Role of thymic- and graft-dependent mechanisms in tolerance induction to rat kidney transplant by donor PBMC infusion

We previously demonstrated the presence of regulatory T cells (Tregs) in lymph nodes (LNs) from rats made tolerant to a kidney allograft by donor peripheral blood mononuclear cell (PBMC) infusion. Here, we investigated the origin of Treg and characterized their phenotype and mechanisms underlying their suppressive effect. At different points after PBMC infusion, thymus, LN, and graft-infiltrating -lymphocyte's (GIL) alloreactivity was evaluated in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype (by fluorescence-activated cell sorting and immunohistochemistry) and cytokines mRNA expression were analyzed. Before transplantation, CD4(+) thymocytes and LN cells from donor PBMC-infused rats showed a reduced anti-donor but a normal anti-third-party proliferation. Anti-donor hyporesponsiveness was reverted by interleukin (IL)-2. CD4(+) thymocytes had no regulatory activity on a na?ve MLR. Treg appeared in LN at 60 days post-transplant. CD4(+)-GIL isolated early (5 days) and late post-transplant (days 60-80) were hyporesponsive and suppressed a na?ve MLR. IL-10 mRNA was upregulated in GIL and an anti-IL-10 monoclonal antibody reverted their inhibitory effect. Cell-to-cell contact potentiated the suppressive activity of CD4(+)-GIL. We suppose that allograft tolerance in this model is mediated by pretransplant generation of anergic cells in the thymus, which may have a permissive role to prevent early graft disruption. The healed graft is a source of donor antigens, which led to early selection of Treg. In the late phase, tolerance is maintained by appearance of Treg in LN.     

The protective effect of CD8+CD28- T suppressor cells on the acute rejection responses in rat liver transplantation

Regulatory T cells (Tr) or T-suppressor cells (Ts), which include CD4+CD25+ T cells and CD8+CD28- T cells respectively, have been shown to be essential for the induction and maintenance of immune tolerance. We have investigated the effect of CD8+CD28- Ts and CD4+CD25+ Tr on acute rejection responses in rat liver transplantation (OLT). METHODS: CD8+CD28- Ts/CD4+CD25+ Tr were obtained from inbred and na?ve rats that show spontaneous tolerance to OLT. Adoptive transfers were performed in acute rejection models of various strain combinations with survival times observed to evaluate suppressive effects. Donor-specific blood transfusion (DST) was used to induce CD8+CD28- Ts in na?ve rats, which were assayed in vitro using carboxyfluorescein diacetate succinimidyl easter-labeled one-way mixed lymphocyte reactions. Secondary adoptive transfers of DST-induced CD8+CD28- Ts were also performed in an acute OLT rejection model. RESULTS: CD8+CD28- Ts from tolerant OLT model rats possessed immunosuppressive activity in allogeneic recipients; adoptive transfers of these cells alleviated the acute rejection responses. However, CD4+CD25+ Tr derived from tolerant or na?ve rats failed to do so. In vitro DST-induced CD8+CD28- Ts inhibited alloantigen T-cell responses in na?ve syngeneic rats in an antigen-specific manner. Secondary adoptive transfer of DST-induced CD8+CD28-Ts further reduced acute rejection but not chronic rejection responses. CONCLUSIONS: CD8+CD28- Ts cells protected allogeneic recipients from acute rejection in rat OLT. Furthermore, this activity was not present in CD4+CD25+Tr. DST was observed to be an effective method to generate functional CD8+CD28-Ts in na?ve rats.     

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens

BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed na?ve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or na?ve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either na?ve or tolerant hosts effected rejection. In contrast neither tolerant nor na?ve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.     

DnIKK2-transfected dendritic cells induce a novel population of inducible nitric oxide synthase-expressing CD4+CD25- cells with tolerogenic properties

BACKGROUND: We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4 T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. METHODS: Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4 T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4 T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. RESULTS: CD4 T cells pre-exposed to dnIKK2-DCs were CD4CD25 and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:10 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). CONCLUSIONS: DnIKK2-Treg is a unique population of CD4CD25 T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.     

The cellular basis of cardiac allograft rejection. IX. Ratio of naïve CD4+CD25+ T cells/CD4+CD25- T cells determines rejection or tolerance

Na?ve CD4+ T cells are central to allograft rejection, but include 3-10% CD4+CD25+ T cells that induce and maintain immune tolerance. Whether increasing the ratio of CD4+CD25+ T cells can inhibit rejection and induce tolerance is not known. This study examined the effects that na?ve CD4+CD25+ and CD4+CD25- T cells have on rejection of MHC incompatible PVG cardiac allografts in whole body irradiated DA rats. The ratio of CD4+CD25+ T cells to CD4+CD25- T cells was increased to examine if this delayed rejection. CD4+CD25- T cells alone restored near first set rejection time of 8-10 days and were significantly faster than unfractionated CD4+ T cells which nearly always took over 10 days to effect rejection. Enriched CD4+CD25+ T cells, either fresh or cultured with IL-2 and donor alloantigen, did not restore rejection. Admixing na?ve CD4+CD25+ T cells with CD4+ T cells at a ratio of 1:10 prevented graft destruction by rejection. Na?ve CD4+CD25+ T cells, either fresh or cultured with IL-2 and donor alloantigen, at a ratio of 1:1, prevented significant episodes of rejection and grafts survived >300 days. These grafts had large areas of normal myocardium but had some foci of CD4+, CD8+ and CD25+ cellular infiltration. This study found CD4+CD25- T cells were the principal mediators of rejection and na?ve CD4+CD25+ T cells partially inhibited the CD4+CD25- T cells in unfractionated CD4+ T cells. Increasing the ratio of na?ve CD4+CD25+ to CD4+CD25- T cells inhibited rejection allowing grafts to survive indefinitely and may induce transplant tolerance, without a need for long-term immunosuppression.     

Tolerance to rat heart grafts induced by intrathymic immunomodulation is mediated by indirect recognition primed CD4+CD25+ Treg cells

BACKGROUND: In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. METHODS: Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. RESULTS: Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. CONCLUSIONS: Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.     

Dendritic cells genetically engineered with adenoviral vector encoding dnIKK2 induce the formation of potent CD4+ T-regulatory cells

BACKGROUND: Immature dendritic cells (DC), characterized by low expression of both major histocompatibility complex class II antigens and co-stimulatory molecules, can be instrumental in the induction of peripheral tolerance. Because nuclear factor (NF)-kappa B is central to DC maturation, the authors engineered DC with an adenoviral vector (Adv) encoding for a kinase-defective dominant negative form of IKK2 (dnIKK2) to block NF-kappa B activation and inhibit DC maturation. METHODS: DC were obtained by culturing bone marrow from Brown Norway (BN) rats with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 11 days. To block NF-kappa B activation, at day 9, cells were transfected with AdV-dnIKK2. At day 11, cells were used as stimulators in primary mixed leukocyte reaction (MLR) with naive Lewis rat lymphocytes as responders. CD4+ T cells were purified from primary MLR and tested in secondary MLR with allogeneic mature DC and in co-culture MLR with naive lymphocytes. The tolerogenic potential of dnIKK2-DC was evaluated in vivo in a model of rat kidney allotransplantation. RESULTS: DnIKK2-DC were immature and lacked any allostimulatory activity. T cells preexposed to allogeneic dnIKK2-DC were hyporesponsive to a secondary stimulation with mature DC and acquired potent regulatory properties, inhibiting naive T-cell proliferation toward allogeneic stimuli. Pretransplant infusion of allogeneic donor dnIKK2-DC prolonged the survival of a kidney allograft from the same allogeneic donor, without the need for immunosuppressive therapy. CONCLUSIONS: Allogeneic DC, rendered immature by dnIKK2 transfection, induce in vitro differentiation of naive T cells into CD4+ T-regulatory cells, effective at low ratios with target cells, rendering them applicable for cellular therapy of immune-mediated abnormalities and for preventing transplant rejection.     

Peripheral regulation of graft-versus-host alloreactivity in mixed chimeric miniature swine

BACKGROUND: Despite the presence of circulating donor-derived T cells during the induction of mixed chimerism across MHC barriers in miniature swine, severe graft-versus-host disease was avoided in the majority of animals. In this study, we investigated the possible roles of recipient and donor lymphoid populations in the regulation of donor-anti-recipient alloreactivity. METHODS: Mixed chimerism across a full MHC-mismatch barrier was established in miniature swine using a high-dose allogeneic peripheral blood stem cell protocol. Peripheral blood mononuclear cells from mixed chimeric swine were co-cultured with na?ve donor-matched responders and na?ve recipient-matched stimulators in mixed lymphocyte reactions. RESULTS: Peripheral blood mononuclear cells from mixed chimeras inhibited donor-anti-recipient proliferation. This suppression was radioresistant to 25 Gy. Suppression of donor-anti-recipient alloreactivity was not observed in mixed lymphocyte co-cultures when donor-derived cells were added in the absence of recipient-derived cells. CONCLUSIONS: These results suggest an association between the presence of an active and relatively radioresistant cell population, demonstrable in vitro, and the regulation of graft-versus-host disease across MHC barriers in mixed chimeric miniature swine.     

Direct versus Indirect Allorecognition: Visualization of Dendritic Cell Distribution and Interactions During Rejection and Tolerization

Interactions of donor and recipient dendritic cells (DCs) with CD4+ T cells determine the alloantigenic response in organ transplantation, where recipient T cells respond either directly to donor MHC, or indirectly to processed donor MHC allopeptides in the context of recipient MHC molecules. The present study evaluates donor and recipient alloantigen-presenting DC trafficking and their interactions with CD4+ T cells in the lymph nodes (LNs) and the spleen under tolerogenic treatment with anti-CD2 plus anti-CD3 mAb compared with untreated rejecting conditions. CX3CR1(GFP) BALB/c (I-A(d)) donor hearts were transplanted into C57BL/6 (I-A(b)) mice and quantification of donor DC direct (GFP+ or I-A(d+)) and recipient DC indirect (YAe+) trafficking and interactions with host CD4+ T cells was performed by fluorescent microscopy. Our data indicate that although both direct and indirect interactions between CD4+ T cells and donor and recipient DCs occur shortly after engraftment, only indirect presentation persists in the LN, but not the spleen, of tolerized recipients. These data suggest that distinct anatomic lymphoid compartments play a critical role in peripheral tolerance induction and maintenance, and persistent indirect presentation to CD4+ T cells within the LNs is an important process during tolerization.     

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. Interleukin-4 does not maintain alloantigen specific CD4+CD25+ Treg

IL-4 is thought to promote induction of transplantation tolerance and alloantigen-specific CD4⁺CD25⁺ T regulatory cells (Treg). This study examined the effect of IL-4 on the induction and maintenance of the CD4⁺ T regulatory cells (Treg) that mediate transplantation tolerance. Tolerance was induced in DA rats with PVG heterotopic cardiac allografts by a short course of cyclosporine. Naïve and tolerant lymphocytes, including the CD4⁺ and CD4⁺CD25⁺ T cell subsets, were assayed in mixed lymphocyte cultures with or without recombinant (r)IL-4 or other cytokines. The proliferation, cell surface and cytokine phenotype of these cells was examined, as was their capacity to adoptively transfer tolerance. rIL-4 enhanced the proliferation of naïve and tolerant lymphoid cells, including CD4⁺ and CD4⁺CD25⁺ T cells, but this was not alloantigen specific. Naïve or tolerant CD4⁺ T cells cultured with rIL-4 and donor PVG antigen effected rapid graft rejection, even though before culture tolerant CD4⁺ T cells transferred antigen-specific tolerance. These rIL-4 cultured CD4⁺ T cells had a phenotype consistent with activated CD4⁺CD25⁺FoxP3⁻ Th2 cells. While naïve natural CD4⁺CD25⁺ T cells (nTreg) cultured with alloantigen and rIL-4 had enhanced proliferation and capacity to suppress rejection in vivo, the culture of tolerant CD4⁺CD25⁺ T cells with alloantigen and rIL-4 could not sustain their proliferation against specific donor, nor their capacity to transfer tolerance to specific donor allograft. Thus, IL-4 promotes both regulatory and effector T cells early in the immune response, but once alloimmune tolerance is established, IL-4 promoted the activation of effector cells to mediate rejection and did not support alloantigen-specific Treg that could transfer specific tolerance.     

CD4-veCD8-ve CD30+ve T cells are detectable in human lung transplant patients and their proportion of the lymphocyte population after in vitro stimulation with donor spleen cells correlates with preservation of lung physiology

INTRODUCTION: Survival following lung transplantation is less than 50% at 5 years, mainly due to immune-mediated chronic rejection. Recently a novel subset of T cells, CD4-veCD8-ve CD30+ve, so-called double negative (DN) CD30+ve T cells, has been described and shown to be responsible for tolerance in an animal model of skin transplantation. METHODS: We investigated 18 lung transplant recipients for the presence of DN CD30+ve T cells in resting peripheral blood and also following in vitro stimulation of recipient peripheral blood mononuclear cells (PBMCs) with donor spleen cells. RESULTS: Small percentages (0.2% to 6%) of DN T cells are detectable in resting PBMCs of human transplant patients (n = 18), but these did not correlate with allograft function, acute rejection episodes, HLA mismatch, or CMV status. On repeated stimulation of recipient PBMCs (two exposures) in vitro by donor spleen cells (2:1 ratio stimulators to responders) the percentage of DN CD30+ve T cells within the lymphocyte pool correlated with preservation of allograft lung function (both for FEV(1), P = .009, and FEF(25-75), P = .036) and was inversely correlated with grade of chronic rejection. On repeated exposure of recipient PBMCs to donor spleen cells with a 1:1 ratio the percentage of DN CD30+ve T cells correlated with the number of acute rejection episodes of grade 2 or greater. The total number of HLA mismatches correlated with the percentage DN CD30+ve T cells present after primary stimulation of recipient PBMCs with donor spleen cells (1:1 ratio). The number of mismatches at the B locus inversely correlated with the percentage of DN CD30+ve T cells after primary stimulation of recipient PBMCs with donor spleen cells (1:1 ratio; P = .031, n = 18). CONCLUSION: Percentages of DN CD30+ve T cells present following repeated stimulation of recipient PBMCs by donor spleen cells correlated with preservation of graft function following lung transplantation.     

Naïve and central memory T-cell lymphopenia in end-stage renal disease

End-stage renal disease (ESRD) is associated with increased propensity to infections, diminished response to vaccination, impaired cell-mediated immunity, and reduced CD4+/CD8+ T-lymphocyte ratio. Four subsets of CD4+ and CD8+ T cells have been recently identified: na?ve cells (as yet uncommitted), central memory (CM) cells (previously programmed), and CD45RA-positive and CD45RA-negative effector memory (EM) cells (programmed to perform specific effector functions). The effect of ESRD on subpopulations of T lymphocytes is unclear and was studied here. Twenty-one hemodialysis patients and 21 age-matched controls were studied. Pre- and post-dialysis blood samples were obtained and analyzed by three-color flow cytometry. CD4+/CD8+ ratio and the numbers of the na?ve and CM CD4+ and CD8+ T cells were significantly reduced, whereas the numbers of EM CD4+ and CD8+ T cells were unchanged in the ESRD group. The reduction of the na?ve and CM T-cell counts in the ESRD group was associated with increased apoptosis of these cells. Negative correlations were found between severity of azotemia, oxidative stress, and hyperphosphatemia with the number of na?ve T cells. Comparison of diabetic with non-diabetic ESRD patients revealed higher numbers of total CD8+ cells and EM CD8+ T cells in the diabetic group. Dialysis did not significantly change the na?ve and CM CD4+ or CD8+ cell counts, but significantly lowered CD8+ EM cell count. Thus, ESRD results in increased apoptosis and diminished populations of na?ve and CM T lymphocytes. This phenomenon may, in part, contribute to the impaired immune response in this population.     

Adoptive transfer of transplantation tolerance mediated by CD4+CD25+ and CD8+CD28- regulatory T cells induced by anti-donor-specific T-cell vaccination

Previous studies have shown that vaccinating rodents with anti-donor-specific T cells significantly prolonged allograft survival; however, the putative mechanism of the tolerance remains unclear. In this study, we used the model of heterotopic heart transplantation between the C57BL/6 donor mice and BALB/c recipient mice vaccinated with anti-donor (C57BL/6) or anti-third party (C3H)-specific T cells to determine whether T cells prolong survival of mouse heart allografts and which cells were involved in induction of allograft tolerance. We observed that the mean survival time (MST) of C57BL/6 heart grafts in BALB/c mice vaccinated with anti-C57BL/6 specific T cells (43.1 +/- 4.7 days) was prolonged from that in untreated BALB/c mice (9.5 +/- 1.1 days) or BALB/c mice receiving anti-C3H-specific T cells (10.4 +/- 1.9 days). These results suggested that alloantigen-specific T-cell vaccination significantly prolonged cardiac allograft survival. The CD4+CD25+ or CD8+CD28- T cells purified from splenocytes of BALB/c mice vaccinated with anti-donor-specific T cells proliferated markedly in response to irradiated anti-C57BL/6-specific T cells in vitro. Adoptive transfer of these CD4+CD25+ or CD8+CD28- T cells to na?ve syngenic mice significantly prolonged the survival of heart allografts. These data suggested that anti-donor-specific T-cell vaccination induced development of CD4+CD25+ or CD8+CD28- regulatory T cells, which in turn mediated allogeneic-specific tolerance.     

Anti-CD28 antibody-induced kidney allograft tolerance related to tryptophan degradation and TCR class II B7 regulatory cells.

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7+, non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.     

A Renewable Source of Donor Cells for Repetitive Monitoring of T- and B-Cell Alloreactivity

A major impediment to repetitive monitoring of alloreactivity or tolerance is the limited supply of donor cells available for assays of host-versus-graft T- and B-cell reactivity. In this paper, we describe the use of CD40L stimulated CD19(+) B cells as targets or stimulators in flow cytometric crossmatching (FXM), mixed lymphocyte reactivity and IFN-gamma ELISPOT assays. Stimulated B cells (sBc) express high levels of MHC class I and II, as well as the costimulatory molecules CD80 and CD86. They can be polyclonally expanded and frozen for later use. We describe the use of sBc in ELISPOT, mixed lymphocyte cultures and FXM. CD4(+) T cells exposed to sBc express a similar cytokine profile as those stimulated with unfractionated PBMC. We further analyzed T- and B-cell responses in 14 patients on the renal transplant waiting list, finding that those with an elevated panel reactive antibody (PRA) (>60%) had higher alloreactive T-cell precursor frequencies as measured by CDFSE MLR and IFN-gamma ELISPOT. We conclude that sBc are a renewable source of donor-specific target/stimulator cells for use in repetitive and coordinate assays of B- and T-cell alloreactivity.     

The Role of Regulatory Cells in Miniature Swine Rendered Tolerant to Cardiac Allografts by Donor Kidney Cotransplantation

To determine the mechanism by which cotransplantation of a kidney allograft induces tolerance to a donor heart in miniature swine, we examined the role of CD25⁺ cells in heart/kidney recipients. Tolerance was induced to class‐I MHC mismatched hearts by cotransplanting a donor‐specific kidney with a 12‐day course of cyclosporine. Peripheral blood leukocytes (PBL) were isolated from tolerant heart/kidney recipients and used in cell‐mediated lympholysis (CML) coculture assays as either unmodified PBL, PBL enriched for CD25⁺ cells or PBL depleted of CD25⁺ cells to assess their ability to suppress CML responses of naïve recipient‐matched leukocytes against mismatched target cells. Primed PBL from tolerant heart/kidney recipients completely suppressed lysis by naïve cells. Complete suppression of the response of naïve recipient‐matched leukocytes against donor‐matched target cells was lost following the depletion of CD25⁺ cells from tolerant heart/kidney animal PBL, but it was reestablished by incubation of naïve cells with small populations of CD25⁺ cells from tolerant heart/kidney animals. These data suggest that peripheral blood from tolerant heart/kidney recipients contains regulatory cells that, upon priming, can suppress the response of naïve‐matched PBL in coculture CML assays, and that suppression appears to be dependent on cells expressing CD25.     

Donor T cells are not required for induction of allograft tolerance in mice treated with antilymphocyte serum, rapamycin, and donor bone marrow cells

BACKGROUND: Postgraft infusion of donor bone marrow cells (BMC) effectively induces tolerance to skin allografts in antilymphocyte serum- and rapamycin-treated recipients in fully major histocompatibility complex-mismatched mouse strain combinations. We used various gene knockout mice to examine the role of donor T cells and B cells in BMC-induced allograft tolerance. METHODS: All recipient mice received ALS on days -1 and 2 and rapamycin (6 mg/kg) on day 7 relative to fully major histocompatibility complex-mismatched skin grafting on day 0. Donor BMC prepared either from mice lacking CD4- and/or CD8a-, or CD3epsilon-expressing cells or B cells, or from corresponding wildtype mice, were given on day 7. The level and phenotypes of chimerism was determined by flow cytometry. RESULTS: All T cell- and B cell-deficient BMC were as effective as wild-type BMC in inducing prolongation of skin graft survival. A low degree of chimerism without donor type T cells was detected in tolerant mice given T cell-deficient BMC or wild-type BMC 60 days after transplantation. Chimeric cells were composed of B cells and macrophages/monocytes. Low level chimerism without donor T or B cells was also present in tolerant mice given B cell-deficient BMC. CONCLUSION: Donor type T cells and T cell chimerism are not required for induction of allograft tolerance by the antilymphocyte serum/rapamycin/donor BMC-infusion protocol. Donor B cells also do not participate in tolerance induction. Thus, infusion of T cell-depleted BMC in conjunction with conventional immunosuppressive regimens will be a simple, safe, and effective way to induce allograft tolerance in clinical organ transplantation.     

Antigen‐specific regulatory T cells can induce tolerance to immunogenic grafts without the need for chronic immunosuppression

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) play an important role in the induction of allospecific tolerance. However tolerance in solid organ transplantation by mere transfer of Tregs has been difficult. Besides this the stability of the differentiation phenotype of Tregs has recently been questioned. We therefore aimed in generating large numbers of stable allospecific Tregs from naïve T cells by retroviral transduction with Foxp3. These were tested in an immunogenic skin transplantation model (C57BL/6→BALB/c). We established a system of transduction of mouse T cells with ecotropic retroviruses expressing Foxp3 and Thy1.1 as a surface marker to follow up transduced T cells. Alloantigen‐specific Tregs were generated by stimulating naïve recipient CD4⁺ T cells with irradiated donor splenocytes. CD25⁺ and/or CD69⁺ allospecific recipient CD4⁺ T cells were isolated and transduced with Foxp3. Alloantigen‐specific Foxp3 T cells (iTregs) showed high expression for the Treg markers Foxp3, CTLA4 and GITR. They could suppress a MLR in an alloantigen‐specific manner. Furthermore, they could be expanded up to 18 fold in vitro while maintaining their Treg phenotype and expression of lymph node homing markers like CCR7 and CD62L. iTregs prevented skin graft rejection without the need for chronic immunosuppression and recipients showed systemic allospecific allotolerance. Alloantigen‐specific Tregs were far more potent than polyspecific Tregs. Mechanisms of tolerance were graft specific homing, expansion and long‐term persistence of Tregs within the graft (>100 days, 90% of intragraft Tregs were alloantigen‐specific). In fact, tolerance could be transferred with re‐transplantation of the tolerant graft onto secondary recipients. Third party grafts were readily rejected demonstrating specificity of tolerance. Due to the Foxp3 transduction, iTregs did not lose their Treg phenotype. The results prove that large numbers of stable alloantigen‐specific Tregs can be generated from a polyclonal repertoire of naïve T cells. This is the first time that allotolerance was achieved in a non‐lymphopenic transplant model using skin grafts in an immunogenic strain combination. Therefore, antigen‐specific Tregs might have a huge therapeutic potential after solid organ transplantation.