction, a cell surface phenotype shown to be a hallmark of Treg cells (24, 25). The regulatory effect observed was not restricted to B10.AKM effectors because CD4⁺CD25⁺ T cells from molecular chimeras were also able to prevent rejection of K^b disparate CBK skin grafts by CBA/Ca effectors. Importantly, CD4⁺CD25⁺ T cells isolated from mice reconstituted with mock-transduced bone marrow were unable to prevent rejection of K^b disparate skin graft in adoptive cell transfer recipients. The regulatory effect observed appeared to be specific for K^b because we were able to achieve permanent survival of K^b disparate but not third party skin allografts. Survival of third party B10.BR skin grafts was prolonged on mice that received CD4⁺CD25⁺ T cells; however, all third party grafts were eventually rejected. We wish to point out that in these experiments, adoptive cell transfer recipients received both third party B10.BR and K^b disparate B10.MBR skin grafts. We suggest that prolongation of third party skin survival could be the result of soluble factors produced by Treg cells when they recognize the B10.MBR skin. Alternatively, because B10.MBR and B10.BR are MHC class II matched, regulatory CD4 T cells may recognize Ags presented by MHC class II of B10.BR and weakly inhibit rejection of the grafts. Consistent with this notion, we observed that survival of fully mismatched third party grafts was not prolonged by regulatory cells in adoptive cell transfer experiments (Fig. 5B).

To use the Tg361 mouse model to define the mechanism by which CD4 T cell tolerance is induced in molecular chimeras, it was necessary to reduce the frequency of alloreactive CD4 T cells by generating bone marrow chimeras. In pilot experiments, we were unable to induce molecular chimerism directly in Tg361 mice, presumably because the frequency of alloreactive CD4 T cells was too high to permit engraftment of bone marrow cells expressing K^b even with transient T cell depletion and whole body irradiation (J. Iacomini, unpublished observation). Using a similar approach, we were previously able to generate mice that contained CD8 T cells expressing a transgenic TCR specific for K^b at a frequency of <10% of CD8 T cells (18). However, in this model, we were unable to generate mice in which the frequency of CD4 T cells expressing the Tg361 TCR was <40% 4 wk after transfer. Interestingly, the frequency of CD4 T cells expressing the Tg361 receptor increased over the next 4 wk in mice receiving mock-transduced bone marrow (Fig. 2B). These data suggest that T cells expressing the Tg361 TCR develop rapidly in these mice and out compete T cells expressing endogenous TCR. Mice expressing K^b on bone marrow-derived cells also contained a relatively high frequency of T cells expressing the Tg361 TCR (20–40%). The observation that these mice are nevertheless able to accept K^b disparate skin grafts demonstrates that the regulatory capacity of CD4⁺CD25⁺ T cells is potent and able to control a relatively large clone size. Based on the observation that T cells expressing the Tg361 TCR in K^b molecular chimeras and controls are CD44⁺CD45RB^high, we suggest that they may have undergone homeostatic expansion, but are otherwise naive. The observation that the majority of T cells expressing the Tg361 TCR in K^b molecular chimeras and controls are CD25^– suggests that expression of K^b in bone marrow-derived cells does not induce T cells expressing the Tg361 TCR to become Treg cells. Therefore, based on the adoptive cell transfer studies we performed, we suggest that Treg cells are most likely derived from B10.AKM bone marrow progenitors.

Based on our data, we propose a model in which expression of K^b on bone marrow-derived cells in molecular chimeras tolerizes developing CD8 T cells that recognize K^b through the direct pathway by negative selection in the thymus (18). In contrast, alloreactive CD4 T cells that recognize K^b through the indirect pathway are controlled through the generation of Treg cells. Based on our data from adoptive cell transfer studies, CD4⁺CD25⁺ cells from molecular chimeras are also able to prevent rejection mediated by CD8 T cell effectors, consistent with the observation that Treg cells can control CD8 T cell function (26). Interestingly, it has been suggested that Ag dose may influence whether mechanisms leading to tolerance are deletional or regulatory, with large doses of Ag favoring deletion (27). The frequency of alloantigen expressing bone marrow-derived cells in molecular chimeras is typically lower than that observed in mixed bone marrow chimeras. Therefore, the level of Ag expressed in molecular chimeras may favor the development of Treg cells, whereas the level achieved in mixed cellular chimeras may favor thymic deletion (5, 28).

Why is expression of K^b on bone marrow-derived cells insufficient to mediate negative selection of CD4 T cells through the indirect pathway, but capable of efficiently negatively selecting CD8 T cells that recognize K^b via the direct pathway? We suggest that differences in the ability to negatively select these alloreactive T cell populations may be related to differences in the amount of Ag encountered in the thymus. It is likely that T cells that recognize Ag through the direct pathway encounter a greater density of Ag in the thymus than T cells that recognize Ag through the indirect pathway. High ligand density may favor deletion, whereas low Ag density may allow for T cells to escape negative selection consistent with an avidity model of thymic selection (29). The ability of T cells to escape negative selection could also be influenced by clone size.

	Acknowledgments

 
We thank Dr. Richard C. Mulligan and Dr. Jeng-Shin Lee for providing the MMP retroviral vector, packaging system, and technical advice. We also thank the Harvard Gene Therapy Initiative Vector Core, supported in part by the Association Francaise contre les Myopathies, for the production of virus preparations. In addition, we thank Dr. Mohammed Sayegh and Dr. Jessamyn Bagley for critical review of the manuscript and members of the Iacomini laboratory for helpful discussions.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Training Grant T32 AI07529 (to D.F.) and Grant R01AI043619-06 (to J.I.).

² Current address: Department of Laboratory Medicine, Ewha Women’s University, Mokdong Hospital, 911-1, YangCheon-Ku, Seoul 158-710, Korea.

³ Address correspondence and reprint requests to Dr. John Iacomini, Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, 221 Longwood Avenue, LM303, Boston, MA 02115. E-mail address: jiacomini{at}rics.bwh.harvard.edu

⁴ Abbreviations used in this paper: Treg, T regulatory; VSV, vesicular stomatitis virus; MST, median survival time.

Received for publication July 20, 2005. Accepted for publication January 10, 2006.

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