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The Journal of Immunology, 2002, 169: 1930-1935.
Copyright © 2002 by The American Association of Immunologists

Induction of Central Deletional T Cell Tolerance by Gene Therapy1

Eun-Suk Kang and John Iacomini2

Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag Kb were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2Kb resulted in lifelong expression of Kb on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2Kb transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with Kb transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to Kb. Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The induction of tolerance to transplantation Ags is a major goal in transplantation because of its potential to allow for replacement of host organs without the need for lifelong immunosuppression. One method to establish stable lifelong tolerance to allogeneic transplantation Ags is through the induction of hematopoietic chimerism following allogeneic bone marrow transplantation (1). The establishment of mixed, host and donor, hematopoietic cellular chimerism through bone marrow transplantation leads to specific tolerance in an otherwise fully immunocompetent host (reviewed in Ref. 2). However, the use of allogeneic bone marrow transplantation to induce tolerance has several potential drawbacks, including the severity of the preparative regimen required to achieve bone marrow engraftment, the potential of inducing graft-vs-host disease, and the possibility of engraftment failure.

We have recently shown that tolerance can be achieved using a gene therapy-based approach to induce molecular rather than cellular chimerism (3, 4, 5). Molecular chimerism is induced by modifying autologous bone marrow cells to express retrovirally transduced Ags. Expression of allogeneic MHC Ags on otherwise autologous bone marrow-derived cells eliminates many of the complications associated with allogeneic bone marrow transplantation used to establish mixed cellular chimerism. Reconstitution of mice with syngeneic bone marrow cells infected with retroviruses carrying the allogeneic MHC class I gene H-2Kb resulted in lifelong expression of Kb on bone marrow-derived cells. T cells from mice receiving H-2Kb transduced bone marrow were unable to kill cells expressing Kb, but were able to kill third party cells. In addition, mice reconstituted with H-2Kb transduced bone marrow exhibited long-term acceptance of H-2Kb mismatched skin grafts, although third party control grafts on the same mice were rapidly rejected. Thus, efficient expression of Kb on bone marrow-derived cells was sufficient to induce tolerance to the product of the transduced MHC gene rather than hyporesponsiveness, which was observed previously in mice expressing low levels of the transduced MHC gene (6, 7, 8).

Our results demonstrate that induction of molecular chimerism by gene therapy results in T cell tolerance; however, the mechanism of tolerance induction remains to be elucidated. We hypothesized that, following reconstitution of mice with H-2Kb transduced bone marrow, tolerance to Kb may be established by a central deletional mechanism resulting from negative selection of alloreactive T cells in the thymus. Alternatively, functional tolerance could be established by peripheral mechanisms involving induction of T cell anergy (9) or the generation of regulatory T cells (10, 11, 12). To address these issues, we made use of BM3.3 TCR-transgenic mice which express an alloreactive TCR on CD8 T cells specific for Kb that can be detected using the anticlonotypic mAb Ti98 (13, 14). Our results suggest that induction of molecular chimerism leads to negative selection of alloreactive CD8 T cells in the thymus. Thus, genetic engineering of bone marrow can be used to fundamentally reshape the T cell repertoire by influencing thymic education.


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

Female B10.AKM/SnJ (H-2Kk, Ik, Dq), B10.MBR (H-2Kb, Ik, Dq), and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The B10.MBR strain was derived from a recombination event that occurred during the back-crossing of B10.AKM to C57BL/10 and differs from the B10.AKM strain in the MHC class I H-2K region (15). BM3.3 TCR-transgenic mice (H-2k, CBA/Ca background (13)) were kindly provided to us by Dr. A. L. Mellor (Medical College of Georgia, Augusta, GA) and bred in our facility. 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.

Retroviruses

VSV-Kb virus has been described previously (3). Briefly, to generate VSV-Kb, the cDNA encoding H-2Kb (Kb) was cloned into the MMP retroviral vector kindly provided by Dr. R. C. Mulligan (Children’s Hospital, Boston, MA) to generate pMMP-Kb. The MMP retroviral vector is a derivative of MFG (16). Vesicular stomatitis virus G envelope protein pseudotyped viruses were prepared by packaging the pMMP-Kb retroviral vector in 293T cells by transient transfection (17) to generate VSV-Kb virus as described in (18). Functional titers of VSV-Kb retroviral supernatants were determined by analyzing expression of Kb on NIH-3T3 cells by cell surface staining and flow cytometry following infection. All virus preparations were made in affiliation with the Harvard Institute for Human Genetics Gene Therapy Initiative. 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 described previously (3, 4). Briefly, bone marrow cells from mice treated 7 days prior with 5-fluorouracil (150 mg/kg) were cultured in Retronectin (Takara Biomedicals, Shiga, Japan)-coated tissue 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, Minneapolis, MN), 100 ng/ml recombinant mouse stem cell factor (BioSource International, Camarillo, CA), 50 ng/ml recombinant mouse thrombopoietin (R&D Systems), and 50 ng/ml recombinant mouse Flt-3 ligand (R&D Systems)). All transductions were performed at 37°C with 5% CO2 for 96 h. Retronectin-coated plates were prepared according to the manufacturer’s instructions. Bone marrow cells were cultured at a density of 6 x 106 cells per milliliter together with VSV-Kb virus to achieve a multiplicity of infection of ~1. Viral supernatants and transduction medium were replaced at 72 h after the start of the transduction. Mock transductions were performed in the same manner, except viral supernatants were replaced with transduction medium. Twenty-four hours later, the cells were harvested, washed twice in HBSS, and counted.

Bone marrow transplantation

BM3.3 bone marrow was harvested from mice treated 7 days prior with 5-fluorouracil as described above. All BM3.3 bone marrow donors also received a depleting dose of anti-CD8 (116-13.1 (19)) and CD4 (GK1.5 (20)) mAbs 4 days before bone marrow harvest to eliminate alloreactive T cells in vivo. Transduced B10.AKM and freshly isolated BM3.3 bone marrow cells were then used to reconstitute B10.AKM mice, which received a lethal dose of irradiation (10.25 Gy), a depleting dose of the anti-CD8 mAbs 2.43 (21) and 116-13.1, and a depleting dose of anti-CD4 (GK1.5). Two different anti-CD8 Abs were used because B10.AKM mice express the Ly-2.2 allele of CD8{alpha}, while CBA/Ca mice express the Ly-2.1 allele of CD8{alpha}, thus allowing us to deplete mature CD8 T cells from both strains.

mAbs and flow cytometry

All cell surface staining and flow cytometry were performed as described previously (22, 23). The hybridoma secreting the BM3.3 anticlonotypic Ab Ti98 was kindly provided by Dr. A. L. Mellor (14). Ti98 Ab was purified in our laboratory and labeled with FITC using standard methods. mAbs specific for CD4 (RM4-5), CD8 (53-6.7), H-2Kb (AF6-88.5), Ly-6G (Gr-1, RB6-8C5), CD19 (1D3), CD11b (Mac-1, M1/70), NK cells (DX5), and CD11c (HL3) were obtained from BD PharMingen (San Diego, CA).

Polymerase chain reaction

DNA was purified from blood using a QIAamp DNA blood mini kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). The BM3.3 V{beta}2 gene segment was amplified using standard PCR conditions using the following primers: V{beta}BM3.3 forward primer 5'-GCAACTACAGTGGCTGTTCAC-3' and V{beta}BM3.3 reverse primer 5'-CGTATTTCCAACCCTGTCTGC-3'. Control primers specific for {beta}-actin are described in Ref. 24 .

Skin grafting

Tail skin grafting was performed and evaluated as previously described (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of a TCR-transgenic model to examine mechanisms of tolerance induced by gene therapy

To create a model in which the fate of alloreactive T cells could be monitored in mice reconstituted with H-2Kb transduced bone marrow, BM3.3 transgenic mice that express an alloreactive TCR on CD8 T cells specific for Kb were used (13). However, because essentially 100% of CD8 T cells in BM3.3 mice are alloreactive against Kb, we first set out to reduce the frequency of BM3.3 CD8 T cells by generating bone marrow irradiation chimeras. This was done to obtain mice containing a population of alloreactive T cells which exists at physiologically relevant frequencies and could be tracked using the anti-BM3.3 clonotypic Ab Ti98.

B10.AKM (H-2Kk, Ik, Dq) bone marrow was harvested from mice treated 7 days prior with 5-fluorouracil and cultured in vitro using mock transduction conditions. This approach was used to ensure that the reconstitution potential of the bone marrow would be similar to that observed after retroviral transduction in later experiments. BM3.3 (H-2k, CBA/Ca background) bone marrow was similarly harvested from 5-fluorouracil-treated mice; however, BM3.3 bone marrow cells were not cultured in vitro in any experiments. In addition, BM3.3 mice also received a depleting dose of anti-CD8 and anti-CD4 mAbs before bone marrow harvest to eliminate alloreactive T cells in the bone marrow. Treatment of BM3.3 mice with anti-CD8 (116-13.1 (19)) and anti-CD4 (GK1.5 (20)) before bone marrow harvest led to complete depletion of T cells from bone marrow (data not shown). Recipient B10.AKM mice were then conditioned with lethal irradiation (10.25 Gy) and a depleting dose of anti-CD8 and anti-CD4 mAbs, and reconstituted the following day with varying ratios of cultured B10.AKM and BM3.3 bone marrow. Mice received 2–3 x 106 B10.AKM bone marrow cells and decreasing doses of BM3.3 bone marrow cells.

By ~9 wk following bone marrow transplantation, mice receiving a ratio of 5–6:1 B10.AKM:BM3.3 bone marrow cells consistently contained a population of CD8 T cells in their peripheral blood that was detectable by cell surface staining and flow cytometry with the anti-BM3.3 clonotypic Ab Ti98. Approximately 2.9 ± 1.2% (n = 5) of CD8 T cells in blood expressed the BM3.3 TCR, detectable using Ti98. At 19 wk after bone marrow transplantation, as high as 10% of CD8 T cells expressed the BM3.3 TCR (Fig. 1Go). Increasing the proportion of BM3.3 cells used for reconstitution led to a dominance of BM3.3 TCR-positive cells (data not shown). Clonotype-positive CD8 T cells were not detectable in the blood of either naive B10.AKM controls or B10.AKM mice reconstituted with B10.AKM bone marrow cells alone (Fig. 1Go).



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  		FIGURE 1. Development of B10.AKM-BM3.3 chimeras. Shown are analyses of CD8 T cells expressing the BM3.3 TCR using the anticlonotypic Ab Ti98 in peripheral blood of control BM3.3 mice (A), control B10.AKM mice (B), B10.AKM mice reconstituted with a mixture of B10.AKM and BM3.3 bone marrow (C), and control B10.AKM mice reconstituted with B10.AKM bone marrow alone (D). Representative mice were analyzed 19 wk after bone marrow transplantation.

 
Generation of B10.AKM-BM3.3 chimeras following retroviral transduction of bone marrow with retrovirus carrying the gene encoding Kb

To examine how expression of a retrovirally transduced MHC class I gene in bone marrow-derived cells affected the development of alloreactive T cells, B10.AKM bone marrow was harvested from 5-fluorouracil-treated mice and either transduced with vesicular stomatitis virus G protein-enveloped retroviruses carrying the gene encoding H-2Kb, hereafter referred to as VSV-Kb as described (3), or mock transduced. Immediately following transduction with VSV-Kb, ~10–20% of B10.AKM bone marrow cells expressed Kb on their cell surface at levels readily detectable by cell surface staining and flow cytometry (Fig. 2Go). As expected, Kb was not detected on the surface of mock transduced cells (Fig. 2Go). Recipient B10.AKM mice were then conditioned with lethal irradiation (10.25 Gy) and a depleting dose of anti-CD8 and anti-CD4 mAbs. The mice were reconstituted the following day with a 5:1 ratio of either Kb or mock transduced B10.AKM (2 x 106 total cells per mouse) and BM3.3 bone marrow cells (0.4 x 106 total cells per mouse) harvested from 5-fluorouracil-treated BM3.3 mice depleted of T cells in vivo as described above.



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  		FIGURE 2. Analysis of bone marrow transduction efficiency and expression of Kb on bone marrow-derived cells in the periphery of reconstituted mice. A, Expression of Kb on the surface of VSV-Kb (solid line) or mock transduced (dashed line) B10.AKM bone marrow before bone marrow transplantation. B, Expression of Kb on the surface of bone marrow-derived cells in the blood of B10.AKM mice reconstituted with either a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow (solid line) or mock transduced B10.AKM and BM3.3 bone marrow (dashed line) 13 wk after bone marrow transplantation. Shown are representative data.

 
Following reconstitution, bone marrow-derived cells expressing Kb on their surface were detectable by flow cytometry in the blood of mice receiving VSV-Kb transduced marrow at all time points analyzed over the 29-wk follow-up period (Figs. 2Go and 3Go). The percentage of cells in the blood expressing Kb appeared to remain stable (Fig. 3Go), suggesting that early progenitor cells had been transduced, as we have observed previously (3). Furthermore, expression of Kb was detectable on T cells (CD4+ and CD8+), B cells (CD19+), macrophages (Mac-1, CD11b+), dendritic cells (CD11c+), granulocytes (Ly-6G, Gr-1+), and NK cells (DX5+) in peripheral lymphoid tissues (data not shown). In control experiments, the frequency of Kb-positive cells in mice receiving a mixture of VSV-Kb transduced B10.AKM bone marrow and BM3.3 cells was similar to that observed in control mice receiving VSV-Kb transduced B10.AKM bone marrow alone (data not shown).



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  		FIGURE 3. Long-term expression of Kb on the surface of blood leukocytes in B10.AKM mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow. Shown are the mean values obtained for five mice per time point. Expression of Kb was not detected on the surface of blood cells from mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow (data not shown).

 
Expression of retrovirally transduced Kb on bone marrow-derived cells prevents the development of CD8 T cells expressing the BM3.3 TCR

Starting at 3 wk after bone marrow transplantation, mice reconstituted with either VSV-Kb or mock transduced B10.AKM and BM3.3 bone marrow cells were bled every other week to examine hematopoietic reconstitution and development of CD8 T cells expressing the BM3.3 TCR. CD8 T cell recovery was complete by ~8 wk after bone marrow transplantation. CD8 T cells expressing the BM3.3 TCR were readily detectable in the blood of B10.AKM mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow cells by cell surface staining with Ti98 (Fig. 4Go). In contrast, we were unable to detect the presence of CD8 T cells expressing the BM3.3 TCR in the blood of B10.AKM mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow cells (Fig. 4Go). The failure to detect CD8 T cells expressing the BM3.3 TCR was not simply due to a delay in reconstitution, because BM3.3 clonotype-positive CD8 T cells remained undetectable in the blood on mice expressing Kb on bone marrow-derived cells for up to 29 wk after transplantation, at which time the experiment was terminated (data not shown).



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  		FIGURE 4. Expression of Kb on bone marrow-derived cells prevents the development of CD8 T cells expressing the BM3.3 TCR. A and B, Analysis of CD8 T cells expressing the BM3.3 TCR using Ti98 in the blood of either mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow (A) or mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow (B). The percentage of Ti98-positive cells is shown. C and D, Analysis of Kb expression in the blood of either mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow (C) or mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow (D). The percentage of Kb-positive cells is shown. Mice were analyzed 15 wk after transplantation. Representative mice are shown.

 
At 19 wk after reconstitution, groups of mice were sacrificed and CD8 T cells from spleen and lymph nodes were analyzed for expression of the BM3.3 TCR by cell surface staining and flow cytometry with Ti98. CD8 T cells expressing the BM3.3 clonotype were readily detected in lymphoid tissues from mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow cells (Fig. 5Go). In contrast, we were unable to detect BM3.3 clonotype-positive CD8 T cells in peripheral lymphoid organs of mice reconstituted with a mixture of VSV-Kb B10.AKM and BM3.3 bone marrow cells (Fig. 5Go). Similar results were observed at earlier time points (data not shown).



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  		FIGURE 5. BM3.3 clonotype-positive CD8 T cells are not present in the periphery of mice expressing retrovirally transduced Kb on bone marrow-derived cells. Shown are analyses of CD8 T cells expressing the BM3.3 TCR using Ti98 in the spleen (A–C) and lymph nodes (D–F) of mice reconstituted with mock transduced B10.AKM and BM3.3 bone marrow (A and D), control mice reconstituted with mock transduced B10.AKM bone marrow alone (B and E), and mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow (C and F). Mice were analyzed 19 wk after transplantation. Representative data are shown.

 
To determine whether the inability to detect BM3.3 clonotype-positive T cells in mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow cells resulted from a failure of BM3.3 bone marrow to engraft, blood cells were harvested from mice reconstituted with either Kb or mock transduced B10.AKM and BM3.3 bone marrow cells and DNA was isolated. Because BM3.3 transgenic mice carry the genes encoding the transgenic TCR in all tissues, the presence of transgene encoded V{beta}2 gene segments in reconstituted mice would be indicative of engraftment, even if CD8 T cells expressing the BM3.3 clonotype failed to develop. Therefore, PCR primers complementary to the framework region of V{beta}2 and the complementarity-determining region 3 of the BM3.3 {beta}-chain were synthesized and used to amplify DNA isolated from chimeric mice in each group. Amplification of DNA isolated from eight of eight mice reconstituted with VSV-Kb transduced B10.AKM and BM3.3 bone marrow cells, and seven of eight control mice receiving a mixture of mock transduced B10.AKM and BM3.3 bone marrow produced a PCR product of the correct size using BM3.3 TCR-specific primers. The amount of PCR product generated was similar in samples from both groups of mice (data not shown). Thus, BM3.3 bone marrow was able to engraft in conditioned hosts, suggesting that the failure to detect T cells expressing the BM3.3 TCR in mice expressing Kb was not due to engraftment failure.

Expression of retrovirally transduced Kb on bone marrow-derived cells induces negative selection of BM3.3 clonotype-positive T cells in the thymus

To examine whether CD8 T cells expressing the BM3.3 TCR underwent negative selection in mice receiving VSV-Kb transduced bone marrow, we analyzed development of BM3.3 clonotype-positive CD8 T cells in the thymus of mice reconstituted with either Kb or mock transduced B10.AKM and BM3.3 bone marrow cells using the mAb Ti98. CD4/CD8 double positive as well as CD8 single positive T cells expressing the BM3.3 clonotype were readily detectable in the thymus of B10.AKM mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow cells (Fig. 6Go). In contrast, B10.AKM mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow cells contained in their thymus relatively few clonotype-positive CD4/CD8 double positive T cells (Fig. 6Go). CD8 single positive T cells expressing the BM3.3 receptor were not detected in the thymus of mice reconstituted with VSV-Kb transduced B10.AKM and BM3.3 bone marrow (Fig. 6Go). These data strongly suggest that expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells results in negative selection of alloreactive CD8 T cells in the thymus.



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  		FIGURE 6. Expression of retrovirally transduced Kb on bone marrow-derived cells induces negative selection of T cells expressing the BM3.3 clonotype. Twenty weeks after transplantation, mice reconstituted with either a mixture of mock transduced B10.AKM and BM3.3 bone marrow cells (A) or a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow cells (B) were sacrificed and thymi were removed. The thymi were then triple stained with Abs specific for CD4, CD8, and Ti98. Shown is expression of CD4 and CD8 on thymocytes from representative mice following gating on Ti98-positive cells. The absolute numbers of cells in each quadrant are indicated. The number of CD8 single positive T cells staining with Ti98 in B is similar to background observed in controls (data not shown).

 
Mice reconstituted with a mixture of VSV-Kb-transduced B10.AKM and BM3.3 bone marrow are tolerant to Kb

To examine the immunological responsiveness to Kb in mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow, mice were grafted with B10.MBR skin. Control mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow cells rejected B10.MBR skin grafts rapidly (Fig. 7Go). In contrast, mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow accepted their B10.MBR skin grafts for >95 days (p < 0.0003 between groups, Fig. 7Go). In control experiments, mice in both groups were able to rejected third party BALB/c skin graft within 15 days, demonstrating that both groups of mice were immunocompetent.



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  		FIGURE 7. Survival of B10.MBR skin grafts on recipients reconstituted with either VSV-Kb transduced B10.AKM and BM3.3 bone marrow (circles, n = 6) or mice reconstituted with mock transduced B10.AKM and BM3.3 bone marrow ({blacksquare}, n = 8). Third party BALB/c skin grafts were rejected within 15 days by mice in both groups (data not shown). Shown are the combined results of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inducing tolerance to transplantation Ags has the potential to overcome rejection without the use of lifelong immunosuppression and therefore remains a major goal in the field. Building on the concept of hematopoietic chimerism, we have developed a gene therapy-based approach designed to induce tolerance following the induction of molecular chimerism. We have shown previously that establishing molecular chimerism results in functional tolerance (3). However, it is important to note that tolerance can be induced by a variety of mechanisms, including negative selection in the thymus, induction of T cell anergy in the periphery, or the generation of regulatory T cells. Central deletional tolerance which occurs in the thymus during T cell development as a result of negative selection is perhaps the most robust form of tolerance, because potentially alloreactive T cells are eliminated from the repertoire (25). In contrast, functional tolerance established by inducing T cell anergy or generating regulatory T cells has the potential of being broken because potentially alloreactive T cells remain, albeit in an unresponsive state. Understanding which mechanisms lead to functional tolerance following the establishment of molecular chimerism is critical in terms of predicting the stability of tolerance induced.

To examine the mechanism by which molecular chimerism induces tolerance, we used BM3.3 mice that express an alloreactive TCR on essentially 100% of CD8 T cells specific for Kb. By generating irradiation bone marrow chimeras using a mixture of B10.AKM and BM3.3 bone marrow, we were able to reduce the frequency of CD8 T cells expressing the BM3.3 receptor to more physiologically relevant levels and generate mice containing a population of alloreactive CD8 T cells that could be reliably tracked using the anticlonotypic Ab Ti98. Expression of Kb on the surface of bone marrow-derived cells prevented the development of clonotype-positive CD8 T cells in mice reconstituted with a mixture of VSV-Kb transduced B10.AKM and BM3.3 bone marrow. Analysis of T cell development in the thymus suggested that expression of Kb on bone marrow-derived hematopoietic cells led to negative selection of CD8 T cells expressing the BM3.3 TCR in the thymus. While relatively few CD4/CD8 double positive thymocytes expressing the BM3.3 clonotype were present in mice expressing Kb, CD8 single positive T cells expressing the BM3.3 receptor were not detected. These data are consistent with negative selection occurring at the double positive stage of T cell development (26, 27).

In control mice reconstituted with a mixture of mock transduced B10.AKM and BM3.3 bone marrow cells, typically 2–4% of CD8 T cells expressed the BM3.3 receptor. Therefore, the frequency of T cells expressing the alloreactive BM3.3 TCR was significantly greater than the frequency of alloreactive T cell observed naturally, which has been estimated to be in the order of ~1 in 1000 (28). Thus, expression of retrovirally transduced Kb on bone marrow-derived cells was able to induce negative selection of a relatively large alloreactive T cell precursor population. In mice reconstituted with VSV-Kb transduced B10.AKM and BM3.3 bone marrow, Kb was expressed long term on multiple hematopoietic cell lineages. Long-term chimeras failed to develop CD8 T cells expressing the BM3.3 TCR (data not shown). We suggest lifelong expression of Kb on bone marrow-derived cells maintains negative selection of Kb-reactive CD8 T cells for the life of the animal. Furthermore, based on our skin graft data, mice receiving VSV-Kb transduced bone marrow exhibited long-term survival of B10.MBR skin grafts. These data suggest that, in addition to BM3.3 clonotype-positive cells, endogenous Kb-reactive T cells derived from B10.AKM bone marrow were also negatively selected in these mice.

In previous studies, inefficient retroviral transduction and expression of retrovirally transduced H-2Kb induced hyporesponsiveness to Kb via peripheral mechanisms that could be broken upon Ag challenge (6, 8). Recent work from our laboratory demonstrated that tolerance, rather than hyporesponsiveness, could be induced by gene therapy following efficient transduction and expression of Kb on bone marrow-derived cells, which was achieved using improved transduction conditions and retroviral vectors (3). T cells from mice receiving H-2Kb transduced bone marrow were unable to kill targets expressing Kb even after rigorous Ag challenge and accepted skin grafts expressing Kb indefinitely (3). We suggest that improved expression of Kb on the surface of bone marrow-derived cells induced stable T cell tolerance, rather than hyporesponsiveness, by inducing negative selection of alloreactive CD8 T cells in the thymus.

Deletion of alloreactive T cells in the thymus as a result of genetic engineering of bone marrow suggests that tolerance induced by this approach is relatively robust and not subject to being broken by the presence of helper determinants on tissues expressing Kb (29, 30). Using genetically modified autologous bone marrow to establish molecular chimerism eliminates many of the complications associated with allogeneic bone marrow transplantation to establish mixed cellular chimerism, but retains the same ability to induce stable and specific tolerance induced by mixed cellular chimerism, including negative selection in the thymus (2). To our knowledge, this is the first example in which gene therapy has been shown to induce negative selection of alloreactive T cells. The observation that efficient expression of retrovirally transduced alloantigens on bone marrow-derived cells is sufficient to induce T cell negative selection in the thymus supports the notion that specific immunological tolerance induce by gene therapy can be used to fundamentally reshape the immunological repertoire. Similar approaches may eventually be applicable for the induction of tolerance in immunological disorders such as autoimmune disease.


   Acknowledgments
 
We thank Drs. Richard C. Mulligan and Jeng-Shin Lee for providing the MMP retroviral vector, packaging system, and technical advice, as well as the Harvard Gene Therapy Initiative Vector Core, in part supported by the Association Francaise contre les Myopathies, for production of viral vectors. In addition, we thank Drs. Shiv Pillai and Jessamyn Bagley for critical review of the manuscript and members of the Iacomini laboratory for helpful discussions. We also thank Hongling Liao for technical assistance, Jessica Lynch for expert secretarial assistance, and Dr. Andrew L. Mellor for providing the BM3.3 transgenic mice and the Ti98 hybridoma.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1 AI43619 (to J.I.). Back

2 Address correspondence and reprint requests to Dr. John Iacomini, Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Building 149, 13th Street, Boston, MA 02129. E-mail address: iacomini{at}helix.mgh.harvard.edu Back

Received for publication May 15, 2002. Accepted for publication June 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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