A novel tool for B-cell tolerance research: characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique

In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.     

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.     

Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients.

The study was designed to compare second heart and skin grafts and in vitro assays as a means of assessing peripheral tolerance in C57BL/6 mice. Vascularized heterotopic BALB/c hearts were placed in C57BL/6 recipients treated with anti-CD4, GK1.5 (1 mg total per 20 g mouse i.p. on days 0, 1, 2, 3). Those mice in which hearts survived for >60 days were challenged with donor and third-party (CBA) skin grafts or with second heart grafts, of donor or third-party origin, attached to the carotid artery and jugular vein. In vitro alloreactivity was assessed by mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) using recipient spleen cells. Parenchymal damage, cellular infiltration and vascular disease were assessed from the histology of long-term allografts and isografts. Allografts in untreated recipients were rapidly rejected while isografts survived > 100 days. Primary allografts in anti-CD4 treated recipients also survived > 100 days, as did donor strain secondary heart transplants given at >60 days after the first graft. Third-party hearts were rapidly rejected, as were donor and third-party skin grafts placed on recipients with long-term allografts. These recipients showed low MLR response to both donor and third-party stimulators and donor-specific suppression of CML at 60 days post graft. Long-surviving heart allografts all showed evidence of parenchymal damage and vascular intimal thickening. Thus in the BALB/c to C57BL/6 donor-recipient strain combination, hearts, but not skin grafts, could be used to demonstrate peripheral tolerance, which seemed to be both organ and major histocompatibility complex (MHC) specific. Despite long survival, BALB/c hearts all showed evidence of parenchymal damage and vascular intimal thickening, a sign of chronic rejection.     

Genetic aspects of the blood transfusion effect

The genetic requirements for the induction of the blood transfusion effect have been investigated in a genetically well-defined model. The survival of fully vascularized, heterotopic murine cardiac allografts was measured after a single preoperative transfusion of blood from mice selected to share MHC and/or minor histocompatibility (miH) antigens with the organ donor. The effect of a transfusion from a donor unrelated to the heart donor (third-party transfusion) on allograft survival was also determined. Five strain combinations were used for these experiments. The results obtained illustrate a number of important aspects of the blood transfusion effect in this model: (1) Donor-specific blood transfusion, where MHC and miH were shared by the blood donor and the organ donor, always induced prolonged graft survival. (2) The sharing of the whole MHC (H-2) by the blood donor and organ donor was found to be sufficient to prolong allograft survival in the five fully allogeneic strain combinations tested. (3) The sharing of miH antigens only was not sufficient to induce prolonged cardiac allograft survival. Special cases were identified showing that several factors could interact to potentiate the action of miH antigens in the induction of the blood transfusion effect. (4) Transfusion with blood from a third-party donor was effective in some strain combinations. In one recipient, blood from several third-party strains of mice, sharing neither MHC nor miH antigens with the organ donor, induced prolonged graft survival. We suggest that the mechanism by which third-party blood has a beneficial effect on graft survival is through crossreaction(s) between the blood donor and the organ donor. The results obtained in this study fit very well with one model for the cellular mechanism by which the transfusion effect may be mediated. This may be the means by which blood from randomly selected donors has a beneficial effect on graft survival in clinical transplantation.     

Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway

BACKGROUND: Inducible co-stimulator (ICOS) is one of the most recently described members of the CD28 family, and it plays an important role in immune responses. To investigate the role of ICOS in allograft rejection, the authors studied graft survival after cardiac transplantation in mice. METHODS: Hearts from BALB/c mice were transplanted into C3H/He mice. Immunohistochemical staining and flow cytometry were performed. Monoclonal antibody to ICOS or ICOS-immunoglobulin (Ig) was injected intraperitoneally. The authors performed mixed lymphocyte reaction (MLR). RESULTS: ICOS was expressed strongly by graft-infiltrating cells during rejection of the allograft. Blockade of the ICOS pathway with anti-ICOS antibody and ICOSIg significantly prolonged graft survival time relative to that in untreated mice; however, all cardiac allografts were eventually rejected by a single treatment. Treatment with both ICOSIg and cytotoxic T-lymphocyte antigen 4 (CTLA4) Ig induced not only long-term acceptance of the cardiac allograft but also donor-specific tolerance, which was shown by acceptance of donor but not third-party skin. Graft arterial intimal hyperplasia in these cardiac allografts was remarkably less than that in cardiac allografts treated with tacrolimus. Addition of anti-ICOS antibody or ICOSIg to MLR resulted in inhibition of T-cell proliferation. CONCLUSIONS: Inhibition of T-cell proliferation with ICOSIg and CTLA4Ig was more effective than that with ICOSIg alone. Thus, ICOS appears to be an important regulator of T-cell activation, and may be an effective therapy in clinical cardiac transplantation.     

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from na?ve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.     

A more persistent tolerance to islet allografts through bone marrow transplantation in minimal nonmyeloablative conditioning therapy

INTRODUCTION: Islet transplantation is a therapeutic approach to prevent diabetes complications. However, the side effects of the required lifelong immunosuppressive regimens to prevent graft rejection restrict the impact of type 1 diabetes. One strategy to overcome these limitations is tolerance induction and graft acceptance through hematopoietic chimerism. In this study we investigated whether tolerance to major histocompatibility complex (MHC) and minor-disparate islet allografts could be induced by minimal nonmyeloablative conditioning and whether more persistent donor-specific islet allografts were accepted if the grafts were implanted with simultaneous bone marrow cells. METHODS: The donor and recipient mice were BALB/c(H-2(b)) and C57BL/6(H-2(d)), respectively. In group 1 streptozotocin-induced diabetic C57BL/6(H-2(d)) mice received only 500 islets of BALB/c(H-2(b)). Group 2 recipients conditioned with antilymphocyte serum, 100 cGy total body irradiation and cyclophosphamide were given islet cells of BALB/c(H-2(b)), but group 3 were simultaneously given 30 x 10(6) BALB/c(H-2(b)) mice BMCs and islet cells similar to group 2. RESULTS: We obtained 5% to 6% allogeneic donor chimerism and 60% graft survival at 80 days after islet transplantation in group 3. We observed lymphocyte infiltration around the islet without destruction of endocrine cells and the presence of strong insulin/glucagon-stained cells in group 3. CONCLUSION: This minimal nonmyeloablative conditioning therapy induced donor chimerism and immune tolerance between MHC- and minor-disparate (BALB/c-->C57BL/6) mice and long-term islet graft survival was obtained through cotransplantation of bone marrow cells.     

Evidence for epitope spreading and active suppression in skin graft tolerance after donor-specific transfusion.

BACKGROUND: To clarify the controversial results in the literature regarding the role of donor-specific transfusion (DST) on allograft survival, we have examined the influence of the following on DST-induced allograft survival in a 2C transgenic mouse model: varying the time between DST and transplantation; the role of MHC disparities between donor and recipient; whether tolerance induced by DST spreads to skin allografts expressing other alloantigens; and whether cyclosporine (CsA) treatment could further modulate skin allograft tolerance after DST. METHODS AND RESULTS: The studies were performed in both 2C anti-Ld (MHC class I) transgenic and normal (nontransgenic) mice. Our data demonstrate that a single infusion of Ld-mismatched lymphocytes 7 days before transplantation leads to permanent acceptance of donor-specific skin allografts in both transgenic (58/58) and nontransgenic (8/8) mice in the absence of any other nonspecific immunosuppressive treatment. Pretransplantation DST from donors mismatched for more than one MHC antigen (Ag) has no beneficial effect on subsequent donor skin allograft survival. However, Ld plus multiple minor histocompatibility (mH) Ag-mismatched DST induced permanent acceptance of donor-specific skin allografts. Tolerance induced by one-locus Ld-mismatched DST spreads to skin allografts expressing either two-locus Ld or one-locus Ld plus multiple mH Ags. Administration of CsA after DST diminished skin allograft survival, rather than enhancing it, suggesting that tolerance in this model system is established by an active immunological process sensitive to CsA. CONCLUSIONS: (1) Pretransplantation infusion of Ld-mismatched lymphocytes in the presence or absence of multiple mH mismatches induces permanent survival of donor-specific skin allografts. (2) CsA abrogates DST-induced transplantation tolerance.     

Organ-specific differences in the function of MCP-1 and CXCR3 during cardiac and skin allograft rejection

BACKGROUND: Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known. METHODS: Fully major histocompatibility complex-mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1-/- (H-2b) and CXCR3-/- (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days. RESULTS: Cardiac allografts in MCP-1-/- recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1-/- grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3-/- recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3-/- donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3-/- donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3-/- skin grafts were used as donors in the SCID model of rejection. CONCLUSION: Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.     

Induction of transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody.

The T-cell-mediated immune response usually results in the rapid destruction of organ allografts transplanted between murine strains incompatible for major and minor histocompatibility antigens. This response may be modified by pretreatment with either donor-specific antigen or anti-CD4 monoclonal antibody. Previous work by others has shown that combined treatment of mice with soluble protein antigens and anti-CD4 monoclonal antibody can produce antigen-specific B cell unresponsiveness that continues long after the nonspecific immunosuppressive effect of the mAb treatment has resolved. Following this principle we have shown that adult C3H/He mice can be made specifically unresponsive to vascularized C57BL/10 cardiac allografts by pretreating the recipient with donor alloantigen under the cover of a brief course of mAb against CD4. A full-dose response analysis shows that the dose of mAb is critically important for the successful induction of tolerance. Tolerance induction using this protocol is dependent on treatment with donor major histocompatibility complex antigens and occurs in the presence of marked depletion but not complete elimination of the CD4+ T cell subset. The unresponsiveness to alloantigen is antigen specific, as determined by the ineffectiveness of third-party (C57BL/10) alloantigen when combined with anti-CD4 mAb to induce long-term survival of BALB/c allografts in C3H/He recipients. The tolerant state is specific and effective in the long-term as indicated by the specific acceptance of C57BL/10 skin grafts in recipients with surviving C57BL/10 cardiac allografts. This study provides a simple method for the successful induction of specific transplantation tolerance in the adult across a full H-2 major and minor antigen mismatch strain combination. The results illustrate the important role of the CD4 molecule in the T cell response to alloantigen in vivo and suggest possibilities for the therapeutic manipulation of complex immune reactions.     

Organ transplant specificity of tolerance to skin grafts with heart or kidney grafts plus nondepleting anti-CD4 monoclonal antibody (RIB 5/2) and intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. Monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating recipient T cells. A single dose of monoclonal anti-rat CD4 antibody RIB 5/2 plus donor splenocytes results in donor-specific unresponsiveness to heart and kidney allografts, but not skin allografts. This study examined whether tolerance to the more resistant skin graft could also be achieved with RIB 5/2. METHODS: Buffalo (RT1(b)) recipients were given a single dose (20 mg/kg) of monoclonal antibody RIB 5/2 IP plus IV Lewis (RT1(l)) splenocytes (25 x 10(6)) 21 days before Lewis heart, kidney, or skin grafts. In addition, Lewis skin was grafted either simultaneously with or after long- term Lewis heart or kidney allograft acceptance (>50 days). RESULTS: While IV alloantigen plus RIB 5/2 results in long-term acceptance of both heart and kidney, skin allografts are rejected when transplanted alone. Simultaneous transplantation with a Lewis kidney, but not with a Lewis heart, resulted in long-term Lewis skin graft acceptance. However, recipients tolerant to Lewis kidney or heart alone will not accept subsequent Lewis skin grafts, while recipients of simultaneous Lewis skin and kidney grafts subsequently accept a second Lewis, but not third-party Brown Norway (RT1(n)), skin graft. CONCLUSION: RIB 5/2 plus Lewis donor splenocytes tolerize for donor-specific heart and kidney but not skin grafts. However, Lewis skin grafted simultaneously with a Lewis kidney, but not Lewis heart, is accepted and protects a subsequent donor-specific Lewis skin graft.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

Intrathymic alloantigen-mediated, tolerant, completely major histocompatibility complex-mismatched mouse hearts are specifically rejected by adoptively transferred anti-class I L(d+)-specific 2C cells

BACKGROUND: Tolerance to cardiac allografts can be induced in mice and rats by the injection of donor alloantigen into the thymus in combination with a CD4 T-cell-depleting antibody. CD8(+) cells in these animals are hyporesponsive to graft-specific alloantigens. Most of the CD8(+) T cells in the transgenic 2C mouse express a T-cell receptor specific for the class I major histocompatibility complex L(d+) locus. This study was designed to determine whether the adoptive transfer of these 2C T cells could precipitate rejection of a tolerant, completely major histocompatibility complex-mismatched L(d+) or L(d-) heart. METHODS: C57BL/6 mice (L(d-)) were given 10 x 10(6) cells of BALB/c (L(d+)) or dm2 (BALB/c background lacking L(d) [L(d-)]) splenocytes intrathymically and GK1. 5 (10 mg/kg) intraperitoneally. Twenty-one days later, BALB/c or dm2 hearts were transplanted. On the day of transplantation or after long-term allograft acceptance, recipients received naive 2C cells or 2C cells sensitized by in vitro mixed lymphocyte culture with BALB/c (L(d+)). RESULTS: Mean survival time of BALB/c cardiac allografts in untreated C57BL/6 mice was 7.3 days, although 73% of the mice that were pretreated with BALB/c splenocytes IT plus GK1.5 accepted the donor antigen-specific heart allografts indefinitely. All recipients that were pretreated with the intrathymic plus GK1.5 and that were injected with naive 2C cells at the time of heart transplantation experienced rejection of the BALB/c (L(d+)), but not the dm2 (L(d-)) hearts. In contrast, naive 2C cells could not reject tolerant (>30 days acceptance) BALB/c (L(d+)) hearts. 2C cells sensitized in vitro against L(d) were able to reject established BALB/c hearts but could not reject the L(d-) dm2 hearts. CONCLUSIONS: L(d)-specific 2C T-cell receptor transgenic T cells that are adoptively transferred to recipients will precipitate the rejection of accepted hearts that express class I L(d+) in mice rendered tolerant by an intrathymic injection of alloantigen plus anti-CD4 monoclonal antibodies.     

The graft helps to define the character of the alloimmune response

Introduction: In mice, kidney and liver allografts may be spontaneously accepted, whereas cardiac and skin allografts in the same strain combinations are rapidly rejected. The reasons for this dichotomy in murine response outcomes remains to be determined. Methods and results: When DBA/2 (H-2d) cardiac allografts were placed in C57BL/6 (H-2b) recipients, they were rejected within 10 days, unless the allograft recipients were transiently treated with gallium nitrate (GN), at which time the allografts were accepted for > 150 days. The cardiac allograft rejector mice displayed DBA/2-reactive DTH responses, whereas the cardiac allograft acceptor mice displayed both TGFbeta- and IL10-mediated inhibition of DTH responses. In contrast, DBA/2 kidney allografts placed at the same location in C57BL/6 mice were spontaneously accepted without immunosuppression. These kidney allograft acceptor mice displayed TGFbeta-mediated, but not IL10-mediated inhibition of donor-reactive DTH responses. Conclusions: In the DBA/2-> C57B1/6 strain combination, cardiac allografts induce pro-inflammatory immunity and allograft rejection, while kidney allografts induce anti-inflammatory immunity and allograft acceptance despite the fact that both organs display the same strong MHC disparities and are implanted at the same location. Anti-inflammatory immunity and allograft acceptance are displayed by cardiac allograft recipients when they are transiently treated with select immunosuppressants. Thus, multiple immune response options are available to the organ allograft recipient, and the choice is determined, to some degree, by the allograft, itself.     

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.     

Rejection of established mouse pancreatic islet allografts by active immunization requires class I (H-2 K, D) antigen disparities

In certain donor-recipient mouse strain combinations with class I (H-2 K, D, and K + D) or with classes I and II (H-2 D + I) disparities the incidence of islet allograft rejection is low. Furthermore pancreatic islet allografts transplanted between strains with class II (H-2 Ia) differences alone are rarely acutely rejected. In this experiment the ability of donor strain or third-party allogeneic splenocytes (active immunization) to induce rejection of established (greater than 100 days) islet allografts when the donor and recipient differed only for class I or class II antigens was tested. Class I disparate islet allografts are rejected if challenged with donor or third-party allogeneic splenocytes. The frequency of rejection is similar (80-89%) if the third-party splenocytes share the class I allele with the islet donor strain. In contrast, class II disparate islet allografts are not rejected after challenge with donor splenocytes or third-party splenocytes even when the third-party strain shares the class II disparity with the islet donor strain as well as class I antigens common to the donor and recipient. Furthermore, rejection of class II disparate islets did not occur following passive transfer of recipient strain splenocytes sensitized in vitro to donor strain lymphocytes. These results show that rejection of established islet allografts can only be induced if (1) the islet graft expresses H-2 K or H-2 D gene products that are different than the recipient strain, i.e., only class I antigens can serve as targets; and (2) challenging splenocytes also have class I disparities with the recipient.     

Donor-specific tolerance in fully major histocompatibility major histocompatibility complex-mismatched limb allograft transplants under an anti-alphabeta T-cell receptor monoclonal antibody and cyclosporine A protocol

BACKGROUND: Recent studies have demonstrated that treatment with alphabeta-T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined alphabeta-TCR-CsA protocol. METHODS: The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of alphabeta-TCR and CsA or a combination of alphabeta-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. RESULTS: Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined alphabeta-TCR-CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (AxC Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. CONCLUSIONS: Combined therapy of alphabeta-TCR-CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.     

'Chimeric' Grafts Assembled from Multiple Allodisparate Donors Enjoy Enhanced Transplant Survival

Certain components of a graft that provoke alloimmunity may not be vital for graft function or critical as targets of rejection. Corneal transplantation is an example of this, because graft epithelium plays a role in allosensitization, whereas corneal graft endothelium—which shares the same alloantigens—is the critical target in allorejection. In this study, we found that exploiting this biology by replacing donor epithelium of an allograft with an allodisparate third-party epithelium yields a marked enhancement in transplant survival. Such 'chimeric' allografts consisted of a C3H/He (H-2^k) corneal epithelium over a C57BL/6 (H-2^b) epithelial-denuded cornea (or v.v. ) and orthotopically placed on BALB/c (H-2^d) hosts. Conventional corneal allografts (C3H/He or C57BL/6) or isografts (BALB/c) were also transplanted on BALB/c hosts. Alloreactive T-cell frequencies (CD4⁺ interferon [IFN]-γ⁺) primed to the graft endothelium were strongly diminished in chimeric hosts relative to conventionally allografted hosts. This was corroborated by a decreased T-cell infiltration (p = 0.03) and a marked enhancement of allograft survival (p = 0.001). Our results represent the first successful demonstration of chimeric tissue, epithelial-denuded allograft plus third-party allodisparate epithelium, in the promotion of allograft survival. Moreover, chimeric grafting can be readily performed clinically, whereby corneal allograft rejection remains a significant problem particularly in inflamed graft beds.     

Distinct requirements for host CD80/CD86 costimulatory molecules in cardiac versus islet rejection

The role of B7 family members CD80 and CD86 in providing costimulatory signals to T cells is well established. Interestingly, previous studies show that host CD80/CD86 expression is required for cardiac allograft rejection. However, the role for host costimulation by CD80/CD86 molecules for the rejection of neovascularized islet allografts and xenografts is unknown. The purpose of this study was to determine whether islet allografts and/or rat islet xenografts required host CD80/CD86 molecules for acute rejection. Streptozotocin-induced diabetic C57Bl/6 (B6, H-2(b)) or B6 CD80/CD86 double-deficient mice were grafted with allogeneic BALB/c (H-2(d)) islet allografts or with WF (RT1(u)) islet xenografts. Nondiabetic B6 mice were grafted with BALB/c heterotopic cardiac allografts. Consistent with previous reports, BALB/c islet allografts were acutely rejected in wild-type B6 mice could survive long-term (>100 days) in B6 CD80/CD86-deficient animals. In stark contrast, both islet allografts and WF rat islet xenografts demonstrated acute rejection in both control B6 and in B6 CD80/CD86 deficient hosts. In conclusion, varied studies imply that the inherent pathways for rejecting primarily vascularized versus cellular allografts or xenografts may be distinct. The present study illustrates this concept by showing a marked difference in the role of host-derived CD80/CD86 costimulatory molecules for cardiac allograft versus islet allograft/xenograft rejection in vivo. Although such costimulation is rate limiting for cardiac allograft rejection, these same molecules are not necessary for acute rejection of either islet allografts or xenografts.     

The natural killer cell activating receptor,NKG2D,is critical to antibody-dependent chronic rejection in heart transplantation

Chronic rejection is among the most pressing clinical challenges in solid organ transplantation. Interestingly, in a mouse model of heterotopic heart transplantation, antibody-dependent, natural killer (NK) cell-mediated chronic cardiac allograft vasculopathy occurs in some donor–recipient strain combinations, but not others. In this study, we sought to identify the mechanism underlying this unexplained phenomenon. Cardiac allografts from major histocompatibility complex (MHC) mismatched donors were transplanted into immune-deficient C57Bl/6.rag^−/− recipients, followed by administration of a monoclonal antibody against the donor MHC class I antigen. We found marked allograft vasculopathy in hearts from C3H donors, but near-complete protection of BALB/c allografts from injury. We found no difference in recipient NK cell phenotype or intrinsic responsiveness to activating signals between recipients of C3H versus BALB/c allografts. However, cardiac endothelial cells from C3H allografts showed an approximately twofold higher expression of Rae-1, an activating ligand of the NK cell receptor natural killer group 2D (NKG2D). Importantly, the administration of a neutralizing antibody against NKG2D abrogated the development of allograft vasculopathy in recipients of C3H allografts, even in the presence of donor-specific antibodies. Therefore, the activating NK cell receptor NKG2D is necessary in this model of chronic cardiac allograft vasculopathy, and strain-dependent expression of NK activating ligands correlates with the development of this disease.