The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts

Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.     

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice

Abstract: Mouse CD4⁺ T cells efficiently develop in fetal pig thymus (FPTHY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8⁺ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of Vβ5.1/5.2⁺ and Vβ11⁺ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of Vβ6, Vβ7, and Vβ8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthyrnic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8⁺ cells can undergo normal functional and phenotypic maturation in FPTHY grafts. Thus, other explanations must be sought for the failure of CD8'cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THYLIV-grafted mice.     

The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice

BACKGROUND: Xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult. METHODS: Neonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts. RESULTS: Repopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice. CONCLUSIONS: NP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.     

Fetal porcine thymus engraftment,survival and CD4 reconstitution in alphaGal-KO mice is impaired in the presence of high levels of antibodies against alphaGal

Xenospecific T-cell tolerance can be induced among murine and human T-cells by porcine thymic grafting. However, anti-alpha 1,3-galactosyltranserase (alphaGal) (Galalpha1-3Galbeta1-4GlcNAc-R) natural antibodies (NAbs) pose a major barrier to porcine xenografts in humans. We used alphaGal knockout (KO) and muchain KO mice to explore the effect of natural anti-alphaGal and other xenoantibodies on porcine thymic engraftment and to examine the potential of thymic tissue to tolerize anti-alphaGal antibody-producing cells. Thymectomized [adult thymectomy (ATX)] non-immunized and rabbit red blood cell (RRBC) pre-transplant immunized alphaGal-KO (knockout), wild-type (WT) and mu chain KO B6 mice were treated with 3Gy total body irradiation (TBI), and T and natural killer (NK) cell depleting monoclonal antibodies (mAbs). These conditioned mice were grafted with fetal porcine thymus and liver (FP THY/LIV) tissue under the kidney capsule. Flow cytometric analysis was performed to follow CD4 reconstitution as a measure of FP THY engraftment and function. Only mice with >10% CD4+ peripheral blood lymphocytes (PBL) were considered successfully engrafted. Enzyme-linked immunosorbent assay (ELISA) was used to assess the kinetics of immunoglobulin M (IgM) and IgG anti-alphaGal antibodies. Anti-pig antibodies were monitored by flow cytometry (FCM). FP THY engrafted successfully in most of the immunoglobulin deficient mice (11 out of 12, 92%) and the outcome was similar in WT B6 controls (8 out of 12, 67%). Non-immunized alphaGal-KO mice grafted with FP THY had a similar success rate (7 out of 11) to that observed in non-immunized alphaGal-WT controls (2 out of 4). In contrast, alphaGal-KO mice immunized pre-transplant with RRBC, then grafted with FP THY/LIV, showed a significant reduction in the success of thymic grafting (2 out of 9, 22%) compared with pre-transplant immunized WT controls (4 out of 7; 57%) and non-immunized alphaGal-KO mice (7 out of 11, 64%). Anti-Gal and anti-pig antibody levels were not markedly augmented by porcine thymus grafts in mice with successful thymus grafts. FP THY engraftment is impaired in the presence of high levels of anti-alphaGal xenoantibodies. However, low levels of anti-alphaGal antibodies and other mouse anti-pig NAbs appear not to play a major role in the rejection of FP THY. Although grafting FP THY expressing the alphaGal epitope did not tolerize B cells producing anti-alphaGal antibodies in a T-cell independent manner, it prevented T-cell dependent sensitization by inducing T-cell tolerance to porcine antigens.     

Highly disparate xenogeneic skin graft tolerance induction by fetal pig thymus in thymectomized mice: Conditioning requirements and the role of coimplantation of fetal pig liver.

BACKGROUND: Highly disparate xenogeneic pig skin graft tolerance and efficient repopulation of mouse CD4+ T cells are achieved in thymectomized (ATX) B6 mice that receive T cell and natural killer (NK) cell depletion by injection of a mixture of monoclonal antibodies (mAbs) (GK1.5, 2.43, 30-H12, and PK136) on days -6, -1, +7, and +14 and 3 Gy total body irradiation (TBI) followed by implantation of fetal pig thymus/liver (FP THY/LIV) grafts on day 0. The requirements for each treatment in this model to achieve pig skin graft tolerance have not previously been defined. Therefore, we performed a series of experiments to address the role of each treatment in achieving maximal skin graft tolerance. METHODS: Peripheral mouse CD4+ T-cell repopulation and pig skin graft survival were followed in this pig-to-mouse model in which recipient B6 mice were treated with modified regimens that omitted thymectomy, 3 Gy TBI, anti-Thy1.2, and anti-NK1.1 mAbs, injection of a mixture of mAbs on day +14, or coimplantation of FP LIV, respectively. RESULTS: Prolongation but not permanent survival of donor MHC-matched pig skin grafts was observed in euthymic B6 mice that received T and NK cell depletion, 3 Gy TBI, and 7 Gy thymic irradiation and FP THY/LIV in the mediastinum, suggesting that full xenogeneic tolerance was not achieved in euthymic mice. However, after grafting FP THY alone to ATX B6 mice treated either with the "standard" regimen, or with a conditioning regimen that omitted all components of the conditioning regimen except treatment with anti-CD4 and anti-CD8 mAbs, efficient peripheral repopulation of mouse CD4+ T cells and long-term donor MHC-matched pig skin graft acceptance were observed. CONCLUSIONS: Highly disparate xenogeneic pig skin graft tolerance can be achieved by grafting FP THY alone in anti-CD4 and anti-CD8 mAb-treated ATX B6 mice, but not in euthymic B6 mice. Additional treatment of ATX recipient mice with anti-Thy1.2 and NK1.1 mAbs and 3 Gy TBI is not essential for donor pig skin graft tolerance induction.     

Presence of Functional Mouse Regulatory CD4+CD25+T Cells in Xenogeneic Neonatal Porcine Thymus-Grafted Athymic Mice

Xenotransplantation with porcine thymus is emerging as a possible means to reconstitute host cellular immunity and to induce immune tolerance in rodents and large animals. However, the presence of regulatory T cells (Treg cells) in this model needs to be determined. We herein demonstrated that efficient repopulation of mouse CD4+CD25+Treg cells was achieved in Balb/c nude mice by grafting neonatal porcine thymic tissue (NP THY). Mouse CD4+CD25+T cells expressed normal levels of Foxp3 in NP THY-grafted nude mice. Furthermore, these CD4+CD25+Treg cells showed significant inhibitory effects on the cell proliferation or interleukin-2 products of syngeneic T cells to alloantigens, Con A or a peptide antigen, although the potent immunosuppressive function might be lower than CD4+CD25+Treg cells in Balb/c mice. CD4+CD25+T cells in NP THY-grafted nude mice showed significantly stronger inhibition on the response to donor porcine antigens of CD4+CD25(-)T cells than CD4+CD25+Treg cells in Balb/c mice. Both CD4+CD25+Treg cells in NP THY-grafted nude and Balb/c mice prevented the development of autoimmune disease mediated by syngeneic CD4+CD25(-)T cells in a similar efficient way in the secondary recipients. These findings provide evidence for the potential involvement of CD4+CD25+Treg cells in keeping self-tolerance and transplant tolerance in this xeno-thymus transplantation model.     

Neutralization of interleukin-2 retards the growth of mouse renal cancer

OBJECTIVE: To examine the significance of the thymus and the neutralization of interleukin-2 (IL-2) in treating renal cancer, as the involvement of immunoregulatory cells in tumour development in vivo is well known, naturally occurring CD25+ CD4+ T cells possess potent immunoregulatory functions, and they are of thymic origin dependent on IL-2. MATERIALS AND METHODS: We first tested activity against mouse renal cell carcinoma (RENCA) cells by adoptively transferring splenocytes of euthymic Balb/c mice depleted of CD25+ cells into athymic Balb/c nude mice bearing established macroscopic RENCA tumours. Second, we tested the anti-RENCA activity in euthymic mice bearing macroscopic RENCA tumours by neutralizing IL-2. RESULTS: The intravenous administration of CD25+ cell-depleted splenocytes of euthymic Balb/c mice initiated the retardation of macroscopic RENCA tumours subcutaneously established in athymic Balb/c mice. The tumour site showed massive lymphocyte infiltration of mainly CD4+ T cells. By eliminating either the CD4+ cells, CD8+ cells, or natural killer (NK) cells with antibodies after the adoptive transfer of CD25+ cell-depleted splenocytes of euthymic Balb/c mice, macroscopic RENCA tumour retardation was abrogated. The growth of macroscopic RENCA tumour established in euthymic Balb/c mice was also retarded with IL-2 neutralization alone by anti-IL-2 monoclonal antibody (mAb), as well as co-administration of anti-IL-2 mAb and anti-CD25 mAb compared with that of the controls given vehicle. After tumour inoculation, peri- and intratumoral infiltration of CD4+ and CD8+ T cells was very prominent in RENCA tumours in hosts given anti-IL-2 mAb, regardless of the administration of anti-CD25 mAb. Two x 10(5) units of recombinant human IL-2 reverted the retardation of RENCA tumour growth caused by the anti-IL-2 mAb. IL-2 neutralization alone in euthymic Balb/c mice with no tumour inoculation did not suppress splenic CD25+ CD4+ T cells. CONCLUSION: Both the intravenous administration of CD25+ cell-depleted splenocytes of euthymic Balb/c mice into athymic Balb/c nude mice and IL-2 blocking with anti-IL-2 mAb in euthymic Balb/c mice retarded the growth of macroscopic RENCA tumours in vivo.     

Extrathymic deletion of CD8+ alloreactive T cells in a transgenic T cell receptor model of neonatal tolerance.

BACKGROUND: Immunological tolerance to foreign antigen is most easily achieved during the neonatal period. Although deletion of T cells has been demonstrated in neonatal tolerance models in which donor and recipient express different MHC class II molecules, the requirement for deletion in MHC class I-disparate models is less clear. To address this issue, we used as recipient the T cell receptor (TCR) transgenic mouse (TgM) strain 2C in which the majority of CD8+ T cells express a single alpha/beta TCR alloreactive to H-2Ld, thus facilitating direct monitoring of the class I alloreactive population. METHODS: Newborn (less than 24 hr of age) 2C TgM received injections i.v.with syngeneic C57BL/6J (H-2b) (B6) or semiallogeneic (B6xDBA)F1 (H-2bd; H-2Ld+) splenocytes. Adults were subsequently analyzed in terms of tolerance, deletion of 2C+ T cells, and chimerism. RESULTS: The results showed that semiallogeneic-, but not syngeneic-, injected neonates were unresponsive as adults to H-2Ld-expressing target cells in vitro and the majority of these mice accepted H-2Ld+ skin grafts. Delaying the injection to 72 hr after birth or reducing the number of cells injected essentially abolished in vivo unresponsiveness in 2C recipients. Thus, the 2C TCR Tg model demonstrates the characteristics typical of neonatal tolerance. Injection of 2C neonates within 24 hr of birth with semiallogeneic versus syngeneic cells led to more than a 12-fold reduction of CD8+ 2C+ T cells in adult spleen and LNCs. In contrast, deletion of CD8+ 2C+ cells in adult thymus was not consistently observed. Based on MHC class II expression to distinguish donor (I-E+) and recipient (I-E-) cells, semiallogeneic-injected mice were chimeric in spleens and lymph nodes (LNs). CONCLUSIONS: These results demonstrate that neonatal MHC class I tolerance in the adult is associated with low level hematopoietic chimerism and extrathymic deletion of alloreactive CD8+ T cells.     

The effect of burn injury on CD8+ and CD4+ T cells in an irradiation model of homeostatic proliferation

BACKGROUND: Homeostatic proliferation of T cells has recently been shown to be an important mechanism in the host response to infection. However, its role in the T cell response to burn injury is unknown. In this study, we examine the effect of burn injury on CD4+ and CD8+ T cell homeostatic proliferation after irradiation. METHODS: Wild-type C57BL/6 female mice were irradiated with six grays ionizing radiation and 48 hours later, syngeneic whole splenocytes or purified CD4+ or CD8+ T cells labeled with carboxy-fluorescein diacetate, succinimidyl ester were adoptively transferred. Two days later, mice underwent a 20% burn injury, followed by splenocyte harvest 3 and 10 days after injury. RESULTS: Burn mice demonstrate increased splenic cellularity and CD8+ T cell proliferation after adoptive transfer of either purified CD8+ cells or whole spleen populations compared with unburned (sham) mice. In contrast, CD4+ T cell proliferation after burn injury is unchanged after adoptive transfer of whole spleen cells and drastically decreased after adoptive transfer of a purified CD4+ population compared with sham mice. Ten days after burn injury CD8+ T cells continue to demonstrate greater proliferation than CD4+ T cells. CONCLUSIONS: CD8+ T cells are more robust than CD4+ T cells in their proliferative response after burn injury. In addition, CD8+ T cell proliferation appears less reliant on other immune cells than purified CD4+ T cell proliferation. These data reiterate the importance of CD8+ T cells in the initial immune response to burn injury.     

Peripheral tolerance in transgenic mice expressing class I MHC L(d) only on cardiac cells

BACKGROUND: The fate of autoreactive T cells exposed to extrathymic self-antigen is examined in a double transgenic (DTG) mouse [(L(d+) cardiacx2C)F1], where cardiac myocytes alone express L(d) and T cells express an antigen receptor (2C TCR) against L(d). METHODS: Na?ve cardiac L(d+) single transgenic (STG) mice (before breeding with 2C) and DTG mice were examined for evidence of autoimmunity. The L(d+) STG hearts were then transplanted to syngeneic L(d-) wild type C57BL/6 to evaluate the heart's immunogenicity. L(d+) skin grafts were transplanted to non-transgenic B6, transgenic 2C, STG, and DTG mice. Phenotype analysis of peripheral 1B2+(identifies 2C T cells), CD4+, and CD8+ T cells was performed by FACS. In vitro MLC and CTL, with and without the addition of IL-2 and suppressor cell assays, were evaluated. RESULTS: Neither STG nor DTG hearts developed any evidence of autoimmunity by histology. In contrast, B6 mice rejected the L(d+) STG heart in 17+/-9.7 days (P<0.01), while a syngeneic B6 heart transplant was accepted indefinitely. Survival of L(d+) skin grafts was prolonged in both STG and DTG mice. FACS quantitation revealed that while there was no deletion of peripheral 2C cells in the DTG, these 2C T cells did have a significantly reduced proliferative and cytotoxic response to H-2L(d). Restoration of the proliferative and cytotoxic response of the DTG cells by the addition of IL-2 was consistent with a state of anergy. CONCLUSIONS: These findings suggest that the expression of extrathymic class I MHC expression alone did not trigger autoimmune reactions but that the T cells can be rendered anergic to the specific 'self' antigen.     

Mediation of skin allograft rejection in scid mice by CD4+ and CD8+ T cells.

We analyzed the role of CD4+ and CD8+ T cells in H-2-disparate skin allograft rejection in the mutant mouse strain C.B-17/Icr scid with severe combined immunodeficiency. On the day of skin allografting, scid mice were adoptively transferred with negatively selected CD4+ or CD8+ splenocytes from normal unsensitized C.B-17/Icr mice. These populations were obtained using a double-mAb--plus--complement elimination protocol using anti-CD4 or anti-CD8 mAb that resulted in no detectable CD4+ or CD8+ cells by FACS and negligible numbers of cytolytic T lymphocytes by limiting dilution analysis in anti-CD8 treated populations. Spleen cells were removed from grafted mice at the time of rejection and were tested in vitro for antidonor reactivity in several assays: mixed lymphocyte culture, cell-mediated lympholysis, and LDA for CTL and for IL-2-producing HTL. The presence of Thy 1.2+, CD4+, or CD8+ cells was determined by FACS. All control C.B-17 mice and scid mice adoptively transferred with nondepleted CD4+, and CD8+ cells rejected skin allografts with similar mean survival times (15.6 +/- 1.5, 18.8 +/- 3.4, 18.0 +/- 5.4, respectively), whereas control scid mice retain skin allografts indefinitely (all greater than 100 days). C.B-17 syngeneic grafts survived indefinitely in all groups. At the time of rejection, splenocytes from scid mice receiving CD4+ cells had negligible donor-specific cytotoxicity in CML and negligible numbers of CTL by LDA, but demonstrated a good proliferative response in MLC and IL-2-producing cells by LDA (frequency = 1/1764). There were no detectable CD8+ cells present by FACS analysis. Conversely, splenocytes from scid mice adoptively transferred with CD8+ cells had strong donor-specific cytotoxicity in CML (58.8% +/- 16.1%) and CTL by LDA (frequency = 1/3448), but no significant proliferation was detected in MLC. There were no detectable CD4+ cells by FACS, but there were small numbers of IL-2-producing cells by LDA (frequency = 1/10,204). These data demonstrate that CD4+ cells adoptively transferred into scid mice are capable of mediating skin allograft rejection in the absence of any detectable CD8+ cells or significant functional cytolytic activity. The adoptive transfer of CD8+ cells also results in skin allograft rejection in the absence of detectable CD4+ cells. The detection of small numbers of IL-2 secreting cells in these mice may indicate that CD(8+)-mediated allograft rejection in this model is dependent on IL-2-secreting CD8+ cells.     

Induction of regulatory T cells from mature T cells by allogeneic thymic epithelial cells in vitro

The ability of thymic epithelial cells (TEC) to re‐educate mature T cells to be regulatory T cells has not been addressed. In the present study, this issue was directly investigated by co‐culturing of mature T cells and allo‐TECs. B6 macrophage cell line 1C21‐cultured BALB/c splenocytes responded to B6 antigens in vitro. However, BALB/c splenocytes precultured with B6‐derived TECs 1‐4C18 or 1C6 did not proliferate to B6 antigens, but responded to rat antigens. Exogenous interleukin‐2 (IL‐2) failed to revise the unresponsiveness of these T cells. Allo‐TEC‐cultured T cells predominantly expressed Th2 cytokines (IL‐4 and IL‐10). B6 TEC‐cultured BALB/c splenocytes markedly inhibited the immune responses of naïve BALB/c splenocytes to B6 antigens, but not to rat or the third‐party mouse antigens. BALB/c nude mice that received naïve syngeneic splenocytes rejected B6 or rat skin grafts by 17 days postskin grafting; however, co‐injection of B6 TEC‐cultured BALB/c splenocytes significantly delayed B6 skin graft rejection (P < 0.01), with the unchanged rejection of rat skin grafts. These studies demonstrate that allo‐TECs are able to ‘educate’ mature T cells to be regulatory cells, and suggest that regulatory cells derived from mature T cells by TECs may play an important role in T cell tolerance to allo‐ and auto‐antigens.     

Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells

BACKGROUND: Donor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natural killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neonatal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell reconstitution, with less reliable tolerance induction. For a number of reasons, it would be advantageous to use neonatal rather than fetal pigs as donors. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved outcomes to be achieved. MATERIALS AND METHODS: Multiple or single fragments of neonatal porcine thymus tissue were grafted with a splenic fragment to different sites (mediastinum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK-cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitoneal injection containing 1 x 10(7) donor splenocytes. Donor-specific skin graft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xenoantibodies were followed by flow cytometry. RESULTS: Peripheral repopulation of CD4+ cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tissue in either the mediastinum or the mesentery, but not in mice grafted under both kidney capsules with the same amount of tissue. The level of CD4 reconstitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five percent of mice with >20% of CD4+ cells among peripheral blood lymphocytes (PBL) by 13 weeks posttransplantation accepted donor porcine skin, while rejecting either non-donor neonatal porcine or mouse BALB/c skin allografts. In contrast, only 29% of grafted mice with <20% CD4+ cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either low or high levels of anti-pig xenoantibodies of the IgM, IgG1, and IgG2a isotypes. Grafted mice with >20% CD4+ cells all had low levels of anti-pig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MLR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators. CONCLUSION: Grafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 compartment than does grafting under the kidney capsule. Good CD4 reconstitution was associated with optimal donor-specific skin graft tolerance and avoidance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.     

The role of leucyl-leucine methyl ester-sensitive cytotoxic cells in skin allograft rejection.

Treatment of murine spleen cells (SpC) with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) depletes L3T4(+) and Lyt2(+) cytotoxic T lymphocyte precursors and the capacity to generate lethal graft-versus-host disease in semiallogeneic class I + II MHC and multiple non-MHC-disparate recipient mice, whereas T helper cell function is preserved. In the present studies the role of Leu-Leu-OMe-sensitive CTL in skin graft rejection was examined. C57BL/6J (B6) mice were serially thymectomized, lethally irradiated, reconstituted with T cell-depleted bone marrow, and treated with intraperitoneal injections of anti-L3T4 and anti-Lyt2 monoclonal antibodies. These adult thymectomized, bone marrow-reconstituted, T cell-depleted (ATXBM, TCD) mice were unable to reject B6xDBA/2F1 (B6D2F1) skin grafts. When such ATXBM, TCD mice were reconstituted with 7 x 10(7) control B6 SpC, acute rejection of B6D2F1 skin was observed. When B6 donor SpC were Leu-Leu-OMe-treated prior to transfer to ATXBM, TCD mice, uniform rejection of B6D2F1 skin grafts was still observed, although a significant delay in the time to rejection was observed. More rigorous T cell depletion of ATXBM, TCD host mice by infusion of antithymocyte globulin did not prevent delayed rejection of B6D2F1 skin initiated by transfer of Leu-Leu-OMe-treated B6 SpC. Despite the lack of complete prevention of skin allograft rejection, Leu-Leu-OMe treatment of B6 donor cells prevented lethal GVHD even in thymectomized B6D2F1 recipients. Precursors of anti-B6D2F1-specific CTL were greatly reduced or undetectable in unreconstituted ATXBM, TCD mice or in irradiated B6D2F1 recipients of Leu-Leu-OMe-treated B6 SpC. By contrast, ATXBM, TCD recipients of Leu-Leu-OMe-treated B6 SpC were found to contain a population of anti-class I MHC-specific CTL precursors of host origin within 28 days of reconstitution. These findings have indicated a number of features of the cells involved in skin graft rejection. First, Leu-Leu-OMe-sensitive CTL play a major role in acute rejection of class I + II MHC and multiple non-MHC antigen-disparate skin grafts. Moreover, the thymus-independent expansion of host-derived CTL precursors in ATXBM, TCD mice reconstituted with syngeneic Leu-Leu-OMe-resistant T helper cells also appears to play a role in mediating rejection of allogeneic skin grafts.     

New experimental model for adoptive transfer of murine autoimmune orchitis

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by subcutaneous (s.c.) injections of viable syngeneic testicular germ cells (TC), without resorting to any adjuvants or immunopotentiators. Using this EAO model, a new and simple protocol was developed for adoptive transfer of EAO. Cell donors were C3H/He mice that received s.c. injections twice with TC alone. Spleen cells from the donors were stimulated in vitro with TC, propagated in interleukin-2 containing medium, then injected i.p. to naive recipient mice. This procedure induced severe orchitis and hypospermatogenesis with or without inflammation in epididymis and vas deferens in the recipients at high incidence. Elimination of all T cells or CD4+ T cells before the transfer produced no histopathological signs in the recipients whereas that of the CD8+ T cells or B cells had no inhibitory effect on the disease transfer, indicating that the effector cells are CD4+ T cells.     

Acceptance of related and unrelated cardiac allografts in neonatally tolerized mice is cardio-specific and transferable by regulatory CD4+ T cells.

BACKGROUND: Non-donor-specific cardiac allograft acceptance is induced in C3H/He (C3H; H-2k) recipients injected as neonates with allogeneic BALB/c (BALB; H-2d) fetal liver cells (FLC). This occurs despite intact reactivity to donor-type and third-party alloantigens in in vitro assays and skin transplants. To investigate a role for regulatory T cells, we performed adoptive transfer studies and specifically assessed CD4+ and CD4- T cells. METHODS: Three cell populations (splenocytes, CD4+, CD4-) derived from neonatally-treated mice with accepted C57BL/6 (B6; H-2b) third-party cardiac grafts were adoptively transferred into sub-lethally-irradiated C3H mice. Reconstituted mice were challenged with B6 cardiac grafts, B6 skin grafts, or unrelated cardiac grafts. Separated cells were assessed in vitro. RESULTS: B6, BALB, and NZW (H-2z) graft acceptance was transferred by unfractionated splenocytes. CD4+ cells transferred B6 graft acceptance (85% survival > 100 days). CD4- cells, unfractionated cells from naive or only irradiated mice, and unfractionated cells from neonatally-treated non-transplanted C3H mice rejected grafts within 35 days. No inoculum induced skin graft acceptance. Co-cultured assays confirmed the suppressive function of CD4+ cells in vitro. CONCLUSIONS: Cardiac allograft acceptance in our model is regulated by CD4+ cells. The regulatory cell population is induced by the cardiac graft itself and mediates in vivo cardiac graft acceptance in a tissue-specific but not donor-strain-specific manner.     

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. IV. Immune reconstitution

Studies of postmyeloablative immune reconstitution have been reported for allogeneic bone marrow transplantation and also for non-T cell-depleted autologous/syngeneic BMT. However, there is a paucity of information regarding immune recovery following T cell-depleted autologous/syngeneic BMT. We have developed a primate transplantation tolerance model in which rhesus monkeys were conditioned with total-body irradiation and extensively T cell-depleted autologous BMT and given a major histocompatibility complex-mismatched heterotopic cardiac allograft. This model provided an opportunity to study peripheral immune recovery following T cell-depleted autologous BMT. Limiting dilution analysis was used to quantify marrow T cells following depletion (2.8% to 25.6% marrow T cells predepletion, 0.00014% to 0.036% residual marrow T cells postdepletion). We found that (1) hematopoietic engraftment was prompt despite extensive marrow T cell depletion, (2) reconstitution of CD4+ helper T cells and CD8+ cytotoxic T cells were substantially delayed (6-12 months) compared with the recovery of CD8+ suppressor T cells, CD16+ NK cells, and CD20+ B cells, (3) distinction between CD8+ cytotoxic T cells and CD8+ suppressor T cells by the CD28 marker was critical in revealing the markedly discrepant recoveries of those subsets, and (4) immune reconstitution resembled that observed in recipients of T cell-depleted allogeneic and non-T cell-depleted autologous/syngeneic BMT, suggesting that the pattern of immune recovery following BMT is not substantially influenced by either allogeneic effects or the number of transferred T cells over a range of values.     

The fate of donor T-cell receptor transgenic T cells with known host antigen specificity in a graft-versus-host disease model.

BACKGROUND: The expansion of transgenic donor CD8+ T-cells with known allospecificity against a host MHC class I alloantigen was examined in a murine graft-versus-host disease (GVHD) model. METHODS: Lethally irradiated, Ld+ BALB/c mice received bone marrow cells and spleen cells from anti-Ld 2C T-cell receptor (TCR)-transgenic B6 mice, alone or with normal B6 spleen cells. Transgenic TCR-bearing T-cell expansion, apoptosis, and function were monitored at various time points and were correlated with clinical outcome. RESULTS: Fifteen-fold clonal expansion of 2C CD8 cells occurred by day 4 after bone marrow transplantation. Between days 4 and 7, increasing proportions of 2C CD8 cells underwent apoptotic cell death, coincident with a 7-15-fold decline in their numbers. CD8 and TCR expression were down-regulated on 2C CD8 cells by day +4 after bone marrow transplantation, and they were anergic to TCR-mediated stimulation. Clinically, the BALB/c recipients of 2C spleen cells exhibited only minimal chronic GVHD. In contrast, lethally irradiated BALB/c mice receiving similar numbers of non-transgenic B6 bone marrow cells and spleen cells exhibited severe GVHD (median survival time: 28 days). The addition of a small number of 2C spleen cells to the inoculum accelerated GVHD mortality, and 2C CD8 cells showed a similar time course of expansion and decline to that observed in recipients of larger numbers of 2C cells alone. CONCLUSIONS: Initial clonal expansion, down-regulation of CD8 and TCR, anergy, and later deletion of graft-versus-host-reactive CD8 cells via apoptosis occurs in lethally irradiated recipients. Expansion of a single CD8 clone produces much less severe GVHD than that induced by a polyclonal, mixed CD4 plus CD8 response. These results have implications for GVHD pathogenesis and its sometimes self-limited nature.     

Depleting anti-CD4 monoclonal antibody cures new-onset diabetes, prevents recurrent autoimmune diabetes, and delays allograft rejection in nonobese diabetic mice

BACKGROUND: The prevention of recurrent autoimmunity is a prerequisite for successful islet transplantation in patients with type I diabetes. Therapies effective in preserving pancreatic beta-cell mass in patients with newly diagnosed diabetes are good candidates for achieving this goal. Anti-CD3 monoclonal antibody (mAb) and antilymphocyte antisera are the only therapies to date that have cured early diabetic disease in the nonobese diabetic (NOD) mouse. We investigated whether other immunosuppressive therapies, including short-term depleting anti-CD4 mAb or costimulation blockade, would affect the disease progression in recently diabetic NOD mice. We also evaluated the effect of the anti-CD4 mAb on syngeneic and allogeneic graft survival in diabetic NOD recipients. METHODS AND RESULTS: We demonstrate that a short course of anti-CD4 mAb early after hyperglycemia onset cured diabetes. Normal islets and islets with CD4+ and CD8+ T-cell peri-insulitic infiltrate were found in the pancreata of cured NOD mice. A similar regimen prevented the recurrence of autoimmune diabetes in NOD/severe combined immunodeficient disease (SCID) islet isografts and delayed the rejection of allogeneic C57BL/6 islet allografts in diabetic female NOD mice. The co-transfer of diabetogenic splenocytes with splenocytes from anti-CD4 mAb-treated and cured NOD mice into 7-week-old, irradiated, NOD male mice was not able to protect from diabetes occurrence. This indicates that an anti-CD4-mediated cure of diabetes is independent of the induction of immunoregulatory T cells. Anti-CD154 mAb and cytotoxic T-lymphocyte antigen 4 immunoglobulin were ineffective in early-onset diabetes. CONCLUSION: Our results provide the first evidence that newly established autoimmune islet destruction in NOD mice responds to a short course of anti-CD4 mAb. In contrast, costimulation blockade is ineffective in this clinically relevant model.