K Lorré

Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production

Although CD28 triggering provides an important co‐stimulatory signal to T cells, blocking the CD80/CD86 – CD28 interaction with CTLA‐4lg fusion protein is not sufficient for tolerance induction in vivo or in vitro. According to more recent data, interruption of the CD40 – CD154 interaction might complement the effect of CTLA‐4lg and induce graft acceptance. We studied the effects of a blocking anti‐CD40 monoclonal antibody (mAb) and/or blocking anti‐CD80/anti‐CD86 mAb in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with allogeneic PBMC. T cells activated by alloantigens in the presence of anti‐CD80, anti‐CD86 and anti‐CD40 entered a state of alloantigen‐specific non‐responsiveness as evidenced upon restimulation by lack of proliferation, cytotoxic activity, and IL‐2, IL‐5 and IL‐13 production. IFN‐γ production during restimulation was less than in the control cultures, while the production of IL‐10 was enhanced. Addition of recombinant IL‐2 during the restimulation rescued alloantigen‐specific activity. We conclude that the simultaneous blocking of the CD40 – CD154 and CD80/CD86 – CD28 interaction during allogeneic T cell activation induces T cell anergy. Since anergic cells induced by this treatment still produce high levels of IL‐10, the latter could contribute to modulation of antigen‐presenting cell activity and to bystander suppression of residual reactive T cells.

Prevention of renal allograft rejection in primates by blocking the B7/CD28 pathway

BACKGROUND There is accumulating evidence that blockade of the costimulatory pathways offers a valid approach for immune suppression after solid organ transplantation. In this study, the efficacy of anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) in combination with cyclosporine (CsA) to prevent renal allograft rejection was tested in non-human primates. METHODS Rhesus monkeys were transplanted with a partly major histocompatibility complex-matched kidney on day 0. Anti-CD80 and anti-CD86 mAbs were administered intravenously daily for 14 days starting at day - 1. CsA was given intramuscularly for 35 days starting just after transplantation. The kidney function was monitored by determining serum creatinine levels. RESULTS The combination of anti-CD80 and anti-CD86 mAbs completely abrogated the mixed lymphocyte reaction. Untreated rhesus monkeys rejected the kidney allograft in 5-7 days. Treatment with anti-CD80 plus anti-CD86 mAbs resulted in a significantly prolonged graft survival of 28+ 7 days (P=0.025). There were no clinical signs of side effects or rejection during treatment. Kidney graft rejection started after the antibody therapy was stopped. The anti-mouse antibody response was delayed from day 10 to 30 after the first injection. No difference in graft survival was observed between animals treated with CsA alone or in combination with anti-CD80 and anti-CD86 mAbs. However, treatment with anti-CD80 and anti-CD86 mAbs reduced development of vascular rejection. CONCLUSIONS In combination, anti-CD80 and antiCD86 mAbs abrogate T-cell proliferation in vitro, delay the anti-mouse antibody response in vivo, and prevent graft rejection and development of graft vascular disease in a preclinical vascularized transplant model in non-human primates.

Effects of anti-IL-4 receptor monoclonal antibody on in vitro T cell cytokine levels: IL-4 production by T cells from non-atopic donors

IL‐4 is a pleiotropic cytokine which is involved in the development of atopic diseases. Only limited data exist on IL‐4 production in humans, and the relative contribution to atopy of either unbalanced IL‐4 production, or increased IL‐4‐responsiveness of target cells, is still unknown. The use of a MoAb to the IL‐4 receptor α‐chain (IL‐4Rα) enabled us to demonstrate that IL‐4 production in vitro is usually underestimated, due to in vitro consumption, even in cultures of purified T cells. When IL‐4 consumption was blocked, it became evident that CD80 and CD86 both provide effective costimulatory signals for high IL‐4 production. Moreover, we found that even stimulation with a soluble antigen (tetanus toxoid) induces IL‐4 production by T cells from healthy non‐atopic donors. Both sets of data imply that IL‐4 is not required for IL‐4 production by memory and/or effector T cells. Our data further show that endogenous IL‐4 activity modulates IL‐10 and interferon‐gamma production by T cells in opposite directions. The use of this receptor‐blocking antibody will thus be helpful for in vitro studies on IL‐4 regulation. Consumption of IL‐4 by different cell types during in vitro cultures might have interfered with previous attempts to quantify IL‐4 production by human T cells.

Prolonged skin graft survival by administration of anti-CD80 monoclonal antibody with cyclosporin A

Costimulation via the B7/CD28 pathway is an important signal for the activation of T cells. Maximal inhibition of T-cell activation and the induction of alloantigen-specific nonresponsiveness in vitro was achieved using anti-CD80 monoclonal antibody (mAb) in combination with cyclosporin A (CsA). Based on this knowledge, the efficacy of the prophylactic treatment of anti-CD80 mAb and CsA on allogeneic skin graft survival was tested in a preclinical rhesus monkey model. No side effects have been observed. Administration of anti-CD80 mAb resulted in high mAb serum levels that decreased to undetectable values around day 7. At the same time, the anti-mouse antibody response started to develop. The anti-CD80 mAb bound to peripheral blood mononuclear cells and was detectable in lymph node and grafted skin during the treatment period. The skin graft survival time of untreated or suboptimally CsA-treated rhesus monkeys was 10 days. Treatment with CsA (blood levels of 100-160 ng/ml) in combination with anti-CD80 mAb (0.5 mg/kg) resulted in a significantly increased skin graft survival time to 14 days. Eventually, skin grafts in all rhesus monkeys were rejected, which coincided with an increase in helper and cytotoxic T-cell frequency and induction of an antibody response directed against the donor antigens. Therefore, treatment of anti-CD80 mAb in combination with CsA has significant immunosuppressive potency, but was unable to induce donor-specific nonresponsiveness in skin graft recipients.

Regulation of the IL-10 Production by Human T Cells

Interleukin (IL)‐10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL‐10 production is therefore highly important for understanding the immunoregulation. The IL‐10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common γ‐chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL‐10 induction. CD80, CD58, rIL‐12 and rIFN‐α all had efficacious and independent costimulatory activities on the IL‐10 production, while PGE2 was inhibitory. Dependence on autocrine IL‐2 signalling was shown by the effects of anti‐IL‐2 and anti‐IL‐2R monoclonal antibodies (MoAb), but the IL‐10 production proceeded partly IL‐2‐independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL‐12 or rIFN‐α, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL‐10 (a cytokine‐inhibitory interleukin) and IL‐2 (a cytokine‐inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL‐10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.

Differences in regulatory pathways identify subgroups of T cell-derived Th2 cytokines

We analysed regulatory mechanisms involved in the production of Th2 cytokines by freshly isolated human T cells. We used an in vitro culture system in which the primary signal was provided by a cross‐linking anti‐CD3 MoAb presented on the Fc receptors of P815 cells. Both CD80 and CD86, expressed on transfected P815 cells, were able to provide efficient costimulation for the production of IL‐4, IL‐5 and IL‐13. IL‐2 was also highly important for induction of all three Th2 cytokines. However, differences between IL‐4 on the one hand and IL‐5 and IL‐13 on the other hand were observed when sensitivity to cyclosporin A (CsA) was studied. CsA (an inhibitor of calcineurin phosphatase activity) strongly inhibited IL‐4 production, but it did either not affect or even increased IL‐5 and IL‐13 production. In accordance with this, CD80 and phorbol myristate acetate (PMA) (without anti‐CD3 or calcium ionophore) were sufficient to induce production of IL‐5 and IL‐13, but not of IL‐4. The subgrouping of Th2 cytokines was further confirmed at another level on the basis of differences in cell sources: IL‐4 was predominantly produced by CD4+ T cells, while IL‐5 and IL‐13 were produced by both CD4+ and CD8+ T cells. Thus, differences in cell sources and in the requirement of the calcium/calcineurin‐signalling pathway allowed us to identify two subgroups (IL‐4 and IL‐5/IL‐13) among human Th2‐type T cell cytokines.