Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F1 offspring (C57BL/6 × C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25+ cell-depleted splenocyte transfer, showing that CD25+ natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6–30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3+ cells among donor CD4+ cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.

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.

Although immunoregulation by several types of regulatory T cells is now clearly established in mice, the demonstration of such regulatory T cells in humans has been proven more difficult. In this study we demonstrate the induction of anergic regulatory T cells during an MLR performed in the presence of blocking mAb to the costimulatory molecules CD40, CD80, and CD86. Despite this costimulation blockade, which totally blocks T cell proliferation and cytokine production, a nonproliferating T cell subpopulation was activated to express inducible costimulator (ICOS). These ICOS+ cells were anergic when restimulated with unmanipulated allogeneic stimulator cells at the level of proliferation and Th1 and Th2 cytokine production, but they did produce IL-10. These ICOS-expressing cells also blocked the capacity of reciprocal ICOS-negative cells to proliferate and to produce cytokines. ICOS+ anergic cells could suppress allogenic responses of either primed or naive T cells through inhibition of IL-2 gene transcription. Suppression was not mediated by IL-10 and did not require ICOS-ICOS ligand interaction, but depended on cell-cell contact. Thus, a subtype of regulatory T cells in human blood can be activated in the absence of costimulatory signals from CD40, CD80, and CD86, and they can be identified by expression of ICOS after activation.

No abstract

No abstract