The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3−class II−CD11b+CD80/86+ plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4+CD25highFoxP3+ regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.

Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the late development of chronic rejection. Blocking the CD28-mediated T cell costimulation pathway is a non toxic alternative immunosuppression strategy that is currently achieved by blockade of CD80/86, the counter receptors for CD28 on antigenpresenting cells.. However interaction of CD80/86 with CTLA-4 is required for immune regulation. Therefore CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and favor immune regulation. By using monovalent antibodies, we identified true CD28 antagonists inducing a CTLA-4-dependent decreased T cell function compatible with regulatory T cell (Treg) suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood regulatory T cells, induced molecular signatures of immune regulation and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 favors CTLA-4-dependent immune regulation and promotes allograft survival.

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7 + , non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7 + non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.

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