Discussion

Successful manipulation of CD40–CD154 signals for therapeutic treatment of autoimmune diseases such as T1D requires a detailed knowledge of the roles of these signals at all stages of the autoimmune process. Here, we provide evidence for previously undescribed roles of CD40 and CD154 in suppression of autoaggressive CD8⁺ T cells after in vivo priming. CD40-transduced signals trigger an increase in T_R cells in inflamed tissue, and CD154-transduced signals sensitize anti-islet CD8⁺ T cells to suppression.

The importance of CD40–CD154 interactions in the regulation of anti-islet CD8⁺ T cells was demonstrated by finding that TNF/CD80.KO mice exhibit rapid diabetes development and a lack of regulation seen in TNF/CD80.WT littermates. The regulation that delays diabetes development in the TNF/CD80 model is characterized by increased numbers of T_R cells in the islets and PLN, and these cells actively suppress islet-specific CD8⁺ T cells (26). Rapid disease progression in TNF/CD80.KO mice and TNF/CD80.WT mice after prolonged islet expression of TNF- is associated with failure of T_R cells to increase in number in the pancreas and associated lymphoid tissue, suggesting a possible association between TNF- and CD40–CD154 signals. It is interesting to hypothesize that prolonged exposure to TNF- may shut down the mechanism whereby CD40–CD154 interactions trigger a tissue-specific increase in T_R cells.

Previous studies have shown that CD154 deficiency not only inhibits priming of anti-islet CD8⁺ T cells but also enhances T_R cell responses in vivo. On a CD154-deficient nonobese diabetic background T_R cells suppressed islet-specific T cell receptor transgenic CD8⁺ T cells indirectly by preventing maturation of dendritic cells (DC) (31). The ability of CD154-deficient T_R cells to suppress CD8⁺ T cells was abrogated by CD40 activation of DCs. This interesting study supports our findings that T_R cells from TNF/CD80.KO mice display a typical regulatory phenotype. However, in the TNF/CD80 model, the presence of APCs activated by TNF- or CD40 stimulation does not compromise the ability of T_R cells to suppress islet-specific CD8⁺ T cells in a CD154-sufficient environment. This finding suggests a requirement for CD40–CD154 interactions in suppression of the effector phase of the CD8⁺ T cell response beyond inhibition of APC maturation. In the absence of CD154, TNF--activated APCs fail to deliver the appropriate signals to trigger an increase in T_R cells in inflamed tissue, and this is restored by in vivo stimulation of CD40. This observation demonstrates a dissociation of TNF- and CD40 stimulation in activation and function of APCs. TRANCE–RANK (15) and TGF-–TGF-R (30) interactions have also been shown to play a role in increasing T_R cell numbers in inflamed tissue. It will be interesting to see whether the CD40–CD154 pathway intersects with these pathways in active suppression of autoimmunity in vivo.

The classical biological effects of CD40–CD154 interactions are mediated by ligation of CD40 on B cells and APCs leading to their activation and maturation (8). Here we show that stimulation of CD40 in the absence of CD154 triggers a systemic and tissue-specific increase in T_R cells. The mechanism of T_R cell increase after CD40 stimulation is unknown. According to the classical APC-activation role of CD40 signals, it is likely that these activated APCs promote migration, expansion, and enhanced survival of T_R cells in the PLN and islets. Alternatively, CD40-stimulated APCs may promote generation of T_R cells from CD4⁺CD25^- T cell progenitors. However, another explanation arises from recent reports documenting expression of CD40 on CD4⁺ and CD8⁺ T cells (36, 37). CD40-transduced signals may therefore act directly on T_R cells, triggering their expansion, or on CD4⁺CD25^- progenitors, triggering their differentiation into T_R cells. It was interesting that CD40 stimulation did not increase T_R cell numbers in TNF/CD80.WT mice (data not shown). This finding highlights the possibility that CD154 may play a role in systemic, as well as tissue-specific, T_R cell homeostasis. An intact CD40–CD154 signaling pathway may prevent aberrant expansion of the T_R population, protecting against inappropriate immune suppression after a strong inflammatory stimulus that may occur, for example, during viral infection. Identifying how CD40 stimulation induces an increase in T_R cells may identify useful targets for the therapeutic manipulation of the T_R cell population

Despite the ability of CD40 signals to increase T_R cell numbers in inflamed tissue, this finding did not translate into protection from T1D. CD40-transduced signals therefore cannot substitute for CD154 deficiency in control of islet-specific CD8⁺ T cells. This finding suggested that CD154-transduced signals might negatively regulate autoaggressive CD8⁺ T cells. In support of this hypothesis, CD154 is up-regulated on CD4⁺CD25^- and CD8⁺ T cells in the PLN of TNF/CD80.WT mice where T_R cells suppress activation of islet-reactive CD8⁺ T cells. However, although there is evidence that CD154-transduced signals lead to CD4⁺ T cell activation in vitro (38) and in vivo (39), the ability of CD8⁺ T cells to receive CD154-transduced signals is less well defined. There are several possible cellular interactions that could result in CD154-dependent suppression of CD8⁺ T cells. T_R cells may express CD40 and interact directly with CD154-expressing autoaggressive CD8⁺ T cells to deliver a suppressive signal. However, the distribution of CD40 expression on T cell subsets in this model remains to be defined. Alternatively, the CD154-transduced signal may be delivered to the CD8⁺ T cell from an APC after conditioning of the APC by interaction with a T_R cell. Understanding the mechanism whereby self-reactive CD8⁺ T cells may resist regulation by T_R cells may shed light on the etiology of autoimmune diseases mediated by CD8⁺ T cells.

Blockade of CD154 by using the MR1 Ab has been shown to prevent development of autoimmunity (10) and transplant rejection (14). Several mechanisms have been reported for the immune suppression induced by MR1. These mechanisms include inhibition of T cell priming (40), induction of novel regulatory cells (11) and deletion of allograft-reactive T cells (41). However, the latter result is controversial (42). Here we show that administration of MR1 Ab after priming of islet-specific CD8⁺ T cells in TNF/CD80.WT mice abrogates regulation of autoreactive CD8⁺ T cells, as assessed by rapid progression to T1D. This finding does not support a role for deletion of autoaggressive CD8⁺ T cells. However, it is possible that CD154 blockade may induce deletion of T_R cells. In light of our previous data, an attractive explanation for our observations is that MR1, acting as a blocking Ab, inhibits CD154-dependent negative regulatory signals and releases CD8⁺ T cells from suppression. We have previously shown that T_R suppression of CD8⁺ T cells requires expression of functional TGF-R on the surface of the CD8⁺ T cell (32). It will be interesting to establish whether the negative regulatory role of CD154 in autoaggressive CD8⁺ T cells is mediated by intersection with the TGF-–TGF-R pathway.

This study demonstrates roles for CD40- and CD154-transduced signals in the regulation of autoreactive CD8⁺ T cells in inflamed tissue. By dual signaling through CD40 and CD154, T_R cells receive the appropriate signals to increase in numbers at the site of autoimmunity, and islet-reactive CD8⁺ T cells are sensitized to suppression by regulatory mechanisms. Therapeutic manipulation of these signals may have important implications for treatment of T1D and possibly other autoimmune diseases.

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