No abstract

Combined treatment with allogeneic small lymphocytes or T-depleted small lymphocytes plus a blocking antibody to CD40 ligand (CD40L) permitted indefinite pancreatic islet allograft survival in 37 of 40 recipients that differed from islet donors at major and minor histocompatibility loci.

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.

Ag-specific immune tolerance results from the induction of cellular mechanisms that limit T cell responses to selective Ags. One of these mechanisms is characterized by attenuated proliferation and decreased IL-2 production in fully stimulated CD4+ Th cells and is denoted T cell anergy. We report the identification of the early growth response gene (Egr-2; Krox-20), a zinc-finger transcription factor, as a key protein required for induction of anergy in cultured T cells. Gene array screening revealed high Egr-2 expression distinctly persists in anergized but not proliferating murine A.E7 T cells. In contrast, Egr-1, a related family member induced upon costimulation, displays little or no expression in the anergic state. IL-2-mediated abrogation of anergy causes rapid depletion of Egr-2 protein. Full stimulation of anergic A.E7 T cells fails to enhance IL-2 and Egr-1 expression, whereas Egr-2 expression is greatly increased. Silencing Egr-2 gene expression by small interfering RNA treatment of cultured A.E7 T cells before incubation with anti-CD3 alone prevents full induction of anergy. However, small interfering RNA-mediated depletion of Egr-2 5 days after anergy induction does not appear to abrogate hyporesponsiveness to stimulation. These data indicate that sustained Egr-2 expression is necessary to induce a full anergic state through the actions of genes regulated by this transcription factor.

Combined treatment with antibody against CD40 ligand and one transfusion of donor splenocytes prolonged survival of fully mismatched BALB/c skin allografts on C57BL/6 recipients, with approximately 20% of grafts surviving > 100 days. In vitro alloresponsiveness in treated animals was reduced in the immediate post-transplantation period, but by day 100 was increased despite the presence of a successful allograft. The presence of alloreactivity on day 100 was confirmed in vivo by adoptive transfer, which suggests that our protocol had induced either a state of "split tolerance" or "graft accommodation." Mice with skin grafts that had survived for > or = 100 days revealed no evidence of lymphoid chimerism. Treatment with donor splenocytes and antibody against CD40 ligand permits long-term survival of highly antigenic donor skin allografts despite the presence of functionally intact alloreactive lymphocytes.