Marked inhibition of transplant vascular sclerosis by in vivo-mobilized donor dendritic cells and anti-CD154 mAb

BACKGROUND: Combination of donor dendritic cells (DC) and anti-CD40 Ligand (L) (CD154) monoclonal antibody (mAb) markedly prolongs heart or skin allograft survival, but the influence of this strategy in models of chronic rejection is unknown. Our aim was to ascertain the influence of in vivo-mobilized immature donor DC plus anti-CD40L mAb on vascular sclerosis in functional murine aortic allografts. METHODS: C3H He/J (C3H;H2k) mice received 2 x 106 freshly isolated, immunobead-purified (>90%) fms-like tyrosine kinase 3 ligand-mobilized C57BL/10 (B10;H2b) CD11c+ DC intravenously (IV), together with 500 microg of anti-CD40L mAb (MR1) intraperitoneally (IP) on days -7, 0, 4, and 10. Controls received either no donor cells, no mAb, or were untreated. B10 aortic grafts were transplanted in the abdominal aorta on day 0. At day 30, antidonor T-cell proliferative and cytotoxic responses and both complement fixing and immunoglobulin (Ig)G alloantibody levels were determined. Grafts were harvested on days 30 and 60 and examined by histology and immunohistochemistry. RESULTS: DC infusion alone enhanced ex vivo antidonor proliferative and cytotoxic T-cell activity. By contrast, complement-fixing alloantibody levels were reduced. Anti-CD40L mAb alone strongly suppressed each of these responses. Graft inflammatory cell infiltration, intimal smooth muscle cell proliferation, fibrosis, and elastic lamina disruption observed in untreated animals were reduced in response to anti-CD40L mAb or donor DC alone. Antidonor immune reactivity, including IgG levels, and intimal proliferation were all markedly suppressed to an overall greater extent in mice given both treatments. CONCLUSION: Whereas blockade of the CD40-CD40L pathway ameliorated transplant vasculopathy, preservation of near-normal vessel architecture was achieved by simultaneous administration of donor DC. This strategy represents a novel application of DC for suppression of chronic rejection.     

Operational tolerance induced by pretreatment with donor dendritic cells under blockade of CD40 pathway.

BACKGROUND: Dendritic cells can mount immune response as competent antigen presenting cells. Recently, it has been reported that immature dendritic cells induce prolongation of allograft survival. However, the ability of mature dendritic cells to induce operational tolerance is unclear. Therefore, in this study, we examined the ability of splenic mature dendritic cells to induce operational tolerance to fully allogeneic antigens using mouse heterotopic heart transplantation model. METHODS: CBA (H2k) mice received i.v. injections with donor splenic dendritic cells or B cells in the absence or presence of monoclonal antibody (mAb) specific for CD40 ligand or CD80/CD86 2 weeks before transplantation of a C57BL/10 (H2b) heart. RESULTS: When donor dendritic cells were injected i.v. 2 weeks before transplantation, rejection response was accelerated compared with that of naive mice [median survival time (MST) = 7 and 8 days, respectively]. However, when CD40 pathway was blocked by anti-CD40 ligand mAb, i.v. injection of donor dendritic cells but not B cells induced indefinite graft survival (MST >100 and 20 days, respectively). Mice treated with anti-CD40 ligand mAb alone rejected their grafts with a MST of 18 days. Intravenous injection of donor dendritic cells and B cells in combination with anti-CD80/CD86 mAbs was less effective to induce graft prolongation (MST = 9.5 and 13 days, respectively). CONCLUSIONS: Therefore, under blockade of CD40 pathway, mature dendritic cells were tolerogens in vivo independent of CD80/86 pathways.     

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.     

霉酚酸酯预处理供者未成熟树突状细胞回输受者延长移植物的存活

目的观察霉酚酸酯(MMF)处理的供者来源的树突状细胞(DC)回输受者延长移植物存活的作用。方法在DC前体的体外培养过程中加入MMF处理,利用同种小鼠异位心脏移植模型,设单纯移植组、供者未成熟DC回输受者组,以及MMF处理的供者未成熟DC回输受者组,观察MMF处理后DC的抗原提呈能力的变化、移植心脏存活时间并做微嵌合和病理学分析。结果MMF处理后DC的抗原提呈能力明显下降,单纯移植组移植心脏的存活期仅为8d,未成熟DC回输受者组心脏存活期为21d,而MMF处理的未成熟DC组移植物存活时间延长为30d,差异有统计学意义(P<0.01);MMF处理的供者未成熟DC在受者体内的嵌合期可达28d以上,且病理分析显示可以明显抑制炎症的产生。结论MMF处理的DC回输受者能够诱导针对移植供者的特异性免疫耐受,进而延长移植物的存活。     

Graft hyporeactivity induced by immature donor-derived dendritic cells

Immature dendritic cells (DCs) are deficient in surface co-stimulatory molecules and have been shown to exhibit a 'tolerogenic' potential. We investigated the allostimulatory activity of immature DCs in one-way mixed leukocyte reactions and their capacity to inhibit anti-donor cytolytic activity in the sponge matrix allograft model. Immature DCs (CD80 and CD86 deficient) were derived from bone marrow cells propagated in GM-CSF and TGF-beta1. Mature DCs (CD80+ and CD86+) were derived from bone marrow cells propagated in GM-CSF and IL-4. Either 2 x 10(6) DBA/2J (DBA, H-2d) immature DCs or 2 x 10(6) mature DCs were injected intravenously into C57BL/6J (B6, H-2b) mice 7 days prior to sponge matrix allograft implantation. On day 12, the sponge was harvested and the graft-infiltrating cells were tested in vitro for cytotoxic T lymphocyte (CTL) activity. Immature dendritic cell (DC) infused significantly and markedly inhibited intra-graft CTL activity compared to mature DCs and syngeneic bone marrow control cells. The administration of immature DCs directly into the sponge allograft failed to induce hyporeactivity. Thus, the only systemic infusion of immature donor DCs was able to recapitulate the donor-specific transfusion effect, and the capacity of donor bone marrow cells to induce donor-specific hyporeactivity in the sponge allograft model.     

Anti-CD4 induced rat heart tolerance: no presence of primed T cells and regulatory mechanisms for cytotoxic T cells

Treatment with anti-CD4 monoclonal antibody (mAb) (OX38) induces heart, but not skin graft tolerance in WF (RT1u) to Lewis (RT1l) rat strain combinations. We examined differences in cellular responses between heart-bearing and skin-rejected hosts that were both treated with anti-CD4 mAb. In the tolerant LEW rats bearing WF heart transplants, the secondary WF heart but not skin grafts were accepted. On the other hand, in anti-CD4 treated WF skin-rejected hosts, both secondary WF heart and skin grafts were rapidly rejected. Spleen cells from anti-CD4 treated WF skin-rejected LEW rats but not from WF heart-bearing LEW rats received the same treatment generated CTL after in vitro stimulation with paraformaldehyde (PFA) treated donor WF stimulator spleen cells. Adoptive transfer of spleen cells from WF skin-rejected LEW rats with or without anti-CD4 therapy into the tolerant LEW rats at the secondary WF heart transplantation blocked the secondary heart graft acceptance. However, transfer of spleen cells from WF heart-rejected rats without immunosuppression failed to block acceptance of the secondary heart graft. Our results indicated the lack of primed T cells and presence of regulatory mechanisms for tissue specific T cells in anti-CD4 treated heart bearing hosts.     

Promotion of long-term heart allograft survival by combination of mobilized donor plasmacytoid dendritic cells and anti-CD154 monoclonal antibody.

Infusion of donor immature dendritic cells (DC) can significantly prolong survival of organ allografts, and this is believed to be due to antigen recognition by T cells in the absence of co-stimulation. In this study we report that a single pre-operative infusion of donor-mobilized immature plasmacytoid dendritic cells (pDCs) is superior to that of other DC sub-sets in suppressing allograft rejection. The combination of pDC infusion with injection of anti-CD154 monoclonal antibody further inhibited graft rejection and, in 50% of the mice, led to indefinite graft survival. This finding suggests a role for the plasmacytoid DC sub-set in facilitating organ transplant survival and also in the treatment of autoimmune disorders.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

Mobilization of T lymphocytes following cardiac transplantation. Evidence that CD4-positive cells are required for cytotoxic T lymphocyte activation, inflammatory endothelial development, graft infiltration, and acute allograft rejection.

Modified limiting dilution analysis (LDA) techniques were used to evaluate the mobilization of antigen-stimulated helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) following allogeneic heterotopic cardiac transplantation. These modified LDA techniques allow a quantitative comparison of T cells that have been stimulated by antigen in vivo versus unstimulated precursor T cells of the same antigen specificity. Endothelial changes associated with mononuclear cell infiltration of the transplant were studied using endothelia-specific monoclonal antibodies and immunohistochemistry. Early (day 3) infiltration of cardiac allografts was characterized by a prevalence of donor alloantigen-specific HTL over CTL. Immunohistology revealed that the day-3 infiltrate was associated with areas of differentiated vascular endothelium, located primarily in the subepicardial region. Though donor-specific precursor HTL and CTL were present in the peripheral lymphoid tissues and blood, very few of them had been stimulated at this early time. During the latter phases of the response (days 6-9), antigen-stimulated HTL and CTL were present in the rejecting heart with CTL dominating the response. Accumulation of large numbers of donor-specific CTL in the allograft correlated with extensive inflammatory endothelial development, myocyte destruction, and loss of graft function by day 9. Stimulated HTL and CTL were detectable in peripheral lymphoid tissues at days 6 and 9. In addition, a marked increase in the number of donor-specific precursor CTL, but not precursor HTL, was observed in the lymphoid tissues at the peak of the response. Depletion of class II MHC-restricted T cells by in vivo treatment with anti-CD4 mAb eliminated HTL activity in all lymphoid compartments assessed and markedly reduced the number of CTL infiltrating the allograft. In addition, no stimulated CTL were detectable in lymphoid tissues, and the number of precursor CTL was not increased. In anti-CD4-treated recipients, cardiac allografts remained functional with minimal histological evidence of rejection for at least 21 days. Though graft-associated inflammatory endothelia were absent in anti-CD4-treated recipients at day 6, endothelial differentiation was observed in day 21 allografts in anti-CD4-treated recipients. These observations indicate that inflammatory endothelial development may precede T cell infiltration and subsequent loss of the cardiac allograft function. Thus, CD4-positive HTL are required for (1) graft-associated inflammatory endothelial development; (2) CTL activation in peripheral lymphoid tissues; (3) CTL accumulation in allografted tissues; and (4) acute cardiac allograft rejection.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Targeting acute allograft rejection by immunotherapy with ex vivo-expanded natural CD4+ CD25+ regulatory T cells

BACKGROUND: Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice. METHODS: Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry. RESULTS: By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection. CONCLUSIONS: Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.     

Graft-versus-leukemia effect in minor antigen mismatched chimeras given delayed donor leucocyte infusion: immunoregulatory aspects and role of donor T and ASGM1-positive cells

BACKGROUND: Previous studies have demonstrated that delayed donor leukocyte infusion (DLI) can increase graft-versus-leukemia (GVL) without increasing graft-versus-host-disease (GVHD) in MHC mismatched bone marrow (BM) chimeras. In our report, the immune status of minor antigen mismatched BM chimeras given DLI was studied. Particularly the role of donor ASGM1 positive or T cells in the graft-versus-leukemia effect (GVL) was investigated. METHODS: AKR mice (H2k, Mls1a, Thy1.1) received TBI (9,5 Gy) and T cell-depleted (TCD) C3H (H2k, Mls2a, Thy1.2) BM alone (BM chimeras), or TCD BM together with immunocompetent C3H spleen cells at the time of BM transplantation (BM+SP chimeras), or TCD BM and 3 weeks later C3H spleen cells (DLI chimeras). Chimerism and T lymphocyte subsets were scored using FACS and anti-Thy, anti-Vbeta6, anti-IL2-beta receptor, anti-CD4, anti-CD3, and anti-CD8 mAbs. Leukemia challenge consisted of 5 x 10(6) AKR T cell lymphoma (BW4157) cells injected i.v. ASGM1 positive (ASGM1+) cells and T cells were depleted using anti-ASGM1 or anti-Thy1.2 antibodies, respectively. Immune tolerance was studied using MLR and CML tests. RESULTS: BM + SP chimeras developed acute and lethal GVHD, whereas DLI chimeras were totally free from GVHD. In DLI chimeras, host-reactive cytotoxic T cells (CTL) could not be induced and host-reactive CD8Vbeta6 cells were deleted whereas CD4Vbeta6 cells and MLR reactivity persisted temporarily. In contrast, in BM+SP chimeras, anti-host CTL were easily generated and an expansion of both host-reactive CD8Vbeta6 and CD4Vbeta6 T cells was found as well as high anti-host MLR reactivity. Depletion of either ASGM1 + cells or T cells from the DLI inoculum resulted in an abrogation of GVL reactivity, suggesting that both cell populations were involved in the protection against BW4157 leukemia. Three weeks after DLI, the GVL effect waned which correlated with the disappearance of host-reactive CD4 cells and MLR reactivity. CONCLUSION: In minor antigen mismatched BM chimeras, anti-host reactivity after DLI is characterized by (1) an absence of clinical GVHD, (2) clonal deletion of host-reactive CD8 cells, (3) an absence of anti-host CTL induction, and ( 4) a temporary persistence of host-reactive CD4 T cells and of MLR reactivity. In addition, either donor ASGM1+ cells or an interaction between these cells and T cells contribute to the GVL effect.     

Costimulatory blockade induces hyporesponsiveness in T cells that recognize alloantigen via indirect antigen presentation

BACKGROUND: Blockade of T cell costimulation by treatment with donor-specific transfusion (DST) and anti-CD154 monoclonal antibody (mAb) induces prolonged allograft survival in mice. This effect is due in part to deletion of host CD8 and CD4 T cells that recognize alloantigen by direct presentation. The fate of host CD4 T cells that recognize alloantigen by indirect presentation, however, is unclear. METHODS: We studied Tg361 TCR transgenic CD4 T cells that recognize alloantigen by indirect presentation. Carboxyfluorescein diacetate, succinimidyl ester-labeled Tg361 cells were adoptively transferred into syngeneic nontransgenic recipients and their fate in the peripheral blood, spleen, and lymph nodes following treatment with DST and anti-CD154 was analyzed. RESULTS: Treatment of mice with DST plus anti-CD154 mAb does not delete Tg361 CD4 T cells, but instead renders them hyporesponsive to rechallenge with alloantigen. Mice circulating hyporesponsive CD4 T cells also fail to reject skin allografts. The hyporesponsive state of the T cells is not reversed by the addition of interleukin-2, anti-CD28 mAb, or an agonistic anti-CD134 mAb in the presence of antigen. These T cells are capable of activation, however, as evidenced by in vitro proliferation in response to anti-CD3 mAb. CONCLUSIONS: These results demonstrate that costimulation blockade can induce hyporesponsiveness of host CD4 T cells recognizing alloantigens by indirect presentation, thus prolonging graft survival by a mechanism that does not involve deletion of alloreactive T cells.     

Role of T and dendritic cells in mouse islet allografts treated with anti-CD45RB monoclonal antibodies

Currently lifelong immunosuppression is required for organ transplant recipients. Anti-CD45RB monoclonal antibody (mAb) prolongs graft survival by mechanisms that are not yet clear. Therefore, we investigated the role of T and dendritic cells (DC) in islet allografts treated with anti-CD45RB mAb after transplantation of 200 allogeneic islets (BALB/c mouse) under the kidney capsules of diabetic C57BL/6 mice treated with intraperitoneal injections of 100 μg of anti-CD45RB mAb on days 0, 1, 3, 5, and 7. We observed a tilt of the ratios of Th1/Th2 and Tc1/Tc2 to Th2 and Tc2. The numbers of naïve and memory T cells were down-regulated in peripheral blood after transplantation. In addition, the maturation, endocytosis, and interleukin-12 secreted by DC derived from bone marrow cells was suppressed in recipient mice. Therefore, anti-CD45RB mAb alleviated, rejection by suppressive effects on T-lymphocyte subsets and DC.     

Decreased alloreactivity using donor cells from mice expressing a CD200 transgene under control of a tetracycline-inducible promoter

BACKGROUND: CD200 delivers an immunsuppressive signal that augments allograft survival following interaction with its receptor, CD200R1. We hypothesized that mice overexpressing CD200 as a trangene would also show a diminished alloresponsiveness and decreased allograft rejection. METHODS: A transgenic mouse on a C57BL/6 background, expressing a murine CD200 cDNA genetically linked to a green fluorescent protein tag (GFP) under control of a tetracycline response element (TRE), was mated with a commercial transgenic mouse carrying the reverse tetracycline regulated transactivator gene under control of a human CMV promoter. F1 mice were examined for induction of alloimmunity in vivo/in vitro, and for their ability to reject skin allografts in vivo. RESULTS: The F1 hybrid expressed CD200 after exposure to doxycyline (DOX), as assessed both by enhanced GFP expression in multiple organs and CD200-GFP expression. Splenocytes from F1 mice stimulated with LPS or allogeneic cells in vitro in the presence/absence of DOX showed reduced production of TNFalpha, and of allospecific CTL. Splenocytes from F1 mice used as stimulator cells in allogeneic MLCs in the presence of DOX were inefficient at induction of cytokines or CTL in vitro from normal allogeneic responder cells. Skin grafts from transgenic mice were inefficient at induction of CTL in vivo. Transgenic mice receiving DOX showed prolonged acceptance of skin allografts, which was abolished by infusion of anti-CD200 mAb. CONCLUSIONS: Our data confirmed that overexpression of CD200 in transgenic mice, or in skin grafts from these mice, decreases alloimmunity. This has potential clinical utility in transplantation and other diseases.     

Prolonged allograft survival in anti-CD4 antibody transgenic mice: lack of residual helper T cells compared with other CD4-deficient mice

BACKGROUND: Investigations of the role of CD4 T lymphocytes in allograft rejection and tolerance have relied on the use of mouse models with a deficiency in CD4 cells. However, in mice treated with depleting monoclonal antibody (mAb) and in MHC class II knockout (KO) mice, there are residual populations of CD4 cells. CD4 KO mice had increased CD4- CD8-TCRalphabeta+ helper T cells, and both strains of KO mice could reject skin allografts at the normal rate. In this study, transgenic mice with no peripheral CD4 cells were the recipients of skin and heart allografts. Results were compared with allograft survival in CD4 and MHC class II KO mice. METHODS: GK5 (C57BL/6 bml mice transgenic for a chimeric anti-CD4 antibody) had no peripheral CD4 cells. These mice, and CD4 and class II KO mice, received BALB/c or CBA skin or cardiac allografts. Some GK5 mice were treated with anti-CD8 mAb to investigate the role of CD8 cells in rejection. CD4 and CD8 cells were assessed by FACS and immunohistochemistry. RESULTS: BALB/c skin on GK5 mice had a mean survival time +/- SD of 24+/-6 days, compared with 9+/-2 days in wild-type mice. Anti-CD8 mAb prolonged this to 66+/-7 days. BALB/c skin survived 10+/-2 days on class II KO and 14+/-2 days on CD4 KO, both significantly less than the survival seen on GK5 recipients (P<0.001). BALB/c hearts survived >100 days in GK5 recipients and in wild-type recipients treated with anti-CD4 mAb at the time of grafting, in contrast to a mean survival time of 10+/-2 days in untreated wild-type mice. Immunohistochemistry revealed that long-term surviving heart allografts from the GK5 recipients had CD8 but no CD4 cellular infiltrate. These hearts showed evidence of transplant vasculopathy. CONCLUSIONS: The GK5 mice, with a complete absence of peripheral CD4 cells, provide the cleanest available model for investigating the role of CD4 lymphocytes in allograft rejection. Prolonged skin allograft survival in these mice compared with CD4 and MHC class II KO recipients was clearly the result of improved CD4 depletion. Nevertheless, skin allograft rejection, heart allograft infiltration, and vascular disease, mediated by CD8 cells, developed in the absence of peripheral CD4 T cells.