Skin Allograft Tolerance Research Articles

Anti-CD4 Monoclonal Antibody Reduces the Dose of Cyclophosphamide Required to Induce Tolerance to H-2 Haplotype Identical Skin Allografts in Mice

Cyclophosphamide (CP)-induced tolerance which consists of a single i.p. injection of 200 mg/kg CP 2 days after priming with 1 × 10 8 donor spleen cells (SC), leads to long-lasting donor-specific skin allograft tolerance in H-2 compatible, multiminor antigen incompatible, murine strain combinations. In this system, the optimal dose of CP has been suggested to be 200 mg/kg, however, such a dose of CP causes strong myelosuppression. In the present study, we therefore attempted to reduce the dose of CP by administering anti-CD4 monoclonal antibody (mAb) before donor cell priming in this toleranceinducing system. When C3H/He (C3H; H-2 k, Mls-1 b) mice were injected i.p. with 200 ltg anti-CD4 mAb on day -3, 1 × 10 8 AKR/J (AKR; H-2 k, Mls-1 a) SC plus 3 × 10 7 bone marrow cells (BMC) i.v. on day -2 and then 100 mg/kg CP i.p. on day 0, a long-lasting donor-specific skin allograft tolerance was established; furthermore, the decreases in the number of leukocytes and the concentration of hemoglobin (Hb) in the peripheral blood were all less in the C3H mice treated with this new combined protocol than in the C3H mice injected with 200 mg/kg CP following the previous protocol. In the periphery of these tolerant mice, the number of donor-reactive Vβ6 +CD4 + T cells decreased and mixed chimerism was observed on both days 14 and 80. On the other hand, in the mice injected with AKR SC, BMC plus 100 mg/kg CP without anti-CD4 mAb, the number of Vβ6 +CD4 + T cells decreased on day 14, and then recovered by day 80 when the mixed chimerism disappeared. These results therefore suggest that the combined use of anti-CD4 . mAb with CP can reduce the dose of CP without affecting the efficiency of inducing donor-specific tolerance, probably due to the enhancement of the destruction effect of donor-reactive T cells by CP.

Priming with donor spleen cells and activated B cells can induce prolonged survival of class I-disparate skin allografts in cyclophosphamide-treated mice.

We have previously reported a method for inducing tolerance using cyclophosphamide (CP) in a murine model, in which 200 mg/kg of CP is administered intraperitoneally 2 days after intravenous priming with donor spleen cells. The CP-induced tolerance method, however, cannot induce long-lasting skin allograft survival in an MHC class I-disparate combination. In this study, we tried to explain this phenomenon based on a costimulatory theory. That is, allo-class I-reactive host CD8+ helper T cells may receive insufficient costimulatory signals from donor spleen cells and, therefore, they are resistant to subsequently administered CP. We demonstrated that activated donor B cells, which can deliver sufficient costimulatory signals, induce a more efficient proliferation of allo-class I-reactive host CD8+ helper T cells than naive B cells in vitro. In addition, we also demonstrated that our idea can also be applied to the CP-induced skin allograft tolerance in an MHC class I-disparate combination.

The Induction of Skin Xenograft Tolerance in Rat-to Mouse Combination could be Affected by DFR Mediating Cells and Antibodies against Rat Bone Marrow Cells as well as NK Cells in the Cyclophosphamide-Induced Tolerance System

We investigated whether the prolongation of skin xenograft survival was obtained by a tolerance-inducing method using cyclophosphamide (CY), by which long-lasting skin allograft tolerance could be induced. The long-lasting skin allograft survival could be obtained in the recipient C3H/HeN (C3H) mice which were given 100 [μg of anti-CD4 mAb on day -3, 1 x 10 8 spleen cells (SC) plus 3 x 10 7 bone marrow cells (BMC) derived from C57BL/6 (B6) mice on day -2, 200 mg/kg CY on day 0, and which were grafted with allogeneic B6 skin on day 14. When the C3H mice were treated with anti-CD4 mAb, 1 x 10 8 SC plus 5 x 10 7 BMC derived from F344 rat and CY, the F344 skin grafts survived slightlv longer (about 15 days) than those in untreated recipients (about 8.4 days). Such a prolongation of skin xenograft survival was considered donor-specific because rejection of 3rd party skin grafts from BN rats occurred significantly earlier than that of F344 skin grafts. In the recipient C3H mice treated with anti-CD4 mAb, F344 SC plus BMC and CY, mixed chimerism in the periphery was detected for a few days after CY administration, although intrathymic chimerism was not detected throughout this study. In these recipient C3H mice, cytotoxic T lymphocytes (CTL) against F344 antigens were completely abrogated though the delayed footpad reaction (DFR) remained at a low but significant level. Moreover, though antibody (Ab) activity against F344 SC was completely abrogated, neither Ab activity against F344 BMC, which seemed to have a background common to natural Ab activity, nor NK activity were abrogated by this treatment. These results suggested that DFR mediating cells directly mediated skin xenograft rejection in the recipient mice treated with anti-CD4 mAb, F344 cells, and CY. Such cells may interfere with establishment of mixed chimerism and longlasting skin xenograft tolerance, presumably in cooperation with CY-resistant Ab activity and NK cells.

TOLERANCE INDUCTION IN A FULLY ALLOGENEIC COMBINATION USING ANTI-T CELL RECEPTOR-αβ MONOCLONAL ANTIBODY, LOW DOSE IRRADIATION, AND DONOR BONE MARROW TRANSFUSION

In a murine strain combination disparate in both H-2 antigens and minor histocompatibility antigens consisting of C57BL/6 (B6; H-2b, Mls-1b) mice as recipients and AKR/J (AKR; H-2b, Mls-1a) mice as donors, we reported that the administration of anti-TCR-αβ mAb nonspecifically suppresses the ability to reject allografts. However, such an effect was only temporary and all grafts were eventually rejected within 40 days in the anti-TCR-αβ mAb-treated mice. In this study, to induce donor-specific tolerance, the transfer of donor bone marrow cells was added to the administration of anti-TCR-αβ mAb but donor bone marrow cells were rejected and failed to cause donor-specific unresponsiveness. After donor bone marrow cell transfer in the anti-TCR-αβ mAb-treated mice, the B cells of the recipients were observed along with the production of antidonor antibody. To abolish the residual lymphocyte populations, low dose irradiation was also added. A long-lasting skin allograft tolerance can be achieved by the tolerance system, in which low dose irradiation was added to the combined treatment with anti-TCR-αβ mAb and transfer of donor bone marrow cells. Such a protocol also established central and peripheral chimerism, which suggests that hematopoietic chimerism is necessary to maintain the tolerance. B cells were completely abolished in recipients given this combined treatment and their antibody production against donor antigens after the transfer of donor bone marrow cells was also completely suppressed. A possible role of B cells in the rejection of donor bone marrow cells before the establishment of chimerism is discussed.

TOLERANCE INDUCTION IN A FULLY ALLOGENEIC COMBINATION USING ANTI-T CELL RECEPTOR-αβ MONOCLONAL ANTIBODY, LOW DOSE IRRADIATION, AND DONOR BONE MARROW TRANSFUSION

In a murine strain combination disparate in both H-2 antigens and minor histocompatibility antigens consisting of C57BL/6 (B6; H-2b, Mls-1b) mice as recipients and AKR/J (AKR; H-2k, Mls-1a) mice as donors, we reported that the administration of anti-TCR-alpha beta mAb nonspecifically suppresses the ability to reject allografts. However, such an effect was only temporary and all grafts were eventually rejected within 40 days in the anti-TCR-alpha beta mAb-treated mice. In this study, to induce donor-specific tolerance, the transfer of donor bone marrow cells was added to the administration of anti-TCR-alpha beta mAb but donor bone marrow cells were rejected and failed to cause donor-specific unresponsiveness. After donor bone marrow cell transfer in the anti-TCR-alpha beta mAb-treated mice, the B cells of the recipients were observed along with the production of antidonor antibody. To abolish the residual lymphocyte populations, low dose irradiation was also added. A long-lasting skin allograft tolerance can be achieved by the tolerance system, in which low dose irradiation was added to the combined treatment with anti-TCR-alpha beta mAb and transfer of donor bone marrow cells. Such a protocol also established central and peripheral chimerism, which suggests that hematopoietic chimerism is necessary to maintain the tolerance. B cells were completely abolished in recipients given this combined treatment and their antibody production against donor antigens after the transfer of donor bone marrow cells was also completely suppressed. A possible role of B cells in the rejection of donor bone marrow cells before the establishment of chimerism is discussed.

Anti-CD3 treatment facilitates engraftment of full H-2-disparate donor bone marrow cells and subsequent skin allograft tolerance.

The aim of the present study was to induce engraftment of full H-2-disparate donor bone marrow cells and the development of subsequent transplantation tolerance. To this end, recipient H-2b mice were treated with anti-CD3 and on the same day received 6 Gy whole body irradiation as well as donor bone marrow cells (H-2d). Anti-CD3 treatment was chosen because it results in suppression of T cell function and in the release of CSF associated with enhancement of donor bone marrow engraftment. Stable, long-term chimerism measured in peripheral blood and mesenteric lymph nodes was obtained using this preparative regimen. In contrast, the use of anti-CD3 F(ab')2 fragments failed to induce donor bone marrow cell engraftment, suggesting indeed an important role of anti-CD3-mediated growth factor production in marrow engraftment. To overcome the side effects of anti-CD3 treatment (cytokine release syndrome), anti-CD4 was given 1 day before the treatment protocol. Omission of anti-CD3 resulted in failure of donor bone marrow engraftment, indicating the essential role of anti-CD3 treatment in marrow engraftment. Skin transplantation performed 2 and 6 months after this well-tolerated conditioning regimen showed indefinite survival of first and second grafts, respectively. In addition, specific CTL nonresponsiveness developed, demonstrating the presence of classical transplantation tolerance across a full H-2 barrier.

Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a nonmyeloablative regimen.

We have investigated the mechanism of tolerance induced by allogeneic bone marrow transplantation (BMT) in mice conditioned with a nonmyeloablative regimen. Permanent mixed chimerism and skin allograft tolerance were achieved when recipient B10 (H-2b) mice were pre-treated with anti-CD4 and anti-CD8 mAbs, thymic irradiation, and 3 Gy whole body irradiation, followed by injection of B10.A (H-2a) bone marrow cells. V beta 11+ T cells are normally deleted in I-E+ B10.A mice, but not in I-E- B10 mice. In spleens of mixed B10.A-->B10 chimeras, V beta 11+ B10 T cells were reduced but detectable in the first 6 wk after BMT, and later became undetectable. Splenic V beta 11+ T cells were unresponsive to receptor cross-linking with V beta 11-specific mAb, demonstrating anergy. B10 V beta 11+ T cells were also detected in spleens of mice that were thymectomized before BMT. In PBL of chimeras, V beta 11+ T cells were undetectable for at least 1.5 yr. Thymocyte chimerism and extensive clonal deletion of mature V beta 11+ B10 thymocytes were observed as early as 10 days post-BMT and at all subsequent time points in thymi of mixed chimeras. Rare I-E+ donor cells were detected in day 10 thymi, and these increased progressively in frequency at later times. In chimeras prepared in the reciprocal strain combination, B10-->B10.A, B10 donor V beta 11+ T cells were undetectable in PBL. Together, our results implicate both central clonal deletion and peripheral clonal anergy in tolerance induced with our regimen. A very low number of intrathymic donor cells or low density of Ag expression is sufficient to mediate extensive clonal deletion of thymocytes that recognize donor Ags.

Interference with cyclophosphamide-induced skin allograft tolerance by cyclosporin A.

In a murine strain combination identical in H-2 Ag but disparate in minor histocompatibility (H) Ag consisting of C3H/He (C3H; H-2k, Mls-1b) mice as recipients and AKR/J (AKR; H-2k, Mls-1a) mice as donors, a permanent skin allograft tolerance can be achieved by the cyclophosphamide (CP)-induced tolerance system that consists of i.v. injection of donor spleen cells (day -2) and i.p. injection of CP 2 days later (day 0). Such permanent take of allografts in CP-induced tolerant mice was interfered with by intramuscular injection of cyclosporin A (CsA) from day -5 to day -1 and their grafts were rejected by 21 days after grafting. Mls-1a-reactive CD4+V beta 6+ T cells in the periphery, as the indicator to follow the kinetics of donor-reactive T cells, increased on day 0 and day 3 in the C3H mice treated with AKR spleen cells alone, whereas they disappeared rapidly from day 0 to day 3 in CP-induced tolerant mice. When CsA capable of interfering with IL-2 production and T cell proliferation was administered before CP treatment in CP-induced tolerance system, the number of CD4+V beta 6+ T cells in periphery did not increase on day 0 and 3, but increased on day 7 in contrast to the decreased number of those in CP-induced tolerant mice. On day 7, MLR against donor cells was decreased in CP-induced tolerant mice, but maintained in CsA-interfered tolerant mice. These result may indicate that the destruction of donor-Ag-stimulated, proliferating T cells by CP is interfered with by CsA, probably because CsA inhibits the proliferation of donor-reactive T cells at the time of CP treatment. Furthermore, these results also implicate that the protocol for immunosuppression with CsA and antimetabolites has to be designed carefully in clinical transplantation.

Similarity and difference in the mechanisms of neonatally induced tolerance and cyclophosphamide-induced tolerance in mice.

The mechanisms of cyclophosphamide (CP)-induced tolerance were investigated by comparing with those of neonatally induced tolerance. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were given i.v. either AKR/J Sea (AKR; H-2k, Mls-1a) or (AKR x C3H)F1 (AKC3F1; H-2k, Mls-1a/b) spleen cells and treated i.p. with CP 2 days later, a long-lasting skin allograft tolerance to AKR was induced in each case without any signs of graft-vs-host disease (GVHD). However, typical signs of GVHD were observed in the C3H mice neonatally tolerized with AKR spleen cells, but not in those tolerized with AKC3F1 spleen cells. The expression of TCR V beta 6, which is strongly correlated with the reactivity to Mls-1a Ag (of donor AKR origin), in the periphery was quite different between the two types of tolerant C3H mice. Namely, in the lymph nodes of the C3H mice tolerized with AKR spleen cells and CP, only CD4(+)-V beta 6+, but not CD8(+)-V beta 6+, T cells selectively disappeared, whereas both of them were abrogated in the lymph nodes of the C3H mice neonatally tolerized of AKR. By contrast, in the thymus of the two types of tolerant C3H mice, both CD4+CD8- and CD4-CD8+ single-positive thymocytes expressing TCR V beta 6 were clonally deleted, suggesting that the thymic involvement was the same in each type of tolerance. These results suggest that the preferential disappearance of the CD4(+)-V beta 6+ T cells (of host origin) and the effector T cells of GVHD (of donor origin) occurred only in the periphery of the C3H mice tolerized with AKR spleen cells plus CP and was attributable to the destruction of Ag-stimulated T cells by the CP treatment. In contrast, the intrathymic clonal deletion of immature V beta 6+ T cells was a common mechanism for both of the tolerance induction systems.

Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system.

The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR.

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.

Evidence for involvement of clonal anergy in MHC class I and class II disparate skin allograft tolerance after the termination of intrathymic clonal deletion.

Mechanisms of cyclophosphamide (CP)-induced tolerance to class I (D) and class II (IE) alloantigens were studied. Transplantation tolerance across H-2D plus IE Ag-barriers has been achieved when B10.Thy-1.1 (Kb,IAb,IE-,Db; Thy-1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) mice (5R; kb,IAb,IEb,Dd; Thy-1.2) and treated i.p. with 200 mg/kg of CP 2 days later. The tolerant state in the early and the late stage was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor Ag. In the tolerant B10.Thy-1.1 mice treated with 5R cells 28 days earlier and followed by CP, intrathymic clonal deletion of V beta 11+ T cells reactive to IE-encoded antigens was observed in association with intrathymic mixed chimerism. 5R skin survived, however, even after the clonal deletion of V beta 11+ T cells terminated by 180 days after tolerance induction. V beta 11+ T cells, which reappeared in the periphery of the recipient B10.Thy-1.1 mice bearing 5R skin at this stage, were not capable of proliferating in response to receptor cross-linking with V beta 11-specific mAb. Furthermore, the CTL activity against class I (Dd) alloantigens of spleen cells from these tolerant mice was restored by the addition of IL-2 to MLC. Thus, our experiments provide direct evidence that tolerance to both class I (Dd) and class II (IEb) alloantigens by clonal allergy occurs during the termination of intrathymic clonal deletion. These results clearly show practical hierarchy of the mechanisms of transplantation tolerance.

Sequential mechanisms of cyclophosphamide-induced skin allograft tolerance including the intrathymic clonal deletion followed by late breakdown of the clonal deletion.

Abstract The cellular basis of the transplantation tolerance in a model system of BALB/c (Mls-1b) mice rendered cyclophosphamide (CP)-induced tolerant to DBA/2 (Mls-1a) skin allograft was investigated by assessing V beta 6+ T cells. From our results, three major mechanisms that are essential to the CP-induced skin allograft tolerance were sequentially elucidated. The first mechanism was destruction of donor-Ag-stimulated T cells in the periphery by CP treatment. The second mechanism was intrathymic clonal deletion of donor-reactive T cells, such as V beta 6+ T cells, correlating strongly with intrathymic mixed chimerism. The clonal deletion, however, was not always essential for the maintenance of the skin allografts, because DBA/2 skin survived even after the clonal deletion terminated and V beta 6+ T cells reappeared in the periphery of the recipient BALB/c mice. The third mechanism was generation of tolerogen-specific suppressor T cells, especially in the late stage of the tolerance. In contrast, the clonal anergy that is evidenced by the specific suppression of mixed lymphocyte reaction in the recipient BALB/c mice after injecting with DBA/2 spleen cells alone was not considered as a significant mechanism in prolonging skin allograft survival because such anergic mice showed accelerated rejection of the skin allografts. These results may suggest practical hierarchy of the mechanisms of CP-induced allograft tolerance.

The requirement of intrathymic mixed chimerism and clonal deletion for a long‐lasting skin allograft tolerance in cyclophosphamide‐induced tolerance

Mechanisms of cyclophosphamide (CY)-induced tolerance were studied. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were primed i.v. with 1 x 10(8) viable spleen cells from H-2-identical AKR/J Sea (AKR; H-2k, Mls-1a) mice and treated with 200 mg/kg of CY 2 days later, a long-lasting skin allograft tolerance to AKR was established. When [C57BL/6 Sea (B6; H-2b, Mls-1b) x AKR]F1 (B6AKF1) cells were used as the tolerogen, however, only a moderate, but not long-lasting, skin tolerance to AKR was observed. In the C3H mice treated with AKR cells and CY, the intrathymic clonal deletion of V beta 6+ T cells, which are strongly correlated with reactivity to Mls-1a antigens, was observed in the chimeric thymus on day 35, although neither the clonal deletion of V beta 6-bearing T cells nor the mixed chimerism was observed in the thymus on day 14. In the C3H mice treated with B6AFKF1 cells followed by CY, however, neither the clonal deletion of V beta 6+ T cells nor the mixed chimerism was observed in the thymus throughout the test period. In the lymph nodes of the C3H mice treated with AKR cells and CY, only CD4+ V beta 6+ T cells, bur not CD8+V beta 6+ T cells, had selectively decreased by day 14, and they were hardly detectable on day 35. The selective decrease of CD4+V beta 6+ T cells in the lymph nodes was also observed by day 14 when B6AKF1 cells were used as the tolerogen, although CD4+V beta 6+ T cells gradually increased on day 35, at which time almost all skin grafts from AKR had already been rejected. These results strongly support the necessity of the intrathymic mixed chimerism and clonal deletion of donor-reactive T cells for a long-lasting skin allograft tolerance in CY-induced tolerance.

Induction of transplantation tolerance by intravenous injection of allogeneic lymphocytes across an H‐2 class II mismatch. Different mechanisms operate in tolerization across an H‐2 class I vs. H‐2 class II disparity

Previously, we have shown that the intravenous (i.v.) injection of allogeneic lymphocytes across an H-2 class I-mutant disparity leads to specific skin allograft tolerance caused by irradiation-sensitive donor T cells, which function as veto cells. In the present study, we show that the i.v. injection of H-2 class II-incompatible spleen cells also results in specific skin allograft tolerance. However, tolerance induction depends on the presence of irradiation-resistant non-T cells in the donor cell inoculum. Thus, different mechanisms operate in tolerance induction across an H-2 class I vs. H-2 class II mismatch. I.v. injection of allogeneic spleen cells across an H-2 class I plus class II disparity does not result in skin allograft tolerance. Finally, our data show that transfusion-induced suppression of the delayed-type hypersensitivity response against alloantigens does not correlate with skin allograft tolerance induced by i.v. injected allogeneic lymphocytes. In conclusion, the type of H-2 mismatch between transfusion donor and recipient not only determines the occurrence of allograft tolerance but also the mechanism leading to tolerization.

Importance of suppressor T cells in cyclophosphamide-induced tolerance to the non-H-2-encoded alloantigens. Is mixed chimerism really required in maintaining a skin allograft tolerance?

Mechanisms of cyclophosphamide (CP)-induced tolerance were studied. When AKR/J Sea (AKR: H-2k) mice were primed i.v. with 5 x 10(7) spleen cells plus 1 x 10(7) bone marrow cells from [C57BL/6 Slc (B6; H-2b) x C3H/He Slc (C3H; H-2k)]F1 (B6C3F1) mice and treated i.p. with 200 mg/kg CP 2 days later, the survival of C3H skin was moderately prolonged, but the survival of either B6 or B6C3F1 skin was not prolonged. By this treatment, however, mixed chimerism of B6C3F1 cells in the AKR mice was not established. When C3H cells were used as the tolerogen, a minimal degree of mixed chimerism associated with profound tolerance to C3H skin was established. Similar results were observed in various donor-recipient combinations. When C3H skin was grafted in the AKR mice 12 wk after the treatment with C3H cells and CP, or B6C3F1 cells and CP, survival of the grafted C3H skin was prolonged remarkably or moderately, respectively, although mixed chimerism was not detectable at the timing of grafting in either of the groups. In this late stage of tolerance, a strong level of tolerogen-specific suppressor cell activity was observed in those tolerant AKR mice. The suppressor activity was mainly attributable to T cells. These results suggest that the role of Ts cells in order to maintain skin tolerance is important in our CP-induced tolerance system, especially in the late stage of tolerance. Moreover, the generation of the Ts cells does not necessarily require the establishment of a long term mixed chimeric state.

Intrathymic clonal deletion of V beta 6+ T cells in cyclophosphamide-induced tolerance to H-2-compatible, Mls-disparate antigens.

When C3H (H-2k, Mls-1b) mice were primed intravenously with 10(8) viable spleen cells from AKR (H-2k, Mls-1a) and treated intraperitoneally with 200 mg/kg of cyclophosphamide (CP) 2 d later, not only a long-lasting skin allograft tolerance but also a tolerance in mixed lymphocyte reaction to Mls-1a-encoded antigens was established. The cellular mechanisms of CP-induced tolerance were examined by assessing the V beta 6-bearing T cells that are strongly correlated with reactivity to Mls-1a-encoded antigens bound to MHC class II molecules. At the relatively early stage (2 or 5 wk) after the CP treatment, CD4+-V beta 6+ T cells of C3H origin were preferentially eliminated in the lymph nodes of the tolerant mice, whereas CD8+-V beta 6+ T cells remained. On the other hand, neither CD4+CD8- nor CD4-CD8+ thymocytes bearing a high density of V beta 6 was detected in the chimeric thymus. Namely, in the thymus of the tolerant C3H mice, neither mixed chimerism nor the clonal deletion of the V beta 6-bearing T cells was observed on day 14, whereas both of them were observed on day 35. The clonal deletion and mixed chimerism in the thymus were lasting for greater than 10 wk after the CP treatment. Expression of V beta 6 on the peripheral T cells in the tolerant C3H mice gradually reduced in the process of time. These results strongly suggested that the clonal deletion in the thymus was one of the essential mechanisms in the CP-induced tolerance system.

Allograft tolerance induction in adult mice associated with functional deletion of specific CTL precursors.

Rejection of H-2 class I bm 1 mutant skin allografts by B6 recipient mice is mediated by a population of CD8+ anti-bm1 cytotoxic T-lymphocytes that produces and consumes its own T helper factor in response to bm1 skin allografts (dual function CTL). Previously we have demonstrated that transfusion of allogeneic lymphocytes across an H-2 class I disparity induces specific long-lasting skin allograft survival. We now show that intravenous injection of allogeneic spleen cells across the bm1 mutant disparity results in a temporary decrease of donor-reactive CTLp in the spleen of recipient mice, lasting for approximately five weeks. The sponge matrix allograft model was used to show that allograft tolerance is caused by a specific functional clonal deletion of CTLp within the allograft. We propose that dual function CTL are vetoed by donor T cells, resulting in skin allograft tolerance.

Induction of Tolerance across Major Barriers Using a Two-Step Method with Genetic Analysis of Tolerance Induction

Using a murine skin allograft tolerance induction system that consists of intravenousinjection of 1 × 10 8 allogeneic spleen cells followed by intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide (CP) 2 days later, sensitivity to tolerance induction was examined across various histocompatibility (H) barriers. Although each group of class I, class II or multiminor H antigens was not by itself a prohibitively strong barrier, resistance to tolerance induction increased when the three types of barriers were combined in various ways. When the donor-recipient combinations were disparate at the entire spectrum of both H-2 plus non H-2 antigens (fully allogeneic), profound tolerance to skin allografts was not induced by this method in any of the combinations examined. Based on these results, induction of tolerance across fully allogeneic barriers was attemptedin C57BL/10SnJ (B10; H-2 b) mice against C3H/HeSnj (C3H; H-2 k) strain by addressing the H barriers as two separate challenges. B10 mice were first given B 10.BR/SgSnJ (B 10.BR; H-2 k) spleen cells plus CP to make them tolerant to the H-2 k component represented among C3H antigens, and then later were given C3H spleen cells plus CP to establish a tolerant state to the remainder of the disparate antigens of the C3H donors. After these two separate manipulations, C3H skin was accepted in the B10 mice, and normal hair growth was observed in the grafted C3H skin. By contrast, B10 mice given C3H spleen cells plus CP and then again another injection of C3H spleen cells plus CP were not rendered tolerant to C3H skin. In B10 mice, tolerance to C3H induced with B10.BR spleen cells plus CP and then C3Hspleen cells plus CP was specific to C3H, and the tolerant B10 mice rejected third-party skin from DBA/2J (DBA; H-2 d) strain in a normal fashion. In transfer experiments, the mechanism of tolerance was found to be based largely on reduction of the effector cells rather than on a mechanism involving active suppression. Assays for chimerism revealed that maintaining the tolerant state required persistence of cells of donor origin. These data indicate that in a primary immune response to a certain dose of allogeneic cells(tolerogen), the existence of a relatively large proportion of potentially reactive clones in the host may trigger proliferation of only a part of the population and some of the potentially reactive cells may differentiate rapidly without a prolonged period of proliferation. The cells which are not responding by proliferation induced by the antigenic stimulation may then escape destruction by CP and ultimately survive to cause rejection of donor-type hematopoietic cells and skin grafts.

The Necessity of Both Allogeneic Antigens and Stem Cells for Cyclophosphamide-Induced Skin Allograft Tolerance in Mice

We have reported that in an H-2 identical murine combination of AKR/J (AKR, H-2 k, Thy1.1) and C3H/HeJ (C3H, H-2 k, Thy1.2), specific tolerance to C3H skin in AKR mice is induced only when both intravenously (i.v.) 1 × 10 8 viable C3H spleen cells and, two days later, intraperitoneally (i.p.) 200 mg/kg cyclophosphamide (CP) have been given. To further examine this mechanism of tolerance, we used 2000R-irradiated C3H spleen cells as an antigen source and bone marrow cells depleted of Thy1.2 + cells and Ia + cells as a stem cell source. When a mixture of 1 × 108 irradiated spleen cells and 3 × 10 7 bone marrow cells was used as tolerogen and 200 mg/kg CP was administered two days later, a profound and specific long-lasting tolerance was induced. This tolerant state, however, was less profound than that induced with spleen cells plus CP. When the number of irradiated spleen cells was fixed at 1 × 10 8, the tolerant state was dose-dependent on the quantity of bone marrow cells. On the other hand, when the number of bone marrow cells was fixed at 1 × 10 6, tolerance induction depended on the dosage of irradiated spleen cells. Tolerance induced with irradiated spleen cells plus bone marrow cells and CP was tolerogen specific. Tolerance was never induced when the bone marrow cells had been irradiated with 2000 R prior to injection. Transfer experiments showed that the tolerant state, in its acute phase, appeared to be predominantly based on reduction of functionally reactive cells. The prolongation of skin allograft survival in tolerant mice could not be attributed directly to suppressor cells, nor was any evidence of suppressive factor induction observed. In the chronic phase, however, the importance of the suppressive mechanisms appeared to be relatively increased. EPICS analysis of the thymocytes using fluorescein-conjugated anti-Thy1.1 and anti-Thy1.2 antibodies showed that a minimal degree of mixed chimerism had been established in the tolerant mice. Moreover, both T cells and Ia + cells had beneficial effects on the induction of tolerance. We conclude that in the tolerance induced by spleen cells plus CP, histocompatibility antigens expressed on the surface of the spleen cells were essential to the antigen-stimulated cell destruction mechanism. Stem cells contained in the spleen cells also appeared to be crucial for maintaining tolerance by establishing a minimal degree of mixed chimerism.