"Infectious tolerance" develops after the spontaneous acceptance of Lewis-to-Dark Agouti rat liver transplants

BACKGROUND: After monoclonal antibody or donor-specific transfusion treatment, infectious tolerance to rat or mouse heart or skin transplants can be passed on to naive recipients by adoptive transfer of tolerant lymphocytes. We examined whether similar regulatory cells develop after the spontaneous acceptance of Lewis-to-Dark Agouti (DA) rat liver transplants without immunomodulating agents. METHODS: After Lewis-to-DA rat liver transplantation, 100 x 10(6) splenocytes were harvested and adoptively transferred into a 450 rad-irradiated naive DA rat 24 hours before Lewis heart transplantation. Adoptive transfer of CD4+ or CD8+ T cells was also examined. In some experiments, splenocytes from recipients with long-term accepted Lewis hearts induced by adoptive transfer were serially transferred to multiple generations of recipients before Lewis rat heart transplantation. In vitro mixed lymphocyte culture response and cytotoxic T lymphocyte generation were measured. RESULTS: When splenocytes from a DA rat recipient >60 days after Lewis rat liver acceptance were transferred into irradiated DA rat recipients, all Lewis rat hearts were accepted, whereas third-party Brown-Norway rat hearts were rejected. However, splenocytes from DA rat recipients 30 days after liver transplantation did not prolong Lewis rat heart survival. Adoptive transfer of 40 x 10(6) CD4+, 10 x 10(6) CD4+ or 10 x 10(6) CD8+ cells from a DA rat bearing Lewis rat liver >60 days resulted in acceptance of 88%, 80%, or 57% acceptance of Lewis rat hearts, respectively. Serial second and third adoptive transfer of long-term survivor splenocytes resulted in the acceptance of all Lewis rat hearts. In mixed lymphocyte culture, splenocytes from a naive DA rat and a DA rat accepting a Lewis rat liver transplant for >60 days showed similar proliferative responses to both Lewis and Brown-Norway rat stimulators. An equivalent level of indirect cytotoxic T lymphocyte activity was exhibited by splenocytes from both a naive DA and a DA rat accepting a Lewis rat liver transplant for >60 days. CONCLUSIONS: Regulatory cells developing after the spontaneous acceptance of a Lewis to DA liver transplant can serially adoptively transfer the acceptance of a Lewis rat cardiac graft in spite of the presence of in vitro antidonor reactivity. Both CD4+ and CD8+ populations have this regulatory activity, although the CD4+ population plays the dominant role.     

Characteristics and function of suppressor T lymphocytes in immunologically unresponsive rats following pretreatment with UV-B-irradiated donor leukocytes and peritransplant cyclosporine

Lewis rats pretreated with UV-B-irradiated donor leukocytes (UV-DL) and peritransplant cyclosporine (CsA...CsA, 20 mg/kg on days 0, +1, and +2) accepted W/F heart allografts permanently. This study of donor-specific immunologic unresponsiveness and its cellular mechanisms shows that the induction phase of unresponsiveness is partially mediated by W3/25+ T cells, while its maintenance is dependent on the presence of 0 x 8+ T cells. In vivo adoptive transfer of either splenocytes, T lymphocytes (T cells), or W3/25+ T cells from ungrafted, UV-DL-transfused rats into unmodified syngeneic Lewis rats that received test grafts 24 hr later led to significant prolongation of donor-specific graft survival. Adoptive transfer of 0 x 8+ cells did not influence donor-type (W/F) test graft rejection. Adoptive transfer of SpL, T cells and 0 x 8+ cells from UV-DL and CsA-treated recipients of W/F heart allografts at 20 or 180 days after transplantation led to significant donor-specific graft prolongation in naive syngeneic hosts, while adoptive transfer of W3/25+ cells, in this group, did not affect test graft survival. However, the adoptive transfer of SpL or of T cell subsets did not influence third-party (ACI) graft survival. In-vitro mixed lymphocyte reaction between thoracic duct lymphocytes obtained at various intervals following grafting from UV-DL and CsA treated Lewis recipients of W/F heart allografts and donor-type SpL resulted in significantly reduced reactivity by 78%, 75%, 69% (P less than 0.001) and 43% (P less than 0.02) compared with controls when responder TDL were obtained at 20, 50, 100, and 180 days after transplantation, respectively. In coculture studies, the MLR response to donor SpL was specifically suppressed by 60%, 57%, 46%, and 50% (P less than 0.01) at 20, 50, 100, and 180 days after transplantation, respectively, compared with controls. These data indicate that the induction of specific unresponsiveness to heart allografts in this model is mediated, in part, initially by the appearance in the host of specific W3/25+ cells either induced or recalled by UV-DL transfusion, and that a stable state of immunologic unresponsiveness is subsequently dependent on the presence of 0 x 8+ suppressor cells.     

The mechanism of the induction of immunologic unresponsiveness to rat cardiac allografts by recipient pretreatment with donor lymphocyte subsets

In continuation of our studies using UV-B-irradiated DST and donor leukocyte (DL) recipient pretreatment to induce specific unresponsiveness to organ allografts, we have examined the relative contributions of splenic lymphocyte populations and T lymphocyte subsets in the induction of immunologic unresponsiveness. Our data show that enriched populations of MHC class II-positive B lymphocytes and the W3/25+ T cell subset obtained from splenic leukocytes using immunoadsorbent columns in conjunction with mAbs led to indefinite graft survival (greater than 100 days) in the Lewis-to-ACI rat cardiac allograft model. In contrast, pretreatment with T lymphocytes or the Ox8+ T subset was relatively ineffective in prolonging cardiac allograft survival. In addition, third-party (W/F) W3/25+ T cell recipient pretreatment did not influence the survival of Lewis cardiac allografts in ACI recipients, thus confirming the specificity of pretreatment with the T cell subset in graft prolongation. Furthermore, we have examined the underlying mechanisms of donor-specific unresponsiveness induced by donor spleen cells, B lymphocytes, and W3/25+ T cells using adoptive transfer assays. Serial adoptive transfer studies demonstrated the presence of 0x8+ suppressor T cells in the spleens of unresponsive recipients bearing well-functioning cardiac allografts and of serum "suppressor factors" that have the capacity for specifically prolonging donor-type test graft survival in naive syngeneic rats. Our findings suggest that the induction of specific unresponsiveness in this model is dependent on a sequential collaboration between the appearance of donor-specific serum factor(s) (humoral phase) and donor-specific suppressor T cells (cellular phase). These results may be potentially useful in planning future strategies for the induction of unresponsiveness to clinical organ allografts by immunologic manipulation of the host with MHC class II-positive B cell and CD4+ T cell clones.     

Effect of ultraviolet-B-irradiated donor-specific blood transfusions and peritransplant immunosuppression with cyclosporine on rat cardiac allograft survival

We have previously demonstrated that pretreatment of ACI recipients with ultraviolet-irradiated donor-specific blood transfusion (UV-DST) leads to permanent cardiac allograft survival without further host immunosuppression (ACI rats are weak responders to Lewis lymphocytes in mixed-lymphocyte reaction). This study examines the effect of UV-DST and the timing of transfusions on ACI cardiac allograft survival in Lewis recipients with and without the addition of peritransplant cyclosporine (CsA) (20 mg/kg i.m.) given on days 0, +1, and +2 in relation to the time of transplantation. The mean survival time (MST) of ACI cardiac allografts in Lewis recipients was significantly increased to 33.6 +/- 5.7 days (P less than 0.001) by CsA treatment alone as compared to 6.5 +/- 0.5 days survival in control. When DST was given on day -3 combined with CsA, graft survival was increased to 42.0 +/- 9.3 days (P less than 0.01), as compared to 5.8 +/- 1.3 days when DST alone was used. When DST was irradiated with ultraviolet B (UV-DST) and administered on day -3 combined with peritransplant CsA, the MST was increased to 68.83 +/- 16.1 days as compared to an MST of 10.0 +/- 1.0 days in controls treated with UV-DST alone. When UV-DST was given on day -7 and combined with peritransplant CsA immunosuppression, the results were similar. However, when UV-DST was peritransplant CsA course, 4 of 6 recipients maintained their ACI heart allografts indefinitely (greater than 300 days) in contrast to the effect of UV-DST alone (MST of 13.5 days). Third-party (W/F) UV-irradiated blood transfusions were ineffective in prolonging ACI cardiac allografts in Lewis rats, regardless of whether the transfusions were given alone or in combination with peritransplant immunosuppression with CsA. In conclusion, these results demonstrate that UV-DST combined with a brief peritransplant immunosuppression with CsA induces prolonged heart allograft survival in a histoincompatible, strong responder host, and that such effect is donor specific. The use of UV-DST combined with peritransplant CsA immunosuppression offers a promising approach to achieving organ transplant unresponsiveness, and decreased sensitization to the donor blood elements, which eventually may have important clinical implications.     

Intrathymic injection of donor alloantigens induces donor-specific vascularized allograft tolerance without immunosuppression.

The induction of donor-specific tolerance could prevent the side effects of immunosuppression while improving allograft survival. Male adult Buffalo (RT1b) rats underwent an intrathymic (IT), portal venous (PV), intrasplenic (IS), or subcutaneous (SQ) injection of 25 x 10(6) major histocompatibility complex (MHC) mismatched Lewis (RT1(1)), UV-B-irradiated Lewis (RT1(1)), ACI (RT1a), or syngeneic Buffalo (RT1b) splenocytes. At the completion of the donor alloantigen injection, 1 mL rabbit anti-rat lymphocyte serum (ALS) was administered intraperitoneally to the Buffalo recipients, and 21 days later a heterotopic Lewis or ACI heart was transplanted. Intrathymic injection of donor alloantigen induced a donor-specific tolerance that allowed the cardiac allograft to survive indefinitely (mean survival time [MST] > 140.7 days) in 84% of the recipients without further immunosuppression, whereas groups receiving antigen injections at other sites (PV, IS, and SQ) plus ALS rejected cardiac allografts in normal fashion (MST approximately 8.0 days). Buffalo recipient rats with long-term surviving Lewis cardiac allografts after Lewis IT injection and ALS subsequently rejected a heterotopic third-party ACI cardiac allograft in normal fashion (MST approximately 7 days), whereas a second Lewis cardiac allograft was not rejected (MST > 116 days). Microchimerism is unlikely because Lewis allograft survival was also prolonged (MST > 38.7 days) in rats receiving UV-B-irradiated splenocytes IT, which cannot proliferate. Survival of Lewis renal allografts was also prolonged, but not indefinitely, in Buffalo recipients possessing a long-term surviving Lewis cardiac allograft (MST approximately 17.6 days versus 7 days for control). This model emphasizes the potential role of exposure of immature thymocytes to foreign donor alloantigens during maturation in the thymic environment for the development of unresponsiveness to an MHC-mismatched donor-specific vascularized allograft.     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Permanent rat cardiac allograft survival induced by ultraviolet B-irradiated donor lymphocytes and peritransplant cyclosporine

This study examines the effect of pretreatment with 10(8) ultraviolet B-irradiated donor leukocytes (UV-DL) with or without peritransplant cyclosporine (CyA) treatment (20 mg/kg on days 0, +1, and +2 relative to transplantation) on rat cardiac allograft survival across major histocompatibility loci. A single UV-DL pretreatment on day -3 or -7 (before transplantation) significantly prolonged survival of heart allografts from Wistar-Furth rats (W/F) in Lewis recipients from 6.8 +/- 0.8 days to 18.4 +/- 2.1 and 17.6 +/- 1.5 days (p less than 0.001), respectively. Multiple UV-DL infusions on days -14 and -7 increased the mean survival time to 20.0 +/- 0.9 days (p less than 0.001). Similarly, UV-DL infusion on day -3 or -7 significantly prolonged the mean survival time of heart allografts from ACI rats in Lewis rats. A single or multiple UV-DL infusions combined with peritransplant CyA led specifically to permanent W/F cardiac allograft survival (more than 200 days) in all recipients. Similarly, UV-DL infusion combined with peritransplant CyA led to indefinite survival of ACI cardiac allografts in two thirds of Lewis recipients. Adoptive transfer of splenocytes from long-term recipients of cardiac allografts, which specifically prolonged donor test grafts in syngeneic hosts, suggests that unresponsiveness to cardiac allografts is, in part, dependent on suppressor cells. This study emphasizes the importance of UV irradiation of DLs in the modulation of alloreactivity and the induction of donor-specific unresponsiveness in adult animals.     

Humoral immunity in allograft rejection. The role of cytotoxic alloantibody in hyperacute rejection and enhancement of rat cardiac allografts

The role of humoral immunity in graft rejection in the rat model remains controversial. Passive transfer of cytotoxic alloantibody (CAA) has resulted either in hyperacute rejection or in graft enhancement. This study examines the effect of transfer of CAA on cardiac allograft survival in three rat strain combinations that are fully mismatched at the major histocompatibility (MHC) loci. Strain-specific immune responsiveness in donor-recipient pairs varied from low (Lewis-to-ACI) to high (ACI-to-Lewis) as measured by mixed lymphocyte reactions. CAA was obtained from rats sensitized by three successive skin grafts at weekly intervals. Group 1 (high responder recipients), which consisted of Lewis rats presensitized to ACI and had a lymphocytotoxicity titer of 1:512 to 1:2048, rejected ACI cardiac allografts in 10.8 +/- 7.2 hr compared with 6.5 +/- 0.5 days in naive controls (p less than 0.001). Injection of 1 ml of high-titer CAA into naive Lewis rats immediately after ACI cardiac grafting led to hyperacute rejection of ACI hears in 2.1 +/- 0.8 hr while 1 ml of CAA followed by 2 ml of guinea pig complement (GPC) resulted in even faster rejection (mean survival time (MST) of 23.8 +/- 4.7 min). Injection of 2 ml GPC alone or in combination with 1 ml naive Lewis serum had no effect on graft survival. Multiple pretransplant injections of 1 ml of CAA on days -3, -2,-1, and 0 relative to transplantation resulted in significant prolongation of allograft survival (MST of 10.3 +/- 0.3 days; P less than 0.01). In group 2 (intermediate responder recipients), where Lewis rats were presensitized to WF strain and where cytotoxicity titer was 1:16 to 1:256, the recipients rejected WF hearts in 23.8 +/- 5.8 hr compared with 6.8 +/- 0.8 days in unsensitized control recipients (P less than 0.001). Injection of 1 ml of Lewis anti-WF CAA resulted in prolonged graft survival of 9.7 +/- 3.5 days, while injection of 1 ml of CAA followed by 2 ml of GPC caused hyperacute rejection in 104 +/- 61.7 min. Pretransplant injections of CAA on days -3, -2, -1, and 0 resulted in enhancement, with an MST of 16.3 +/- 1.3 days (P less than 0.001). In group 3 (low responder recipients), ACI presensitized to Lewis developed a cytotoxicity titer of 1:2 to 1:32 and rejected Lewis hearts in 5.3 +/- 0.4 days compared with 10.6 +/- 1.0 days in naive recipients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanisms of immunologic unresponsiveness induced by ultraviolet-irradiated donor-specific blood transfusions and peritransplant cyclosporine

Recipient pretreatment with UV-B irradiated donor-specific blood transfusions (UV-DST) combined with peritransplant cyclosporine on days 0, +1, and +2 leads to permanent cardiac allograft survival in the ACI-to-Lewis rat strain combination. This study investigates the mechanisms of immunologic unresponsiveness induced by UV-DST and CsA by examining several in vitro and in vivo parameters in long-term cardiac allograft recipients. The results of the in vitro studies demonstrate that thoracic duct lymphocytes (TDL) of treated and allografted Lewis rats respond less in a mixed lymphocyte reaction to donor splenic lymphocytes (SpL) by 69%, 75%, and 73% (P less than 0.001) at 30, 50, and 100 days after transplantation, respectively, compared with controls, while the response to a third-party (W/F) SpL is unimpaired. In coculture experiments, the TDL from treated recipients specifically suppressed the response of unmodified Lewis TDL to ACI SpL by 59% and 40% (P less than 0.01) at 30 and 50 days after transplantation, respectively, while responses to W/F SpL were suppressed by only 3-6%. The sera obtained from ungrafted rats transfused with UV-DST suppressed the MLR between unmodified Lewis TDL and ACI SpL by 31% (P less than 0.05) while the sera from UV-DST and CsA-treated and allografted rats specifically suppressed the MLR by 75%, 80% (P less than 0.001) and 37% (P less than 0.01) at 10, 30, and 50 days after transplantation, respectively. In vivo adoptive transfer of 10(4) donor-type dendritic cells (DC) into recipients of beating cardiac allografts at 40 or 60 days after transplantation led to rapid and acute allograft rejection, while the adoptive transfer of 10(8) unseparated SpL obtained at 50 days after transplantation from treated Lewis recipients to syngeneic naive hosts led to a modest but significant prolongation of ACI test cardiac allografts. The transfer of serum from treated and allografted recipients at 10, 30, and 50 days after transplantation led to specific and significant prolongation of test grafts in syngeneic naive hosts. These findings suggest that the mechanisms underlying the in vivo immunologic unresponsiveness induced by pretreatment with UV-DST and peritransplant CsA include inactivation without elimination of class II-antigen presenting cells (APC), generation of specific serum suppressor factor(s) and/or antiidiotypic antibody, and induction of donor-specific suppressor cells.     

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.     

Simultaneous blockade of co-stimulatory signals, CD28 and ICOS, induced a stable tolerance in rat heart transplantation

An inducible co-stimulator (ICOS), a recently identified co-stimulatory receptor with a close structural homology of CD28 and CTLA4, is expressed on activated T cells. Anti-ICOS antibody was demonstrated to be effective on prolongation of graft survival after liver transplantation in rats. In this study, we investigated the potency of tolerance induction using the antibody combined with a recombinant adenovirus vector containing CTLA-4Ig cDNA (AdCTLA-4Ig) in rat heart transplantation model. Using a DA-to-Lewis rat heart transplantation model, an anti-rat ICOS antibody and AdCTLA-4Ig were simultaneously administered i.v. into recipients. The tissue specimens from the grafts were removed on various days after transplantation for histological evaluation. Donor-strain skin and heart grafts, and third-party heart allografts were challenged in the recipients with a long-term surviving graft. Splenocytes from the tolerance-induced recipients were used for adoptive transfer study. Anti-ICOS antibody alone did not prolong the survival of heart allograft. AdCTLA-4Ig monotherapy significantly prolonged the survival of heart allograft (Group 4). With a combination of Anti-ICOS antibody and AdCTLA-4Ig, all recipients were resulted in a long-term allograft acceptance for more than 200 days (Group 8). When challenged donor-strain skin grafts in the tolerant rats of Group 4, the skin was rejected, which also lead to a rejection of primary heart allografts. The recipients in Group 8 also rejected donor-strain skin grafts with no rejection of the primary heart grafts. These recipients accepted secondary heart grafts from donor-strain but not third-party. In Group 8 long-term survival recipients showed a high population of CD4+CD25+ regulatory T cell in peripheral blood, and in adoptive transfer study subtraction of these CD4+CD25+ T cells accelerate the rejection of heart graft in secondary irradiated recipients. The present results demonstrated that anti-ICOS antibody combined with AdCTLA-4Ig potently induces a stable immune tolerance after heart allografting in rat, which is mediated by the induction of CD4+CD25+ regulatory T cells. This strategy may be attractive for clinical employment to induce transplantation tolerance.     

Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) regulatory T cells ameliorates rejection of DA-to-Lewis rat liver transplantation

BACKGROUND: Adoptive transfusion of splenocytes from long-term survivors of a tolerance model of rat orthotopic liver transplantation can induce acceptance of liver allografts in a rejection model preconditioned with donor gamma-irradiation before liver transplantation. Recent studies suggest that the regulatory T cells (Treg cells) in splenocytes from long-term survivors play an important role in the induction of liver graft tolerance, but this observation was made from a rejection model preconditioned with donor gamma-irradiation; little is known about the role of Treg cells in liver graft rejection using a naive rejection model. In this study, we examined the therapeutic potential of CD4(+)CD25(+) Treg cells in a naive rejection model of rat liver transplantation. METHODS: Freshly isolated or ex vivo alloantigen-stimulated CD4(+)CD25(+) Treg cells (1 x 10(6) cells) from naive Lewis RT(1) (LEW) rats were adoptively transferred into another LEW rat on days 1 and 7 after liver transplantation from a Dark Agouti RT1(a) (DA) rat. Recipients were treated with or without oral tacrolimus (FK506) (0.1 mg/kg/day) from days 1 to 7 after transplantation. For ex vivo alloantigen-stimulation, CD4(+)CD25(+) Treg cells from LEW rats were cocultured with mitomycin C-treated DA (donor alloantigen specific) or Brown Norway (BN)(RT1(n), third party) splenocytes for 72 hours. Ex vivo alloantigen-specific CD4(+)CD25(-) T-cell proliferation responses were assessed with fresh and stimulated CD4(+)CD25(+) Treg cells. RESULTS: Freshly isolated, donor alloantigen-stimulated and third-party alloantigen- stimulated CD4(+)CD25(+) Treg cells suppressed antigen-specific CD4(+)CD25(-) T-cell proliferation ex vivo, and adoptive transfusion of these 3 kinds of CD4(+)CD25(+) Treg cells prolonged survival of the liver allografts. The group transfused with the donor alloantigen-stimulated CD4(+)CD25(+) Treg cells had the greatest mean survival among the 3 groups (fresh Treg cells, 21 +/- 2 days, n = 6; third-party alloantigen-stimulated Treg cells, 20 +/- 2 days, n = 6; donor alloantigen-stimulated Treg cells, 30 +/- 2 days, n = 6). When combined with short-term tacrolimus administration, adoptive transfusion of donor antigen-stimulated Treg cells induced the greatest survival time in recipients (greater than 60 days; n = 6). CONCLUSION: Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) Treg cells combined with short-term tacrolimus treatment may represent a new strategy for preventing rejection after liver transplantation.     

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.     

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.     

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 effects of donor-specific blood transfusion enhancement of rat renal allografts on host NK cell responses.

Donor-specific blood transfusion (DSBT) given 1-2 weeks prior to transplantation prolongs the survival of fully allogeneic ACI (RT1a) renal allografts in PVG (RT1c) recipients from 6-8 days to greater than 100 days. We have previously demonstrated that ACI kidneys transplanted to autologous blood transfusion (ABT)- or DSBT-pretreated PVG recipients stimulated an increase in CD8+ (OX8+) cells in the peripheral blood by 6 days after transplantation. To determine whether this increase represents a general expansion of the entire CD8+ population or only a subpopulation of CD8+ cells, subset analysis was performed on peripheral blood lymphocytes depleted of cells reactive with monoclonal antibodies against rat alpha beta T cell receptor (TCR), CD8, or NK cells (R7.3, OX8, or 3.2.3, respectively). Phenotypic studies of PBL depleted of CD8+ cells demonstrated that all 3.2.3+ NK cells coexpressed CD8; depletion of 3.2.3+ PBL revealed a second subpopulation of CD8+3.2.3- cells comprised predominantly of alpha beta TCR+ T cells. In naive PVG rats the prevalence of these two CD8+ subpopulations was approximately equal. Both ABT- and DSBT-pretreated renal allograft recipients demonstrated a significant and equivalent expansion of the CD8+ cell subpopulation that coexpresses the 3.2.3 NK marker. In contrast, the second subpopulation of CD8+3.2.3- cells did not change significantly after allografting. There were also no differences between DSBT and ABT pretreated rats in activity of PBL against the NK targets YAC-1 and Doxie at 6 days after renal transplantation, though the level of activity was modestly increased compared with naive controls. These findings indicate that renal transplantation in the rat is associated with a significant increase in PBL with the NK phenotype (CD8+3.2.3+) and a modest increase of NK activity, but that DSBT enhancement does not affect this NK cell response.     

Regulatory cells develop after the spontaneous acceptance of rat liver allografts

BACKGROUND: After donor-specific transfusion, tolerance to heart transplants is serially passed to naive rats by the adoptive transfer of long-term survivor (LTS)-tolerant splenocytes (SC). We examined whether regulatory cells similarly develop after the spontaneously accepted Lewis (LEW) to Dark Agouti (DA) liver transplants. METHODS: SC from a LTS DA rat with a LEW liver were adoptively transferred to a naive DA 1 day before transplantation of an irradiated (1000 rad) LEW liver. RESULTS: Untreated LEW to DA liver allografts were uniformly accepted; whereas all irradiated LEW liver grafts were rejected. In contrast, when 1.5 x 10 8 DA LTS SC were transferred to a naive DA recipient, all irradiated LEW liver grafts were accepted. When decreased to 1.0 x 10 8 LTS DA SC, only 1 of 4 irradiated LEW grafts was accepted. However, if 1.5 x 10 8 DA SC harvested only 30 days after liver transplantation were transferred, only 2 of 5 irradiated LEW liver grafts were accepted. The serial second and third adoptive transfers of 1.5 x 10 8 DA LTS SC also resulted in the uniform acceptance of irradiated LEW livers. CONCLUSION: Regulatory cells that develop after the spontaneous acceptance of a LEW to DA liver transplant can serially transfer tolerance to new naive LEW liver allograft DA recipients. This "infectious tolerance" is dependent on the time of cell harvest after transplantation and on the cell dose given.     

Optical tissue window: a novel model for optimizing engraftment of intestinal stem cell organoids

BACKGROUND: The pre-transplant administration of donor antigens to recipients is reported to prolong transplanted organ survival. We investigated the effect of pre-transplant intraportal administration of recipient blood on rat hepatic allograft survival. MATERIALS AND METHODS: Male LEW (RT1l) and ACI (RT1a) rats were used as transplant recipients and donors, respectively. Before transplantation, donors were transfused with recipient blood. Experimental animals were divided into groups as follows: group I, no treatment; group II, pre-treatment with recipient blood via the penile vein 7 days before transplantation; group III, pre-treatment with recipient blood via the portal vein 5 days before transplantation; and group IV, pre-treatment with recipient blood via the portal vein 7 days before transplantation. Serum interferon (IFN)-gamma concentrations were measured post-operatively. RESULTS: Animals in group I survived a mean of 10.1 +/- 0.7 days. The survival of groups II and III was 10.6 +/- 1.6 and 13.1 +/- 0.9 days, respectively. The survival rate in group IV was prolonged significantly to 33.7 +/- 2.6 days. Serum concentrations of IFN-gamma were increased significantly in group IV, as compared with group I. The ratio of OX76+CD4+ or OX76+CD8+ T cells to OX76-CD4+ or OX76-CD8+ T cells was greater in group IV, as compared group I. OX76+CD8+ T cells from hepatic allografts in group IV expressed IFN-gamma and interleukin (IL)-10, but not IL-2 mRNA. Apoptotic hepatic infiltrates were greater in group IV, as compared to group I. CONCLUSION: The cytokine profile of donor CD8+ T cells from allografts treated by the intraportal administration of recipient blood is associated with apoptosis of graft-infiltrating cells and the prolonged survival of hepatic allografts in rats.     

Prolongation of survival of fully allogeneic cardiac grafts and generation of regulatory cells by a histamine receptor 2 antagonist

BACKGROUND: The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation. METHODS: CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined. RESULTS: CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4 cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days). CONCLUSION: In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4 regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).