Prolonged allograft survival but no tolerance induction by modulating CD28 antibody JJ319 after high-responder rat heart transplantation

BACKGROUND: Allograft rejection depends on T cell immune responses requiring antigen recognition and costimulatory signals through accessory T cell receptors, including CD28. Inhibition of CD28 signaling with a CTLA-4-immunoglobulin (Ig) fusion protein has resulted in immunosuppression and occasional T cell anergy in mouse transplant models, but not in rats. Because this approach also inhibits a potentially tolerizing signal through CTLA-4, selective blockade of CD28 ligation might induce more profound immunosuppression and transplant tolerance. METHODS: The effects of escalating doses of the rat CD28 monoclonal antibody JJ319 on allograft survival were studied after vascularized heterotopic heart transplantation in a high responder strain combination (DA to Lewis). CD28 antigen modulation and circulating antibody levels were monitored by flow cytometry. RESULTS: CD28 antibody JJ319 markedly prolonged cardiac graft survival compared with untreated controls (7 days, range: 6-8). A strictly dose-dependent increase in median graft survival time was demonstrated with a maximum of 36 days (range: 30-40; p <0.001) after the administration of 8 x 1 mg JJ319 i.p. (days -1 to +6 before/after transplantation). However, indefinite graft survival and tolerance could not be induced by JJ319 treatment. At the maximal dose, flow cytometry showed complete down modulation of the CD28 receptor for 10-14 days without T cell depletion in close temporal relation to antibody presence in serum. In vitro, CD28-modulated T cells showed significantly reduced responses to activation. CONCLUSIONS: CD28 antibody JJ319 induces profound immunosuppression after rat heart transplantation, however without development of transplant tolerance. The underlying mechanism seems to be receptor modulation during primary alloantigen recognition. While still potentially applicable clinically, there are no qualitative or quantitative differences to the treatment with CTLA-4/lg or the blockade of CD2 or LFA-1, as reported elsewhere. Thus, a CD28-modulating approach seems not to allow therapeutic exploitation of a tolerizing signal delivered by CTLA-4 but may still be clinically applicable, especially in combined immune interventions.     

Transfer of "infectious" cardiac allograft tolerance induced by donor-specific transfusion

BACKGROUND: "Infectious tolerance" has been defined as the tolerance induced in a new recipient by the adoptive transfer of cells from a recipient accepting an allograft after anti-CD4 and anti-CD8 monoclonal antibody treatment. A clear understanding of the mechanisms responsible for graft acceptance after donor-specific blood transfusion (DST) has remained elusive. We examined the development and "infectious" nature of immunologic changes resulting in indefinite survival of LEW to DA rat cardiac allografts after DST alone without the need for antibody. METHODS: One hundred x 10(6) LEW splenocytes (SC) as DST were injected intravenously into DA recipients 7 days before LEW cardiac transplantation. Subsequently, 100 x 10(6) SC harvested from a DA recipient 30, 60, or 100 days after graft acceptance were adoptively transferred into lightly gamma-irradiated (450 rad) na?ve DA recipients 24 hours before a second LEW cardiac allograft. Subsequent graft function was determined. RESULTS: Adoptive transfer of SC from the DST-treated DA rats 30 days after LEW heart transplant acceptance into na?ve gamma-irradiated DA rats failed to transfer tolerance to LEW cardiac allografts. However, SC from DA rats bearing LEW hearts for more than 60 days induced indefinite tolerance to all LEW hearts. This infectious tolerance could be adoptively transferred again to a second DA recipient. CONCLUSIONS: DST-generated regulatory cells can downregulate na?ve lymphocytes to promote allograft acceptance. This tolerance can be expanded and serially transferred to a subsequent na?ve cardiac recipient.     

Mediation of antigen-induced suppression of renal allograft rejection by a CD4 (W3/25+) T cell

The mechanism of induction of specific immunosuppression by preoperative donor-specific blood transfusion in the rodent remains unclear. In a previous study we demonstrated that a single LEW blood transfusion in a DA rat results in the generation of donor-specific suppressor cells in the lymph node and thoracic duct lymph, detectable both in vivo and in vitro 7 days after DST. In contrast, no such activity was found in the splenic compartment. In this study lymphoid cells were harvested from either untreated DA rats or DA rats transfused 7 days previously with LEW blood and purified by rosette depletion using appropriate monoclonal antibodies. The purified lymphocyte subpopulation from transfused rats, of splenic or TDL origin, was adoptively transferred into syngeneic, lightly irradiated (200 rads) DA rats, where the adoptive transfer of 4.25 x 10(7) T (OX12-negative) cells (98.9% pure) or 2.5 x 10(7) CD4 (i.e., W3/25-positive, OX8 and OX12-negative) cells (97.9% pure), both of TDL origin, resulted in the indefinite survival of a LEW kidney (median survival time [MST]greater than 100 days), but not a PVG kidney (MST = 11 days). In contrast lymphocytes of splenic origin, regardless of the phenotype, did not prolong graft survival. Thus we have phenotypically characterized a CD4 (W3/25+) T suppressor cell that is generated after a single DST and is detectable 7 days later in the TDL but not the splenic compartment. Although there was no significant difference in the lymphocyte subset composition of lymphoid organs from transfused and untreated hosts, as determined by flow cytometry, the mean channel fluorescence of the lymphocyte population markers of cells harvested from transfused animals was significantly less than that observed with untreated controls, a finding which remains unexplained.     

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).     

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.     

Differential effects of interleukin 2 receptor-targeted therapy on heart and kidney allografts in rats. Depression of effectiveness of ART-18 monoclonal antibody treatment by uremia

ART-18, a mouse antirat IL-2R mAb inhibits IL-2 binding and IL-2-dependent T cell growth. Although both (LEW x BN)F1 kidney and heart allografts survive ca. 3 weeks in ART-18-treated LEW rats (acute rejection occurs within 10 days, P less than 0.001), the host responses against the two organs vary. In the heart model, the splenic CD4:CD8 ratio as determined by FMF was similar both in untreated and treated animals, but decreased significantly in kidney recipients conditioned with ART-18. In both mAb-modulated animal groups, splenocytes inhibited test MLR and prolonged test cardiac allograft survival in a donor-specific fashion upon adoptive transfer, suggesting that ART-18 mediates "sparing" of Ts. However, both CD4+ and CD8+ cells from kidney-grafted hosts conferred suppression in vivo; only the CD8+ subset was effective in the heart model. Immunohistologically, IL-2R+ cells were absent in the heart grafts of treated hosts; a significant proportion of the kidney cell infiltrate remained IL-2R+ despite continuous mAb administration. Although ART-18 therapy prolonged renal graft survival significantly, function was poor and the rats remained uremic. However, when one of the native kidneys was retained and the rat continued to enjoy normal renal function, IL-2R+ cells were abolished from the graft infiltrate, as shown by FMF and immunohistology. Thus, ART-18 treatment influences host responses differentially against kidney and heart allografts (modulation and depletion of IL-2R+ cells, respectively) despite increasing their survival comparably. The uremic state in the kidney model prevents elimination of infiltrating IL-2R+ mononuclear cells by a mAb directed specifically against them.     

Posttransplant infusion of donor-specific blood induces immunological unresponsiveness in rat hepatic allografts

BACKGROUND: We previously reported that pretransplant donor-specific blood transfusion (DST) induces CD45RC-CD4+ T cells, Th2-like effector cells, and prolongs rat hepatic allograft survival. Our study investigated the effects of posttransplant DST on rat hepatic allograft survival. METHODS: Three days after transplantation, LEW (RT1(1)) recipient rats with ACI (RT1a) livers were injected i.v. with freshly heparinized donor-specific blood. The time kinetics of CD45RC-CD4+ and CD45RC+CD4+ T cell subsets in hepatic infiltrates were examined. RESULTS: Posttransplant DST significantly prolonged rat hepatic allograft survival. Interferon (IFN)-gamma, interleukin (IL)-12, and IL-18 mRNA levels in hepatic allografts of untreated recipients were significantly greater than in recipients treated with posttransplant DST. However, hepatic allografts of recipients treated with posttransplant DST showed significantly higher IL-4, IL-10, and transforming growth factor (TGF)-beta mRNA levels than untreated recipients. The ratio of CD45RC-CD4+ T cells to CD45RC+CD4+ T cells was significantly higher in hepatic allografts treated with posttransplant DST than in untreated animals. Immunostaining with anti-rat dendritic cell (OX-62) monoclonal antibody revealed that OX-62+ cells were distributed to the splenic red pulp of animals treated with posttransplant DST and to the splenic white pulp in untreated animals. Most OX62+ cells isolated from the spleen of recipients treated with posttransplant DST expressed donor RT1Ba class II major histocompatibility complex antigens, suggesting that OX-62+ cells were of donor origin. CONCLUSION: Posttransplant DST was associated with persistent infiltration of CD45RC-CD4+ T cells, Th2-like effector cells, in rat hepatic allografts, causing immunologic unresponsiveness and establishment of microchimerism in the spleen.     

Induction of carbon monoxide in donor animals prior to organ procurement reduces graft immunogenicity and inhibits chronic allograft dysfunction

BACKGROUND: Nonspecific inflammatory damages occurring prior to organ transplantation reduce long-term graft survival. Here, we tested the beneficial effects of carbon monoxide (CO) induction by methylene chloride (MC). METHODS: Fischer-344 (F-344 Rat) or Dark Agouti (DA Rat) donor animals were either treated with MC four hours prior to organ removal or remained untreated. Kidneys were transplanted into Lewis (LEW) recipients. The low responder strain combination (F-344-->LEW) was studied for long-term graft changes. Dendritic cells (DCs) migration and early changes were followed in additional groups of a high responding donor/recipient strain combination (DA-->LEW). Native kidneys of uninephrectomized, age-matched normal animals served as controls. RESULTS: Following MC application COHb peaked within two hours in donor animals. Renal function and morphology improved significantly in renal allografts of CO induced donor animals and were comparable to native controls long-term (24 wks). Early after transplantation (24 hr) donor-derived DCs, CD4+ T-cells and alloreactive T-cells were significantly reduced following the engraftment of organs from treated donors. In addition, a trend towards a Th1/Th2 shift and a significant intragraft reduction of CD3 mRNA expression was observed. CONCLUSION: Donor treatment for the induction of CO reduced graft immunogenicity and inhibited chronic allograft nephropathy.     

Adoptive transfer of primed CD4+ T-lymphocytes induces pattern of chronic allograft nephropathy in a nude rat model

BACKGROUND: Chronic allograft nephropathy (CAN) remains the most common cause of late graft loss in renal transplantation. Presensitized patients have a specifically increased risk to lose their graft. To analyze the immunological factors involved, a new experimental rat model was created with nude athymic LEW.RNU rats as recipients of F344 renal allografts. METHODS: Adoptive transfer of CD4+ T-lymphocytes (2x, 3.5x, or 5 x 10(7) cells) primed against donor skin grafts was performed one week after transplantation. The animals were monitored for renal function, graft infiltrating cells, and the development of donor specific alloantibodies for 20 weeks or until graft loss. RESULTS: Survival of the animals was dose dependent; rats suffered from renal failure with severe albuminuria and developed various lesions typical for CAN including interstitial fibrosis and tubular atrophy. The cell infiltrate in the graft increased with the amount of CD4+ T-cells transferred and consists predominantly of CD4+ T-cells and macrophages/monocytes. More than half of the grafts showed histological signs of glomerulopathy consistent with CAN. 9/12 rats with CAN had antibodies against the donor major histocompatibility complex (MHC)-I and in all rats donor specific anti-glomerular basement membrane (GBM) antibodies were detected. CONCLUSION: Adoptive transfer of primed CD4+ T-cells results in a severe infiltrate of CD4+ cells in the graft and production of anti-MHC and GBM antibodies in this nude rat model. Histological changes are consistent with CAN with frequent glomerular changes. In conclusion, the induction of donor specific alloantibodies by primed CD4+ T-lymphocytes may play an important role in the pathogenesis of CAN.     

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.     

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.     

Rat xenograft survival in mice treated with donor-specific transfusion and anti-CD154 antibody is enhanced by elimination of host CD4+ cells

BACKGROUND: Treatment with a donor-specific transfusion (DST) and a brief course of anti-mouse CD154 (anti-CD40-ligand) monoclonal antibody (mAb) prolongs the survival of both allografts and rat xenografts in mice. The mechanism by which allograft survival is prolonged is incompletely understood, but depends in part on the presence of CD4+ cells and the deletion of alloreactive CD8+ T cells. Less is known about the mechanism by which this protocol prolongs xenograft survival. METHODS: We measured rat islet and skin xenograft survival in euthymic and thymectomized mice treated with combinations of DST, anti-CD154 mAb, anti-CD4 mAb, and anti-CD8 mAb. Recipients included C57BL/6, C57BL/6-scid, C57BL/6-CD4null, and C57BL/6-CD8null mice. RESULTS: Pretreatment with a depleting anti-CD4 mAb markedly prolonged the survival of both skin and islet xenografts in mice given DST plus anti-CD154 mAb. Comparable prolongation of xenograft survival was obtained in C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb. In contrast, anti-CD8 mAb did not prolong the survival of either islet or skin xenografts in mice treated with DST and anti-CD154 mAb. Thymectomy did not influence xenograft survival in any treatment group. Adoptive transfer of splenocytes from C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb and bearing long-term skin xenografts revealed the presence of residual xenoreactive cells. CONCLUSIONS: These data suggest that treatment with DST and anti-CD154 mAb induces a state of "functional" transplantation tolerance. They also support the hypothesis that both the induction and maintenance of graft survival based on this protocol depend on different cellular mechanisms in allogeneic and xenogeneic model systems.     

Evidence that therapeutic strategies targeted at CD4+ cells modulate accelerated rejection of cardiac allografts in sensitized rats by different mechanisms.

(LEW x BN)F1 cardiac allografts are rejected within 36 hr in LEW rats presensitized with BN skin grafts 7 days earlier (acute rejection occurs within 8 days). We have previously described the effects of individual CD4 (BWH-4), CD25 (IL-2R, ART-18) mAbs, and CsA therapeutic regimens upon cardiac allograft survival in sensitized hosts. The present studies were designed to probe an adjunctive use of ART-18 or CsA upon BWH-4-mediated suppression of accelerated graft injury. Sequential therapy with BWH-4 and ART-18 in the sensitization phase (days -7 to -1) and effector phase (from day 0, the day of cardiac transplant), respectively, prolonged graft survival additively to c. 22 days. Treatment with BWH-4 markedly diminished host humoral response against ART-18 preparation. BWH-4 given in concert with subtherapeutic dose of CsA produced graft survival comparable to that induced by mAb alone (c. 13 days) with concomitant decreased host anti-BWH-4 response. None of the combined regimens affected the frequency of circulating CD4+ cells, as compared with that exerted by BWH-4 monotherapy. Thus this study defines principles and some mechanistic aspects of optimal immunosuppressive strategies potentiating the effects of CD4-targeted therapy.     

The mechanism of synergistic interaction between anti-interleukin 2 receptor monoclonal antibody and cyclosporine therapy in rat recipients of organ allografts

Untreated anephric LEW rats die ca. 9 days following transplantation of LBNF1 kidney allografts. Although treatment with ART-18, a mouse antirat IL-2R mAb (300 micrograms/kg/day x 10 days), prolonged graft survival to ca. 3 weeks, the severely impaired renal function was comparable to untreated controls (creatinine levels 3-5 mg/dl). In contrast, simultaneous infusion of ART-18 and a very low dose of CsA (0.75 mg/kg x 10 days), marginally effective on its own, resulted in survival of greater than 45 days; the grafts exhibited relatively good function comparable to that in rats treated with full-dose (15 mg/kg/day) CsA. This beneficial biological effect did not depend upon elevated CsA trough levels in animals conditioned with both modalities. The CD4:CD8 ratio at the graft site was lowest (0.3-0.4) in recipients treated with ART-18 + CsA. Synergy between the two agents has been demonstrated by adoptive transfer studies in which nonspecific suppression has been conferred selectively by cells infiltrating kidney grafts in rats given ART-18 and CsA in concert but not separately (LBNF1 and WF test cardiac allograft survival ca. 12 days). In contrast, suppression in the recipient spleens was donor-specific; both CD4 and CD8 cells prolonged test graft survival. Immunohistological evaluation of renal allografts revealed that therapy with ART-18 or low-dose CsA alone failed to deplete IL-2R+ cells and prevent production of IL-2, IFN-g, and TNF. In contrast, the frequency of infiltrating IL-2R+ cells and elaboration of endogenous cytokines in non-uremic hosts receiving combination therapy was greatly depressed, stressing again synergistic interaction between ART-18 and CsA. Additionally, markedly reduced class II antigen induction, XL-fibrin deposition, and glomerulitis may also contribute to prolonged survival and satisfactory function of kidney allografts in this animal group.     

输注供者CD8+CD28-调节性T淋巴细胞抑制大鼠肝移植急性排斥反应的作用

目的 评价具有免疫抑制作用的CD8+CD28-调节性T淋巴细胞(Treg)体内输注在抑制大鼠肝移植急性排斥反应中的作用.方法 建立近交系大鼠肝移植自发耐受及急性排斥反应模型.从肝移植自发耐受模型受者脾脏中分离CD8+CD28-Treg,于急性排斥反应模型建立前1 d输注给受者,比较不同的输注组间受者的存活时间和移植肝病理学表现.结果 来自自发耐受模型(LEW大鼠为供者,DA大鼠为受者)的CD8+CD28-Treg输注可以延长急性排斥反应模型(LEW大鼠为供者,BN大鼠为受者)受者的存活时间,由(14.0±2.2)d延长至(24.0±3.0)d(P＜0.01),移植肝病理学显示排斥反应程度减轻.结论 大鼠肝移植自发耐受模型受者体内诱导的CD8+CD28-Treg具有抑制急性排斥反应的作用,该免疫抑制作用具有抗原特异性.     

Effect of blocking the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in a rat small intestinal transplantation model

The effect of blocking the expression of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in a graft by an antibody, and immunohistochemical changes in the graft were monitored, using a rat small intestinal transplantation model. Dark Agouti (DA) rat small intestines were heterotopically transplanted into Lewis (LEW) rats. The graft was treated with or without an anti-MAdCAM-1 antibody, F(ab')(2), during the operation. The survival of the grafts and histological changes, such as lymphocyte infiltration and destruction of the intestinal architecture in epithelium villus thickness, villus height and submucosal thickness of the graft, were examined. The expression of MAdCAM-1 and beta 7 integrin in the graft was also checked by immunostaining. Furthermore, graft infiltrating lymphocytes, in mesenteric lymph nodes (MLN) and Peyer's patches (PP) were measured by FACS analysis. Survival was prolonged in the DA graft with anti-MAdCAM-1 F(ab')(2) treatment; DA to LEW: 7.0+/-3.3, DA to LEW with the antibody: 24.6+/-8.4 days (p<0.05). Histological findings and scoring of the grafts were consistent with this conclusion. Moreover, MAdCAM-1 expression itself was suppressed in grafts of the antibody-treated group. While a FACS analysis showed no difference in the % of CD4+ T cells and CD8+ T cells in the PP of the graft, CD4+ T cells in the MLN of the antibody-treated graft were significantly low. A strategy directed at blocking the adhesion molecule, MAdCAM-1, in the small intestinal grafts could be useful in the prevention of acute rejection.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.     

An increase in the survival of murine H-2-mismatched cultured fetal pancreas allografts using depleting or nondepleting anti-CD4 monoclonal antibodies, and a further increase with the addition of cyclosporine

Depletion of CD4+ T lymphocytes with monoclonal antibodies (mAbs) has been shown to prolong allograft survival in mice. In this study, two rat anti-CD4 mAbs, H129.19 and GK1.5, were administered either alone or in combination with cyclosporine (CsA) to recipients of MHC-mismatched (H-2k to H-2d) cultured fetal pancreas allografts to determine their effect on graft survival. When compared with control mice, splenic CD4+ cells of GK1.5-treated mice were depleted by greater than 95%, but in H129.19-treated mice no depletion of CD4+ cells occurred. Instead, rat Ig was present on the surface of CD4+ cells in H129.19-treated mice. Anti-CD4 therapy with either H129.19 or GK1.5 prolonged fetal pancreas allograft survival to a similar extent, but did not lead to indefinite survival. Blockade of the CD4 antigen by the mAb H129.19 was as effective as the depletion of CD4+ cells by GK1.5 in prolonging allograft survival. Rejection of grafts by day 28 posttransplantation occurred in the absence of CD4+ cells, as determined by both flow cytometric examination of spleen cells and immunoperoxidase staining of the graft site. CsA alone did not prolong graft survival, but its addition to either H129.19 or GK1.5 mAb treatment significantly increased the survival rate of grafts at 28 days compared with mAb treatment alone. These results suggest that CD4+ cell depletion is not essential for effective anti-CD4 mAb therapy--and, further, that CsA may have a direct inhibitory effect on CD8+ cells during allograft rejection.