Elmara Graser

Anti-CD4 monoclonal antibody-induced allograft tolerance in rats despite persistence of donor-reactive T cells.

Although CD4-targeted therapy abrogates acute rejection and may induce permanent graft acceptance in rodents, little is known about the mechanisms of long-term graft survival in these models. Recently, we have shown that treatment with a nondepleting anti-CD4 monoclonal antibody (mAb) (RIB-5/2) induces long-term survival of renal, heart, and skin allografts in strong major histocompatibility complex I/II incompatible rat strains. Here, we demonstrate that the development of major histocompatibility complex-specific and tissue-nonspecific tolerance rather than graft adaptation is responsible for long-term anti-CD4 mAb-induced transplant survival. Donor-specific but not third-party heart and pancreatic islet grafts were accepted permanently without adjunctive therapy in long-term kidney allograft recipients, and infusion of naive or alloimmune splenocytes failed to break the tolerant state. Interestingly, alloreactive T cells were not depleted in these long-term survivors, as ex vivo donor-specific mixed lymphocyte reaction was largely unaffected. The reverse transcriptase-polymerase chain reaction analyses of long-term renal allografts before and after donor-specific antigen challenge revealed no changes in CD3 mRNA level, but showed up-regulation of CD25, interleukin (IL) 2, interferon (IFN) gamma, IL-4, and IL-10 mRNA in the early phase, suggesting the presence of alloreactive T cells in tolerant rats. At later time points, the expression of IFN-gamma declined rapidly, whereas IL-4 persisted, resulting in a reversal of IFN-gamma/IL-4 ratio. Our data demonstrate the stability of anti-CD4 mAb-induced tolerance despite persistence of alloreactive T cells, suggesting the role of active tolerance-maintaining mechanisms. The T helper (Th) 1/Th2 shift may be involved in this regulatory process, as anti-CD4 mAb prevents acute graft-deteriorating rejection by effectively blocking Th1 responses, and well-functioning grafts may tolerize themselves by inducing regulatory cells.

The effects of nondepleting CD4 targeted therapy in presensitized rat recipients of cardiac allografts

The immunosuppressive effects of RIB-5/2, a nondepleting anti-rat CD4 monoclonal antibody (mAb), were analyzed in a well-defined model of accelerated cardiac allograft rejection. (LEW x BN)F1 hearts are rejected within 24 hours in LEW hosts presensitized with BN skin grafts at day -7. Treatment with RIB-5/2 mAb (3.5 mg/day i.v.) at days -7 and -1, prolonged cardiac allograft survival to the median of >62 days. The long-term recipients rejected acutely third-party (Wistar-Furth) test skin grafts, without an adverse effect on the survival of the original cardiac transplants. Lymphocytes harvested from mAb-treated hosts significantly decreased proliferative responses of donor cells in mixed leukocyte reaction. The cell activation and cytokine elaboration patterns were evaluated at the mRNA and protein levels by competitive template reverse transcriptase polymerase chain reaction and immunohistochemistry, respectively. Cardiac allografts in CD4 mAb-treated rats at 24 hours displayed reduced CD3, CD25, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-2, interferon (IFN)-gamma, and IL-10 mRNA levels as compared to those in rejecting grafts. Equal amounts of IL-4 mRNA were detected throughout in both animal groups; the expression of IL-10 mRNA increased progressively in the treated hosts. In contrast, IFN-gamma was consistently depressed after mAb therapy. The mRNA levels coding for CD3, CD25, tumor necrosis factor-alpha, IL-1-beta, and IL-2 genes were comparable in long-surviving and rejecting allografts. The staining for IL-2R, IL-2, and IFN-gamma was diminished, whereas the staining for IL-4 was either unaffected or enhanced in well-functioning grafts in RIB-5/2 mAb-treated hosts. The untreated recipients elicited strong circulating IgM allo-Ab response, which peaked around the time of cardiac rejection and then switched to IgG allo-Ab 4-7 days after heart transplantation. Treatment with RIB-5/2 mAb decreased IgM and prevented the switch into the IgG allo-Ab response. In conclusion, the ability of RIB-5/2 mAb treatment to combat accelerated rejection and to produce long-term graft acceptance is unprecedented in our experience in this model. These data provide new insights into the complexities of the cellular and humoral responsiveness, contributing to the the induction of donor-specific unresponsiveness in sensitized hosts. This study, along with our previous reports, indicate that an immune deviation in which intragraft Th1-type cytokines (primarily IFN-gamma) are diminished and Th2-type cytokines (IL-4 and IL-10) are maintained represents the common effector mechanism of CD4 mAb regimens in recipients of vascularized organ allografts.

Blockade of very late antigen-4 integrin binding to fibronectin in allograft recipients: I. Treatment with connecting segment-1 peptides prevents acute rejection by suppressing intragraft mononuclear cell accumulation, endothelial activation, and cytokine expression.

BACKGROUND Allograft rejection is associated with infiltration of inflammatory cells and local deposition of fibronectin (FN). This study was carried out to examine the hypothesis that peptides known to specifically block adhesive interactions between the connecting segment-1 (CS1)-binding domain of FN and alpha4beta1 integrin on circulating cells may interfere with the immune cascade, which would lead to acute rejection in transplant recipients. METHODS AND RESULTS Cardiac allografts from Lewis x Brown Norway F1 hybrids were rejected in 7+/-1 days in Lewis rats. Treatment with bioactive CS1 peptides (4 mg/kg/day i.v. for 7 days) abrogated acute rejection and prolonged cardiac allograft survival to 13+/-1 days (P<0.001). This effect correlated with decreased expression of total fibronectin and cell adhesion molecules, such as alpha4beta1, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, as well as reduced infiltration by CD4+ and CD8+ T cells at the graft site. Treatment with CS1 peptides decreased alloantigen activation, as evidenced by decreased intragraft infiltration by CD25+ cells, and diminished expression of mRNA coding for Th1 (interleukin [IL]-2, interferon-gamma)- and Th2 (IL-4, IL-5, IL-6)-type cytokines. CS1-mediated immunosuppressive effects could be reversed and acute rejection recreated after adjunctive treatment of rats with recombinant IL-2. CONCLUSION Our data are consistent with the model in which in vivo interaction between the alpha4beta1 integrin receptor and the cell-associated CS1 motif of FN is critical for rejection cascade. The novel therapeutic approach of selectively blocking the alpha4beta1-FN activation pathway with CS1 peptides prevents acute allograft rejection by inhibiting expansion of antigen-specific T cells and inducing a transient state of cytokine-responsive anergy in the residual T-cell population.

Cytokine mRNA Levels and Lymphocyte Infiltration in Pancreatic Tissue During Experimental Chronic Pancreatitis Induced by Dibutyltin Dichloride

There is little information available regarding the role of inflammatory cells in the pathogenesis of chronic pancreatitis. Therefore, we analyzed the local cytokine profile and infiltrating lymphocytes in a rat model of chronic pancreatitis. Experimental pancreatitis was induced by a single intravenous application of dibultyltin dichloride (DBTC). During a time course of two months we observed the mRNA expression of cytokines using competitive RT-PCR. Lymphocytes were characterized by immunohistochemistry, FACS analysis, and the lymphocyte proliferation test. IL-1β, IL-6, IL-5, and IL-10 were immediately up-regulated in the acute phase of disease, while lymphocyte-restricted expression of IL-2, IL-2R, and IFN-γ was only found in the chronic course. Among the infiltrating lymphocytes, CD4+ cells dominated, but during the chronic process there was an increase of CD8+ cells, resulting in a reduced CD4/CD8 ratio. Mitogen-induced activation of isolated mesenteric lymph node cells increased during the chronic inflammation. Our results suggest that in experimental pancreatitis acute inflammatory reactions are followed by a T-lymphocyte-mediated process.

Downregulation of intragraft IFN-gamma expression correlates with increased IgG1 alloantibody response following intrathymic immunomodulation of sensitized rat recipients.

A single intrathymic injection of donor-specific spleen cells (2 x 10(7)) abrogates accelerated (24 hr) rejection of LBNF1 cardiac allografts in presensitized LEW rats and prolongs graft survival to about 11 days. This effect is donor-specific, gamma-irradiation-sensitive, thymus-dependent, and requires no concomitant therapy. We have recently shown that following intrathymic alloantigen administration, there is an earlier and increased systemic production of alloreactive IgM, and subsequently a premature isotype switching to IgG with the predominant IgG1 and IgG2a alloantibody responses. There is also a preferential binding of these IgG subclasses to the endothelium of well-functioning allografts. In this work, we analyzed the early cell activation and related cytokine elaboration patterns at the mRNA and protein levels by competitive template RT-PCR and immunohistochemistry, respectively. We found that prolonged cardiac allograft survival following intrathymic administration of donor spleen cells in presensitized rats was associated with markedly depressed intragraft IFN gamma mRNA and protein expression. Moreover, intrathymic allostimulation has led to a defect in the IL-2 pathway as the expression of IL-2 and IL-2R protein at the graft site was inhibited despite stable IL-2 mRNA levels. The inhibition of cell activation was also demonstrated by reduced MHC class II and the lack of ICAM-1, and TNF-alpha expression by immunohistochemistry. The expression of biologically active IL-12 (p70) by mononuclear and endothelial cells was detected in rejecting grafts between 3 and 12 hr. The well-functioning grafts after intrathymic allostimulation were devoid of IL-12 (p70), which in turn may have contributed to the downregulation of IFN-gamma mRNA and protein. Treatment with r.IFN-gamma, but not with r.IL-2, recreated the rejection response, and the characteristic IgG subclass pattern associated with accelerated graft loss. Hence, intrathymic immunomodulation with alloantigen results in selective inhibition of IFN-gamma-producing cells and a preferential upregulation of IgG1 alloantibodies. These data support the notion of the interlocked immunoregulatory roles of cytokine and alloantibody networks in rat allograft recipients.

Intragraft overexpression of interleukin-4 is neither sufficient nor essential for tolerance induction to cardiac allografts in a high-responder strain combination.

BACKGROUND Recently we have demonstrated that the nondepleting anti-CD4 monoclonal antibody (mAb) RIB5/2 induces long-term acceptance of kidney and heart allografts in all rat strain combinations tested. Cytokine gene expression studies by reverse transcriptase-polymerase chain reaction revealed a reversed intragraft interleukin (IL)-4/interferon-gamma ratio. Whether IL-4 mediated immune deviation contributes to transplantation tolerance is not clear so far. METHODS To learn more about the functional relevance of the relative IL-4 up-regulation, IL-4 was overexpressed in rat heart allografts by using ex vivo adenoviral gene transfer. The efficiency of gene transfer was analyzed by reporter gene assays as well by reverse transcriptase-polymerase chain reaction analysis of IL-4 mRNA expression. RESULTS The intragraft overexpression of IL-4 did not prolong the allograft survival compared with controls. Moreover, neutralization of IL-4 by OX81 mAb did not prevent tolerance induction by RIB5/2 treatment. CONCLUSIONS Anti-CD4 mAb-induced tolerance is associated with an intragraft type1/type2 shift, however, the up-regulation of IL-4 alone is neither sufficient nor essential to induce tolerance to cardiac allografts in a high-responder strain combination.