CD154 Blockade Prevents Allosensitization through Inhibiting Both T- and B-Cell Activation and Decreasing Expression of IFN-γ and IL-10 in T-Cells.

Recipient sensitization to MHC antigens from transfusion therapy and prior graft rejection is among the most critical of problems in clinical transplantation. Sensitized patients reject vascularized organ or bone marrow transplants within minutes to hours as a result of preformed anti-donor Abs. Preventing allosensitization at the time recipients are exposed to donor alloantigens would be of obvious clinical benefit. B cell activation and the generation of memory B cells depends upon T cell responses via signaling from the co-stimulatory molecule CD154 (on activated T cells) to CD40 (on B cells). We have demonstrated in an allogeneic mouse model [BALB/c (H2K_d) to B6 (H2K_b)] that blockade of T and B cell interactions with anti-CD154 induces B cell tolerance, as defined by complete abrogation of the generation of donor-specific Ab after skin grafting. Furthermore, anti-CD154 treatment promotes successful subsequent bone marrow transplantation in these recipients, confirming that sensitization was prevented. In this study, we evaluated the effect of anti-CD154 mAb on T- and B-cell populations, activation state, and cytokine expression by T cells. B6 recipients were treated with anti-CD154 (day 0 and +3) or isotype hamster IgG control around the time receiving BALB/c skin grafts (day 0), and the number of T-cells (CD4⁺ and CD8⁺), total B-cells (CD19⁺), immature B-cells (CD19⁺CD24^highCD23^low), and follicular B-cells (CD19⁺CD24^lowCD23^high) in the spleen was enumerated by 4 color flow cytometry at day 7, 15 and 25 after skin grafting. No significant difference in absolute number of T- and B-cell subpopulations was seen between anti-CD154 and control IgG treated groups at the time points tested. By measuring the percentage CD71⁺ cells in the CD8⁺ or CD4⁺ gate or CD69⁺ in the CD19⁺ gate, activated T and B cell populations were evaluated. In vivo blockade of CD154 resulted in a significantly reduced activation of alloreactive T- and B-cells: the percentage of CD8⁺/CD71⁺ T cells was significantly lower at day 7 and the percentage of CD4⁺/CD71⁺ T cells was significantly lower at all time points compared with control mice (P < 0.05). The percentage of CD19⁺/CD69⁺ B cells at day 7 and 25 was significantly lower compared with control IgG treated mice (P < 0.05). To determine the effect of anti-CD154 treatment on Th1 and Th2 cytokine production, intracellular IFN-γ and IL-10 expression was analyzed. The IFN-γ expression in both CD8 and CD4 T-cells was inhibited at day 7 and reached significance (P < 0.01) by day 15 compared with control IgG treated group. IL-10, a cytokine which promotes B-cell activation and differentiation expression, was similar at day 7 between the two groups, but significantly decreased in both CD8 and CD4 T-cells at day 15 in mice treated with anti-CD154. Therefore, these data suggest that blockade of CD154 during initial antigen exposure mechanistically interferes with activation of both allo antigen-specific T and B-cells and inhibits the generation of allogeneic Ab (allosensitization). These effects are associated with suppression of IFN-γ and IL-10 cytokine secretion.

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