We have shown a role for RAGE in adaptive immune responses to allo- and autoantigens. A small molecule inhibitor of RAGE reduced the destruction of syngeneic islets that were transplanted into NOD mice with spontaneous diabetes, and RAGE-deficient B6 mice rejected allogeneic BALB/c islet grafts at a reduced rate compared with WT B6 mice. RAGE−/− T cells were not anergic. Their response in a MLR was reduced, but they showed the same proliferative responses to activation with full doses of anti-CD3 and anti-CD28 mAbs as WT T cells. However, there were markedly reduced responses of the RAGE−/− T cells to CD28 co-stimulatory signals with anti-CD3 mAb that was not explained by differences in the expression of CD28 on the surfaces of RAGE−/− T cells. In response to anti-CD3 and anti-CD28 mAbs, RAGE−/− T cells produce relatively greater amounts of IL-10, IL-5, and TNF-α
in response to TCR ligation, which is similar to our findings previously in which there was higher levels of IL-10 in the pancreata of NOD/SCID mice that were treated with sRAGE and showed reduced development of diabetes after receiving diabetogenic splenocytes (16
). The small molecule RAGE inhibitor TTP488 also inhibited responses of T cells in a MLR, and inhibition was not seen with RAGE−/− cells. Moreover, the responders from MLR’s that had been cultured with TTP488 secreted reduced levels of IFN-γ
in response to activation with PMA/ionomycin consistent with a role of RAGE in differentiation of Th1 cells. The discrepancies between the cytokine pattern in the RAGE−/− T cells activated with anti-CD3 and anti-CD28 and responders in a MLR in the presence of the RAGE inhibitor are likely due to the different culture systems used to activate the cells – one involving the direct activation of naive cells and the other the secondary activation after differentiation in the presence of the RAGE inhibitor. Indeed, in a previous report, we found that OTII cells on a RAGE−/− background produced reduced levels of IFN-γ
and IL-2 when activated with PMA/ionomycin after priming with peptide pulsed DC’s (21
). Finally, our finding of increased RAGE expression on activated clonal populations of cells under conditions that promote development of Th1 compared with Th2 cells is consistent with the role of this receptor in early events that lead to development of T cell phenotypes, specifically polarization toward a Th1 phenotype.
A number of recent studies have highlighted the role of RAGE on the activation and maturation of DCs and other APCs, but few have addressed the role of RAGE on T cells. Exposure of neutrophils, monocytes, or macrophages to the RAGE ligand HMGB1 enhances expression of proinflammatory cytokines by these cells, but RAGE was shown to play only a minor role in activation of these cells by HMGB1 (22
). Likewise, these same investigators found that HMGB1 interacts with TLRs 2 and 4 but they were unable to show binding of HMGB1 to RAGE by fluorescence resonance energy transfer and immunoprecipitation. However, Tian et al. (14
) recently reported that DNA-containing immune complexes in systemic lupus erythematosus bind HMGB1 and RAGE activating TLR9 on plasma DCs causing them to secrete IFNα
. A role for RAGE in T cell activation was suggested by previous studies by Yan et al. (15
) in which a dominant-negative RAGE, expressed on CD4+
T cells, blocked induction of experimental allergic encephalomyelitis. We previously showed that sRAGE attenuated the adoptive transfer of diabetes by diabetogenic T cells into NOD/SCID recipients (16
). However, sRAGE did not prevent diabetes induced with a clonal population of CD4+
T cells, which raised the possibility that the role of RAGE was indirect, rather than directly on T cells, or alternatively that RAGE played a role in earlier stages of T cell differentiation. These new studies, however, establish a role of RAGE signaling on the early stages of T cell differentiation. Moreover, in other studies, we have also found that RAGE−/− OVA-reactive OTII cells show diminished proliferative responses to Ag when transferred into WT recipients (21
Studies in RAGE null mice have indicated that RAGE is involved in perpetuation of cellular responses (9
). This conclusion is consistent with our previous studies in autoimmune diabetes and the present studies of recurrent diabetes in diabetic NOD recipients of syngeneic islet grafts in which the responses of previously activated effector cells were inhibited. However, in the current studies, involving responses to allografts, alloantigens, and TCR signaling, the differences in cytokine production and RAGE expression on T cell phenotypes were in primary responses. There was negligible expression of RAGE on T cells studied after secondary activation (not shown).
CD28 expression is required for the formation of the mature immunologic synapse – central supramolecular activation cluster localization of PKC θ that is found in CD28+
T cells is absent in CD28−
T cells (23
). CD28 engagement leads to the redistribution and clustering of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagements. This results in higher rates and more stable tyrosine phosphorylation of several substrates and higher consumption of Lck (24
). These important events in TCR signaling may be absent in the RAGE−/− mice, but further studies of TCR signaling will help to define the basis for the loss of CD28 responsiveness in the absence of RAGE. RAGE ligands, such as HMGB1, have previously been shown to affect differentiation of DCs and HMGB1 released by DCs is needed for clonal expansion, survival, and functional polarization of naive T cells (25
). Release of HMGB1 by human DCs was necessary for up-regulation of CD80 and CD86, ligands for CD28. RAGE was required for this effect of HMGB1 on DCs, suggesting that the innate HMGB1/RAGE signaling pathway results in adaptive immune responses (25
Our findings differ from those of Liliensiek et al. (11
) who found that deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture, but their studies did not support a role of RAGE in adaptive immune responses. However, these previous studies were done in mice on a mixed genetic background, whereas our studies were done on mice in which the RAGE mutation was backcrossed to B6 for more than 10 generations so that differences between WT and KO mice could be identified more clearly (11
). Consistent with our findings, Dumitriu et al. (25
) found that HMGB1 signaling through RAGE was needed for clonal expansion, survival, and functional polarization of naive T cells. These findings also support a direct effect of RAGE on T cell activation and differentiation rather than just T cell migration or localization as has been suggested in other model systems (15
). Likewise, we have also found that clonal populations of RAGE−/− T cells show reduced responses when adoptively transferred into WT recipients, indicating that the role of RAGE on T cell activation is primary (21
Our findings suggest a novel testable hypothesis for the relationship between metabolic control of Type 1 diabetes and the immunologic progression of disease in patients. Studies from the Diabetes Control and Complications Study showed that individuals with new onset Type 1 diabetes, who maintained intensive glycemic control, had reduced loss of C-peptide responses compared with individuals with conventional metabolic treatment (26
). Reduced availability of RAGE ligands with strict glycemic control would be expected to modulate adaptive responses that may be responsible for destruction of β
cells. Indeed, the results in these experimental settings indicate that RAGE activation can affect even previously primed responses.
In summary, we have demonstrated a role of RAGE in the activation of adaptive immune responses to auto- and alloantigens. RAGE expression on T cells affects the early events its absence is manifest by reduced activation of T cells to alloantigens in vivo and in vitro. RAGE is involved in the differentiation of T cells along a Th1 phenotype and RAGE mRNA is more abundant in Th1 compared with Th2 cells. The reduced rate of recurrent diabetes and prolonged allograft survival by RAGE blockade and in RAGE-deficient mice suggests that RAGE may be an important new target for therapeutic strategies to prevent adaptive immune responses. The activation of RAGE by ligands, such as HMGB1, released from dying cells implies a broad role for this pathway in immune homeostasis. Moreover, the increased availability of RAGE ligands during hyperglycemia suggests a hypothesis for the amplification of autoimmune responses in Type 1 diabetes that may be tested in future clinical settings.