K Risch

Identification of gene markers for the prediction of allograft rejection or permanent acceptance.

The clinical success of new treatment strategies aiming on inducing permanent graft acceptance will rely on the ability to determine whether specific unresponsiveness to donor alloantigens has developed and for how long it is maintained. To identify markers for such posttransplant monitoring, genes differentially expressed by graft infiltrating leukocytes during tolerance induction or rejection after kidney transplantation in rats were compared. A subsequently performed full kinetic analysis in two different transplant models, kidney and heart, in two species, rat and mouse identified two markers (TOAG‐1, α‐1,2‐mannosidase) with high specificity and reproducibility, which are highly expressed during induction and maintenance of acceptance, and downregulated during rejection. Expression level of these markers showed a strong positive correlation with graft function. In addition, expression of both genes was downregulated in the peripheral blood and the graft prior to rejection, suggesting that these markers may be useful for monitoring in clinical transplantation where peripheral blood is the most easily accessible patient sample. Interestingly, downregulation of TOAG‐1 and α‐1,2‐mannosidase expression occurred in graft infiltrating cells and expression of both genes was also downregulated after T‐cell activation in vitro.

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.

IFN-γ Regulation in Anti-CD4 Antibody–Induced T Cell Unresponsiveness

ABSTRACT. The anti-rat CD4 mAb RIB5/2 is very potent in inducing allospecific tolerance in vivo . It is interesting that the unresponsiveness is breakable by exogenous IL-2 applied during the induction phase of tolerance. The molecular mechanisms underlying anti-CD4 antibody–mediated inhibition of allospecific T cell activation and how this is antagonized by exogenous IL-2 were investigated. Anti-CD4 treatment, in vivo and in vitro , completely abrogated IL-2 production by alloreactive T cells. In contrast, anti-CD4–treated alloactivated T cells showed similar IFN-γ mRNA expression as untreated alloactivated T cells but did not secrete any protein. Thus, the anti-CD4 antibody cannot prevent IFN-γ mRNA expression but is interfering with posttranscriptional mechanisms that control IFN-γ production during alloactivation of T cells. Addition of IL-2 but not IL-15 to anti-CD4–treated alloactivated T cells restored IFN-γ protein production without leading to enhanced IFN-γ mRNA expression. Further investigations revealed a diminished activation of translation initiation factor eIF2α in anti-CD4–treated T cells, which was restored by exogenous IL-2. As activated eIF2α is essential for the translation of IFN-γ mRNA, the results may explain the reversibility of anti-CD4–induced unresponsiveness by exogenous IL-2. Furthermore, these results not only shed further light onto the molecular mechanisms of tolerance induction but also reveal the possible weaknesses of anti-CD4 antibody–induced unresponsiveness.

Bag-1 up-regulation in anti-CD4 mAb treated allo-activated T cells confers resistance to apoptosis

The nondepleting anti‐CD4 mAb RIB5/2 is a powerful inducer of tolerance to MHC‐incompatible renal and heart allografts in rat recipients. In vitro the mAb blocks the proliferation andcytokine production of alloreactive T cells. To learn more about the mechanism of anti‐CD4‐mediated suppression, we applied differential display reverse transcription‐PCR to identify differences atmRNA level between T cells stimulated by alloantigen in the presence or absence of anti‐CD4 mAb. A sequence alignment of a 550‐bp DNA fragment appearing only in anti‐CD4 mAb‐treated cells resulted in at least 95% homology to a mouse cDNA encoding for the anti‐apoptotic protein Bag‐1. Further investigation of Bag‐1 expression during mixed lymphocyte reactions revealed a three‐ to fourfold up‐regulation of Bag‐1 mRNA expression in anti‐CD4 mAb‐treated allogeneic cultures which was confirmed at protein level. Bag‐1 up‐regulation was associated with an increase resistance to apoptosis of T cells from anti‐CD4 mAb‐treated cultures. Application of antisense oligonucleotides specific for Bag‐1 reduced Bag‐1 protein expression and restored susceptibility to apoptosis. In addition, up‐regulationof Bag‐1 mRNA could also be detected in graft‐infiltrating T cells from anti‐CD4 mAb‐treated rats in vivo. Thus, the expression of Bag‐1 in a subset of anti‐CD4 mAb‐treated alloreactive T cells conferred resistance against apoptosis, potentially contributing to the long‐term survival of these cells.

Ischemia/reperfusion injury-mediated down-regulation of adenovirus-mediated gene expression in a rat heart transplantation model is inhibited by co-application of a TNFRp55-Ig chimeric construct.

E1-deleted adenoviral vectors are efficient vectors for somatic gene therapy. Recently, we have shown that intratracheal application of an adenoviral reporter construct leads to significant reporter gene expression in rat lungs within 24 h. In contrast, reporter gene expression in syngeneic rat heart transplants after adenovirus-mediated gene transfer was delayed. Since the adenovirus cannot replicate, down-regulation of the hCMV-IE promoter controlled reporter gene expression in initially infected cells by cytokines, which are released as a result of ischemia/reperfusion injury, might be involved. In order to investigate the role of proinflammatory cytokines, eg TNF-α in affecting hCMV-IE promoter-driven reporter gene expression, transient blockade of TNF-α was achieved by local co-application of an Ad-construct encoding for a soluble TNFRp55-Ig chimeric molecule in a syngeneic rat heart transplantation model. Co-application of the reporter construct together with the TNFRp55-Ig chimeric molecule significantly increased the early reporter gene expression after transplantation. Moreover, infiltration of inflammatory cells (T cells, macrophages, NK cells) and production of TNF-α in the transplant was markedly reduced. Our results indicate that: (1) proinflammatory cytokines are involved in down-regulation of reporter gene expression in ischemia/reperfusion injured tissues; and (2) inhibition of TNF-α might be a useful tool to increase early gene expression in gene therapy protocols, particularly in transplantation.

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.

FTY720 Prevents Anti‐CD4 mAb‐Induced Tolerance but Cannot Reverse Established Tolerance in a Rat Kidney Transplantation Model

FTY720 is highly effective in various models of transplantation and autoimmunity. In order to find drugs that act synergistically with a tolerance‐inducing nondepleting anti‐CD4 mAb we studied this combination in a strong DA to LEW kidney transplantation model. Rats were treated with 0.3 mg/kg of FTY720 for 14 days and anti‐CD4 mAb RIB5/2, alone or in combination. After kidney transplantation serum creatinine and blood lymphocyte counts were monitored. Immunohistology, ELISPOT and TaqMan™‐PCR analysis of biopsies were performed.

Bag-1 up-regulation in anti-CD4 mAb-treated allo-activated T cell confers resistance to activation-induced cell death (AICD)

The non-depleting anti-CD4 monoclonal antibody (mAb) RIB5/2 is a powerful inducer of tolerance to major histocompatibility complex (MHC)-incompatible allografts in rat recipients. The unresponsiveness induced is characterized by the persistence (over 300 days) of donor-reactive regulatory T cells within the graft. We applied differential-display reverse-transcription polymerase chain reaction (RT-PCR) to identify differences at the mRNA level between graft-infiltrating cells of anti-CD4 mAb-treated and non-treated control rats at day 5 after kidney transplantation. A 550-bp DNA fragment appearing only in anti-CD4 mAb-treated rats is identical with the anti-apoptotic protein Bag-1. A further investigation of Bag-1 expression during mixed lymphocyte reactions (MLR) revealed a three-four-fold up-regulation of Bag-1 mRNA expression in anti-CD4 mAb-treated allogeneic cultures. Bag-1 up-regulation is associated with higher protection against apoptosis of anti-CD4 mAb-treated cultures. Application of antisense oligonucleotides specific for Bag-1 leads to both a reduction in Bag-1 expression and sensibility against apoptosis. Thus, the expression of Bag-1 in anti-CD4 mAb-treated alloreactive T cells conferred resistance against apoptosis, which may contribute to the long-term survival of tolerance-mediating T cells in vivo.

Abolition of anti-CD4-induced allotransplantation tolerance by exogenous IL-2.

THE CD4-targeted therapy is a potent inhibitor of immune responses following transplantation of MHCincompatible allografts. We and others have demonstrated that the nondepleting anti-rat CD4 monoclonal antibody (MAb) RIB5/2 induces allotransplantation tolerance in models of acute and accelerated rejection. At present, anergy is considered to be one of the main immune mechanisms of anti-CD4-induced peripheral tolerance. The characteristic feature of this unresponsive cellular state is a downregulated IL-2 gene transcription (reviewed by Schwartz). Because of the central role of IL-2 gene regulation for the development of a responsive/unresponsive immune state, several experiments were designed to analyze the effects of exogenous IL-2, applied during induction and maintenance of peripheral tolerance. In contrast to other models, we have previously shown that administration of high-dose exogenous hrIL-2 does not reverse tolerance induced by intravenous injection of donor-specific splenocytes together with anti-CD4 MAb RIB5/2 treatment 3 weeks before transplantation. In this report, we analyzed another experimental model of RIB5/2-induced tolerance, in which exogenous IL-2 administration is able to break developing or established specific immune nonresponsiveness.