Infectious tolerance develops after intrathymic alloantigen-induced acceptance of rat heart allografts can be adoptively transferred

BACKGROUND: We have shown that intrathymic (IT) injection of alloantigen with antirat lymphocyte serum (ALS) treatment can induce donor-specific allograft acceptance. The purpose of this study was to investigate whether T-regulatory (T-reg) cells play a role in the maintenance of donor-specific heart graft tolerance that develops after IT injection of Lewis (LEW, RT1(l)) alloantigen into a Dark Agouti (DA, RT1(a)). METHODS: Na?ve DA rats were injected IT with 2.5 x10(7) LEW donor splenocytes and injected intraperitoneally with 1 mL ALS. Twenty-one days after pretreatment, a LEW or Brown Norway (BN, RT1(n)) heart was transplanted into a treated DA recipient. Splenocytes (1 x 10(8) or 5 x 10(7)) from a LEW heart-tolerant long-term survivor (LTS; >60 days) DA recipient were harvested and adoptively transferred (AT) into an irradiated (450 rad) na?ve DA rat 24 hours before transplanting a LEW heart. RESULTS: All LEW heart allografts were rejected by untreated DA rats in a mean survival time (MST) of 7.4 +/- 1.7 days (n=7). In contrast, 66.7% of LEW heart grafts into IT+ALS-pretreated DA recipients were accepted indefinitely (n=24). When either 1 x 10(8) (n=5) or 5 x 10(7) (n=5) splenocytes from a LEW heart graft-tolerant LTS (>60 days) DA recipient were AT into a new na?ve DA rat, all new LEW heart grafts were accepted indefinitely. CONCLUSIONS: The donor-specific tolerance that develops after IT+ALS-induced LEW heart acceptance by DA recipients can be transferred adoptively to new na?ve DA recipients, thus indicating that it is infectious tolerance.     

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.     

Combination of donor-specific blood transfusion with anti-CD28 antibody synergizes to prolong graft survival in rat liver transplantation

Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to na?ve LEW rats 7 days before LTx [DST(-7d)], 14 and 7 days before LTx [DST(1 x 2)], twice a week for 2 weeks prior to LTx [DST(2 x 2)] and once a week for 4 weeks prior to LTx [DST(1 x 4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1 x 2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4+ CD45RC- but not CD4+ CD25+ foxp3+ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.     

The persistence of regulatory cells developing after rat spontaneous liver acceptance

BACKGROUND: We have previously reported that the spontaneous acceptance of Lewis (LEW, RT1(l)) to Dark Agouti (DA, RT1(a)) rat orthotopic liver transplant (OLT) is eliminated by donor gamma-irradiation. The acceptance of the irradiated LEW liver is also reestablished in a na?ve rat after the adoptive transfer of T regulatory (T-reg) cells from a LEW to a DA liver-tolerant long-term (>60 days) survivor (LTS) into a na?ve DA rat. However, little is known about the growth conditions required to maintain T-reg function. In this study, we examined the need for continued donor-specific alloantigen stimulation for the maintenance and function of T-reg cells. METHODS: Splenocytes (SCs; 1.5 x 10(8) cells) from a LEW liver allograft-tolerant LTS DA recipient were adoptively transferred fresh or after in vitro stimulation into another naive DA rat on day 1, 4, or 7 before an irradiated (1000R) LEW liver transplant. For in vitro alloantigen stimulation, SCs from LEW to DA LTS were co-cultured with mitomycin-C (MMC)-treated na?ve LEW (donor alloantigen-specific) or Brown Norway (BN) (RT1(n); third party) SCs for 72 hours. Graft rejection, as defined by death of the recipient, was confirmed histologically. RESULTS: All LEW liver grafts were accepted spontaneously by DA recipients for more than 60 days (n=32), while all irradiated LEW livers were acutely rejected (n=9; mean survival time [MST]=12.8 +/- 4.0 days). When LTS DA SCs were adoptively transferred into a naive DA rat 1 day before OLT, all irradiated LEW grafts were accepted greater than 60 days (n=9). However, when fresh LTS DA SCs were transferred to a new na?ve DA rat on 4 or 7 days before OLT, all irradiated LEW liver grafts were acutely rejected (MST=10.2 +/- 0.5 days [n=4] and MST=13.5 +/- 5.0 days [n=4], respectively). When LTS DA SCs were stimulated in vitro before adoptive transfer, irradiated LEW liver grafts after 4 days (n=5) were then accepted. In vitro culture of LTS DA SCs with MMC-treated BN SCs (third-party) for 72 hours before adoptive transfer resulted in 3 of 5 irradiated LEW livers at day 4 being accepted (n=5). CONCLUSIONS: The maintenance of T-reg function requires continuous LEW donor-specific alloantigen stimulation.     

328. Mechanismen der langzeitakzeptanz allogener rattenlebertransplantate: makrophagenaustausch und suppressorzellaktivierung

Summary After orthotopic rat liver transplantation in the fully allogeneic BN (RT-1n) to LEW (RT-11) combination, the phenomenon of spontaneous tolerance of donor antigen occurs. We demonstrate two different immune mechanisms that may account for this process. Using adoptive transfer assays we show the presence of donor-specific T-suppressor lymphocytes in the spleens of long-term surviving liver graft, recipients. These cells prolong - adoptively transferred into irradiated syngeneic hosts — the survival of donor-specific (BN) but not third-party (DA) renal allografts (I00 days vs 1I days in control groups). Secondly, we demonstrate the replacement of Kupffer cells in the graft by recipient macrophages using polymorphic monoclonal antibodies in an immunoperoxidase technique. This may contribute to graft adaptation and thus to long-term graft acceptance.     

成年大鼠联体共生诱导肾移植耐受及其机制研究

目的：建立异基因大鼠联体共生的动物模型,探讨其诱导肾移耐受机理,方法对DA和LEW大鼠实施联体手术并诱导耐受,然后进行DA→LEW的肾移植。FACS检测术后脾脏、脾胸中嵌中状态态;检测体内、外过继转移耐受大鼠脾细胞对正常大鼠单向混合淋巴细胞反应（MLR）和迟发性过敏反应（DTH）的抑制作用,以及T细胞克隆不应答在耐受中的作用。结果：联体共生诱导耐受可使DA→LEW的肾移植存活明显延长。耐受组脾脏、胸腺中供体细胞的嵌合比例明显高于排斥组。耐受可通过体内,外过继转移给正常大鼠。外源性IL－2可部分逆转耐受状态。结论嵌合状态态与肾移植耐受存在明显相关性,克隆不应答、抑制细胞参与了耐受的形态。     

Alloreactive T suppressor cells in the rat. I. Evidence of three distinct subsets of splenic suppressor T cells resistant to cyclosporine

In this study we examined the effect of cyclosporine on three distinct subsets of T suppressor (Ts) cells identified in a rat renal allograft model. Ts inducer (Ts1) cells having the CD4 marker are found in the spleens of DA rats undergoing acute rejection of LEW kidneys. Transducer (Ts2) and effector (Ts3) cells both carry the CD8 marker and are found in the spleens of long-term surviving DA rats bearing LEW kidney allografts made tolerant by donor-specific blood transfusions or by cyclosporine (in most cases). These latter cells are distinguished by their susceptibility to cyclophosphamide (CY), Ts2 cells being resistant while Ts3 cells are sensitive to CY. When Ts cells from DA rats undergoing acute graft rejection of LEW kidneys or bearing long-term-surviving LEW kidneys that had been treated with cyclosporine (10mg/kg/day) for 2 or 10 days, respectively, were adoptively transferred into lightly irradiated DA recipients, these cells were still able to specifically induce long-term survival of LEW kidneys. LEW kidney survival was not prolonged in DA rats given no cells or cells from rats treated with cyclosporine for 10 days. Thus it would appear that the three functional subsets of Ts cells demonstrated in this renal allograft model by adoptive transfer of spleen lymphocytes are not inhibited by cyclosporine, suggesting that this resistance of Ts cells to cyclosporine may be partly responsible for the immunosuppressive effect of this agent.     

Analysis of cellular events in hepatic allografts: Donor progenitors induce intragraft chimerism

Abstract Long‐term graft acceptance and tolerance induction after allogeneic rat liver transplantation are well described. However, the underlying mechanisms remain unclear. In this study we investigated the cellular events within the liver graft during initial immunosuppression and long‐term acceptance. Orthotopic liver transplantation was performed in the Dark Agouti (DA)‐to‐Lewis (LEW) and LEW‐to‐DA rat strain combination. In order to achieve long‐term acceptance, LEW recipients of DA livers were treated with two different short‐term therapies. Non‐parenchymal cells (NPC) were isolated from liver allografts on days + 10 and + 100 after transplantation and donor‐specific leukocytes were immunophenotyped by flow cytometry. Both the monotherapy and triple therapy prolonged graft survival (> 100 days). Liver allografts from LEW donors into DA recipients were spontaneously accepted across a complete MHC mismatch without immunosuppression. Liver allograft rejection was induced by infiltrating alloreactive immunocompetent cells. But the intensities of cell infiltration in the early and late phases after transplantation did not correlate with eventual outcome. Donor‐specific NPC decreased to 18‐25% on day + 10 in both therapeutic groups, but had rebounded to up to 40% by day + 100. Recurrence of donor‐specific cells was caused almost exclusively by rising T cell counts. The persistence of dendritic cells in the late phase after transplantation could be clearly demonstrated. Repopulation by donor‐specific T lymphocytes was observed in long‐term accepted liver grafts. This recurrence may be based on the differentiation of liver‐derived progenitor cells. The persistent coexistence of donor and recipient cells within the liver allograft (intrahepatic chimerism) appears to be characteristic and may be important for long‐term acceptance.     

Involvement of autoimmunity against nuclear histone H1 in liver transplantation tolerance

BACKGROUND: Our recent studies suggested that anti-histone H1 autoantibody (auto-Ab) plays an important role in experimental and clinical liver allograft tolerance as a natural immunosuppressive factor. The present study aimed to explore how the autoimmune response against histone H1 is involved in tolerance induction. METHODS: The measurement of anti-histone H1 auto-Ab and immunohistochemical analysis were performed in serum and liver allografts after orthotopic liver transplantation (OLT). To compare the auto-Ab response against histone H1 between the recipients of rejector (DA-LEW) and tolerogenic (DA-PVG) OLT models, na?ve recipients were immunized with calf thymus histone H1. The immunosuppressive state of histone H1-immunized rats was assayed by mixed lymphocyte reaction (MLR). RESULTS: Anti-histone H1 Ab titer was transiently increased during the rejection phase after OLT (days 7-21) in the DA-PVG combination, while no such response was confirmed in the DA-LEW acute rejection model. Nuclear histone H1 antigens were found in the cytoplasm and the extracellular environment in liver allografts at the rejection phase in the tolerogenic model but not in the rejector model, resulting from the transient induction of anti-histone H1 auto-Ab in recipient PVG rats after OLT. Low dose and short-term immunization with histone H1 upregulated the anti-histone H1 Ab titer in na?ve PVG rats, which exhibited a low allogeneic immune response, while no such response was found in na?ve LEW rats. CONCLUSIONS: These results suggest that the sensitivity to nuclear antigens such as histone H1 may be a key factor determining the acceptance or rejection of donor liver grafts, at least in DA-PVG and DA-LEW combinations.     

Peritransplant streptavidin recipient treatment prolongs rat cardiac allograft survival

BACKGROUND: Because streptavidin shows high localization in inflamed tissues, it might also interfere with the proliferation of cells involved in allograft rejection. METHODS AND RESULTS: Treatment of na?ve ACI recipients with 20 mg/kg streptavidin i.p. alone significantly prolonged Lewis cardiac allografts from a mean survival time of 9.8+/-0.7 days in controls to 19.8+/-6.5 days, with one recipient accepting the graft permanently (>250 days). Peritransplant streptavidin treatment combined with 0.5 ml of antilymphocyte serum (ALS) transient immunosuppression led to permanent graft survival (>250 days) in 6 of 10 recipients. Second-set skin grafts performed 60 days after the primary cardiac allograft were prolonged to 45 days, whereas the third party Wistar-Furth (WF) skin grafts were rejected in 15 days without the rejection of the primary Lewis cardiac allografts. Pathology of transplanted cardiac allografts at 100 days showed no mononuclear cell infiltration or chronic allograft vasculopathy. Streptavidin given for 5 days at 20 mg/kg caused a moderate initial weight loss but had no effect on hematologic, biochemical, and histologic parameters in the treated recipients. CONCLUSION: This study demonstrates that peritransplant recipient treatment with streptavidin combined with peritransplant ALS induces prolonged cardiac and second-set skin allograft survival. We conclude that recipient peritransplant streptavidin treatment may provide a new strategy for the induction of transplant tolerance.     

"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.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

目的 联合阻断OX40/OX40L和CD28/B7双协同信号途径和供体特异性脾细胞输注,诱导预存同种反应性记忆T淋巴细胞大鼠对同种心脏移植物的耐受.方法 建立大鼠预存同种反应性记忆T淋巴细胞的移植模型,采用免疫磁珠法分选CD8+CD44-记忆性T细胞;对过继转移供体特异性CD8+记忆性T细胞3 d的Lewis大鼠分别/合并输注AdCTLA4Ig、AdOX40Ig、供体脾细胞(DST),同时移植来至DA大鼠的心脏;48 h后取出心脏进行组织学分析、细胞因子表达分析,同时观察不同处理移植心脏存活时间.结果 AdCTLA4Ig+AdOX40Ig+DST组分别与AdCT-LA4Ig,AdOX40Ig和DST比较,心脏组织病理级别较低,白细胞介素(IL)-2及干扰素(IFN)-γ的表达水平也明显比其他组低很多,而心脏存活时间显著延长.结论 联合阻断OX40/OX40L和CD28/B7和供体特异性脾细胞输注能较好地诱导大鼠心脏移植物耐受.     

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.     

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.     

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.     

The role of B7 ligands (CD80 and CD86) in CD152-mediated allograft tolerance: a crosscheck hypothesis

BACKGROUND: The regulatory mechanism by which the B7 ligands (CD80 and CD86) direct the CD28/CD152 costimulatory pathways is unclear. This study investigated the role of CD80 and CD86 in a CD152-mediated allograft tolerance model. METHODS: A low-responding cardiac transplant model (BALB/c-->B10.A) with possible long-term acceptance was used. Immunocytochemical and flow cytometric analyses of the graft-infiltrating cells were conducted to characterize this transplant model. The influence of anti-CD80 and anti-CD86 treatments on the proliferation and interleukin (IL)-2 productions of the tolerated splenocytes (SC) was analyzed. The role of CD80 and CD86 in the induction and maintenance of the graft acceptance in this transplant model were also tested. RESULTS: B10.A mice could accept the BALA/c cardiac allografts (11/22), and an anti-CD152 antibody blocked the graft acceptance (10/10). Immunocytochemical and flow cytometric analyses showed that CD152+ cells were predominant among the CD4+ cells infiltrating the 100-day grafts of the B10.A recipients (B10.A-100). Either anti-CD80 or anti-CD86 treatment significantly enhanced polyclonal proliferation and IL-2 production of the B10.A-100 SC. Blockade of either CD80 or CD86 prohibited the tolerance transmitted by adoptive transfer, and anti-CD80 or anti-CD86 plus skin grafting undermined the established allograft tolerance. CONCLUSIONS: Both CD80 and CD86 were essential for the induction and maintenance of the CD152-mediated allograft tolerance.     

Mediation of the induction of immunologic unresponsiveness following antigen pretreatment by a CD4 (W3/25+) T cell appearing transiently in the splenic compartment and subsequently in the TDL

We have previously demonstrated in a rat renal allograft model, using adoptive transfer studies, that a single blood transfusion results in the generation of blood donor-specific W3/25+ (CD4) T suppressor cells detectable in the thoracic duct lymph, but not in the spleen, 7 days after transfusion. In this study, again using the LEW-to-DA strain combination, we have investigated the time of appearance of suppressor activity in different lymphoid compartments in vivo and in vitro following a single blood transfusion. Cells (5 x 10(7) unfractionated TDL or 1 x 10(8) unfractionated spleen cells) were harvested from DA rats on days 1-6 after a single LEW blood transfusion and were adoptively transferred into syngeneic (DA), lightly irradiated (200 rads) hosts. The following day these animals received a kidney allograft (LEW or PVG). Specific suppressor activity, as demonstrated by the prolongation of survival of LEW, but not PVG, renal allografts was noted in the spleen 4 days after transfusion (MST greater than 100 days). Suppressor cells were not found in the TDL at this stage--but 6 days after transfusion suppressor activity could be detected in the TDL (median survival time [MST]greater than 100 days), while at this time no such activity was demonstrable in the spleen. The suppressor activity in the spleen on day 4 after transfusion was shown to be mediated by a W3/25+ (CD4) T cell. The proliferative response in a mixed lymphocyte culture of DA spleen cells, prepared 4 days after transfusion, or TDL cells prepared 6 days after transfusion, to LEW stimulator cells was significantly suppressed when compared with the response of cells harvested from untreated controls. In contrast, the proliferative response of spleen cells examined 6 days after transfusion, when no suppressor activity was detected in vivo, was significantly augmented in comparison with untreated controls. Thus we have demonstrated that a DST results in the generation of W3/25-positive (CD4) T cells capable of suppressing renal allograft rejection that appear transiently in the splenic compartment (4 days after transfusion) and subsequently in the TDL (six days after transfusion).     

Specificity of cellular migration into cardiac allografts in rats.

The question has not been previously investigated as to whether host lymphocytes infiltrate vascularized organ allografts in indiscriminate fashion, or whether they are retained selectively because of specific antigenic recognition sites on their surfaces. By using a dual cardiac allograft model in LEW rats, we have examined this problem by two methods: (1) detection of preferentially accumulating cells selectively cytotoxic to allografts bearing specific transplantation antigens as compared with "third-party" allografts; and (2) examination of trafficking patterns of separate radiolabeled populations of sensitized cells adoptively transferred into double heart-grafted recipients. In the first series of experiments, using BN and BUF rats as donors, differences in specific cytotoxicity mounted by infiltrating lymphocytes harvested from the appropriate and inappropriate graft were moderately significant (P less than 0.05). Because the question of cross-reactivity betweeen BUF and BN antigen was raised, lymphocytes sensitized to BN and WF donors were differentially labeled in vitro with 3H- or 14C-thymidine. After mixture and adoptive transfer, the ratio of specific to third-party labels was measured in each graft. In this second series of experiments, significant (P less than 0.001) preferential accumulation of specifically sensitized cells were found in the appropriate vascularized organ allograft. These experiments confirm the results of other experimental models, and demonstrate that sensitized lymphocytes accumulate selectively in specific vascularized organ allografts.     

Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector

There is a need to achieve donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and the side-effects of long-term immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it was "self". The AdCTLA4Ig-mediated gene transfer (SC) + cyclophosphamide (CP) treatment alone prolongs allograft survival but does not induce tolerance. However, in our study, the AdCTLA4Ig-mediated gene transfer combined with SC + CP treatment yielded significantly prolonged mean survival times (149.7 +/- 18.0 days), while those in the untreated or AdLacZ treated mice were rejected in normal fashion (5.3 +/- 0.5 and 5.2 +/- 0.4 days, respectively), and survival in the AdCTLA4Ig or SC + CP treated groups were 45.7 +/- 9.6 or 50.2 +/- 5.3 days, respectively. In conclusion, a protocol of AdCTLA4Ig + SC + CP improved the survival of DA-->LEW cardiac allografts.