Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.     

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.     

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.     

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.     

Critical Role for IL-6 in Hypertrophy and Fibrosis in Chronic Cardiac Allograft Rejection

Chronic cardiac allograft rejection is the major barrier to long term graft survival. There is currently no effective treatment for chronic rejection except re-transplantation. Though neointimal development, fibrosis, and progressive deterioration of graft function are hallmarks of chronic rejection, the immunologic mechanisms driving this process are poorly understood. These experiments tested a functional role for IL-6 in chronic rejection by utilizing serial echocardiography to assess the progression of chronic rejection in vascularized mouse cardiac allografts. Cardiac allografts in mice transiently depleted of CD4+ cells that develop chronic rejection were compared with those receiving anti-CD40L therapy that do not develop chronic rejection. Echocardiography revealed the development of hypertrophy in grafts undergoing chronic rejection. Histologic analysis confirmed hypertrophy that coincided with graft fibrosis and elevated intragraft expression of IL-6. To elucidate the role of IL-6 in chronic rejection, cardiac allograft recipients depleted of CD4+ cells were treated with neutralizing anti-IL-6 mAb. IL-6 neutralization ameliorated cardiomyocyte hypertrophy, graft fibrosis, and prevented deterioration of graft contractility associated with chronic rejection. These observations reveal a new paradigm in which IL-6 drives development of pathologic hypertrophy and fibrosis in chronic cardiac allograft rejection and suggest that IL-6 could be a therapeutic target to prevent this disease.     

Posttransplant interleukin-4 treatment converts rat liver allograft tolerance to rejection

BACKGROUND: Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection. METHODS: DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition. RESULTS: IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition. CONCLUSIONS: IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.     

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.     

Effect of FTY720 in rat small bowel transplantation: apoptosis of crypt cells and lymphocytes in gut-associated lymphoid tissues

AIM: We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 during rejection. METHODS: Orthotopic small bowel transplantations (SBTs) were performed from BN to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, and 7 after operation: isograft, untreated allograft, allograft with FTY720. FTY720 was orally administered by gavage (1 mg/kg/d) to allograft recipients on 7 consecutive days. Cryostat sections were prepared from grafts, including PPs. An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas/Fas-L. RESULTS: Graft survival was prolonged in the FTY720-treated group. The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7 compared with the isograft group. In the FTY720-treated group, the number of ISEL-positive enterocytes in the allografts was down-regulated significantly on days 3, 5, and 7 compared with untreated allograft group. In the PPs, the number of ISEL-positive mononuclear cells increased significantly in the allografts compared with the isograft group. In the FTY720-treated groups, the number of ISEL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. The number of Fas/FasL-positive enterocytes were increased significantly in allografts compared with isograft group. In FTY720-treated groups, the number of Fas/FasL-positive enterocytes were down-regulated significantly on day 7 compared with the untreated allograft group. In the PPs, Fas/FasL-positive mononuclear cells also increased significantly on day 7 in the allografts compared with isografts. In the FTY720-treated groups, Fas/FasL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. CONCLUSIONS: The number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes increased during small bowel graft rejection. FTY720 prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes while also prolonging small bowel allograft survival.     

Differential effects of transferrin receptor blockade on the cellular mechanisms involved in graft rejection

Since transferrin receptor (TfR) appears on activated T cells following the interaction of the antigen-major histocompatibility complex (MHC) with the T cell receptor (TCR) and the appearance of interleukin (IL)-2R, we therefore hypothesize that in vivo blockade of TfR prolongs allograft survival by altering the cellular mechanisms involved in graft rejection. Previous results in our laboratory have demonstrated that anti-TfR monoclonal antibody (mAb) at 100 microg on days 0 and 1 of transplantation significantly prolonged allograft survival to 25.7 +/- 0.9 days in a murine heterotopic, nonvascularized cardiac allograft model. In the current studies, administration of anti-TfR mAb at the time of maximal TfR expression, on days 2 and 3 post-transplantation, failed to prolong allograft survival (13.0 +/- 0.0 days) compared to the isotype controls (10.5 +/- 0.5 and 10.7 +/- 0.4 days) (p < 0.01, Wilcoxon rank sum). A 4-day course of anti-TfR mAb significantly prolonged allograft survival compared to the isotype controls, but was no more effective than a 2-day course of the mAb. Anti-TfR mAb suppressed the mixed lymphocyte response to donor-specific and third-party alloantigen by 78.7% (p < 0.05) and 80.8% (p < 0.05), respectively, while stimulating the CTL response to donor-specific (16.3%, p < 0.05) and third party (49.3%, p < 0.01) alloantigen. Anti-TfR mAb suppressed IL-15 and increased IL-4 intragraft mRNA expression when compared to the isotype controls. Examination of cell surface receptors important during T cell activation revealed alterations in expression following anti-TfR mAb treatment. Anti-TfR mAb is an effective immunosuppressant prolonging allograft survival by altering cell-mediated immune responses and the intragraft cytokine micro-environment.     

Effect of FTY720 and Ex Vivo Graft Irradiation in Rat Small Bowel Transplantation: Expression of Mucosal Addressin Cell Adhesion Molecule-1

Purpose We performed a semiquantitative analysis of the expression of Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and gut-associated tissues (GALT) during small bowel graft rejection in the rat to confirm the effect of FTY720 and ex vivo graft irradiation. Methods Small bowel transplantations (SBT) were performed from BN rats to LEW rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations (untreated, FTY720, ex vivo graft irradiation, FTY720+ex vivo graft irradiation). Indirect immunoperoxidase staining was performed against CD4 and MAdCAM-1. The number of CD4-positive cells in the allografts was also analyzed by flow cytometry. Results The graft survival was prolonged only in the FTY720-treated groups. The SBT allografts treated by FTY720 demonstrated less infiltration, but the ex vivo graft irradiation group did not show any effect on its expression. In the FTY720-treated groups, MAdCAM-1 expression on high endothelial venules (HEVs) in Peyer's patches (PPs) was upregulated and its expression on endothelial cells of vessels in the lamina propria was downregulated in comparison with the allograft group without FTY720. Conclusions It is important to prevent the infiltration of CD4-positive cells, the downregulation of MAdCAM-1 expression on HEVs in PPs and the upregulation of MAdCAM-1 expression on endothelial cells of vessels in the lamina propria for the prolongation of graft survival.     

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND: Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS: Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS: Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION: Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.     

LFA‐1 Antagonism Inhibits Early Infiltration of Endogenous Memory CD8 T Cells into Cardiac Allografts and Donor‐Reactive T Cell Priming

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts and produce IFN‐γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti‐LFA‐1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti‐LFA‐1 mAb given to C57BL/6 6 (H‐2^b) recipients of A/J (H‐2^a) heart grafts on days –1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN‐γ‐induced genes. Donor‐specific T cells producing IFN‐γ were at low/undetectable numbers in spleens of anti‐LFA‐1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti‐LFA‐1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti‐LFA‐1 mAb inhibits early donor‐reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.     

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.     

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.     

Transient Lymphopenia Breaks Costimulatory Blockade‐Based Peripheral Tolerance and Initiates Cardiac Allograft Rejection

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long‐term graft acceptance. After vascularized MHC‐mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti‐CD4 plus anti‐CD8 mAb. Long‐term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44^high effector/memory T cells in lymphatic organs and strong recovery of donor‐reactive T cell responses, indicating lymphopenia‐induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4⁺ Foxp³⁺) cell and B cell numbers as well as donor‐specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor‐reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.     

IL-4 deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of MHC class II disparate skin grafts

BACKGROUND: Acute rejection of MHC class II-disparate bm12 skin grafts by C57BL/6 recipient mice is characterized by massive graft infiltration by eosinophils, together with increased intragraft amounts of IL-4 and IL-5 mRNA. IL-5 blockade prevents the intragraft eosinophil infiltration and prolongs the survival of skin allografts. As the differentiation of T cell precursors into Th2 cells is largely driven by IL-4, we investigated the role of IL-4 in MHC class II-disparate allograft rejection. METHODS: We performed skin grafts from MHC class II incompatible bm12 mice into wild-type C57BL/6 mice (IL-4) or C57BL/6 IL-4 deficient mice (IL-4). Graft survival, in vitro T cell reactivity, and histology were compared. RESULTS: We observed that 50% of IL-4 mice rapidly rejected their bm12 allograft, whereas the other 50% retained their graft 60 days after transplantation. Histological examination of bm12 allografts retained by IL-4 mice showed a normal appearance with no inflammatory infiltrate and no eosinophils. Among IL-4 mice that acutely rejected their bm12 skin graft, we observed a dense polymorphonuclear infiltrate. The depletion of neutrophils significantly prolonged bm12 graft survival. CONCLUSIONS: Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.