Aims/Hypothesis The CD40–CD154 interaction directs autoimmune inflammation. Therefore, a longstanding goal in the treatment of autoimmune disease has been to control the formation of that interaction and thereby prevent destructive inflammation. Antibodies blocking CD154 are successful in mouse models of autoimmune disease but, while promising when used in humans, unfortunate thrombotic events have occurred, forcing the termination of those studies. Methods To address the clinical problem of thrombotic events caused by anti-CD154 antibody treatment, we created a series of small peptides based on the CD154 domain that interacts with CD40 and tested the ability of these peptides to target CD40 and prevent type 1 diabetes in NOD mice. Results We identified a lead candidate, the 15-mer KGYY15 peptide, which specifically targets CD40-positive cells in a size- and sequence-dependent manner. It is highly efficient in preventing hyperglycaemia in NOD mice that spontaneously develop type 1 diabetes. Importantly, KGYY15 can also reverse new-onset hyperglycaemia. KGYY15 is well tolerated and functions to control the cytokine profile of culprit Th40 effector T cells. The KGYY15 peptide is 87% homologous to the human sequence, suggesting that it is an important candidate for translational studies. Conclusions Peptide KGYY15 constitutes a viable therapeutic option to antibody therapy in targeting the CD40–CD154 interaction in type 1 diabetes. Given the involvement of CD40 in autoimmunity in general, it will also be important to evaluate KGYY15 in the treatment of other autoimmune diseases. This alternative therapeutic approach opens new avenues of exploration in targeting receptor–ligand interactions.
The BDC2.5 T cell clone is highly diabetogenic, but the transgenic mouse generated from that clone is surprisingly slow in diabetes development. While defining pathogenic effector T cells in autoimmunity has been inconsistent, CD4+ cells expressing the CD40 receptor (Th40 cells) are highly diabetogenic in NOD mice and NOD.BDC2.5.TCR.Tg mice possess large numbers of these cells. Given the importance of CD40 for pathogenic T cell development, BDC2.5.CD40−/− mice were created. Tregs, CD4+CD25hiFoxP3+, develop normally but pathogenic effector cells are severely reduced in number. Th40 cells from diabetic BDC2.5 mice rapidly induce diabetes in NOD.scid recipients but Th40 cells from pre-diabetic mice transfer diabetes very slowly. Demonstrating an important paradigm shift, effector Th40 cells from pre-diabetic mice are FoxP3+. As mice age, moving to T1D development Th40 cells lose FoxP3. When Th40 cells that are FoxP3+ are transferred to NOD.scid recipients disease is delayed. Th40 cells that are FoxP3− rapidly transfer disease. Th40 cells from BDC2.5.CD40−/− mice do not transfer disease nor do they lose FoxP3 expression. Mechanistically, FoxP3+ cells produce IL-17 but do not produce IFNγ while FoxP3− Th40 cells produce IFNγ and IL-2. This poses a new consideration for the function of FoxP3, as directly impacting effector T cell function.
CD40-CD154 interaction is decisive in directing autoimmune inflammation; therefore, controlling this interaction and hence destructive inflammation is a major goal in a number of autoimmune diseases including Type 1 Diabetes (T1D). Initial attempts to block CD40-CD154 interaction used antibody, random peptides, and small organic molecules, which all were problematic. Therefore an efficacious treatment remains a major goal. Here we reveal a unique targeted peptide, KGYY15, based on the domain of CD154 that binds to the CD40 receptor. Immunoprecipitation utilizing the peptide demonstrates that it binds directly to the CD40 receptor protein which in turn alters the CD40 signaling outcome. KGYY15 prevents T1D in 87% of non-obese diabetic (NOD) mice and histology reveals significant decreases in β-islet infiltration. Interestingly, KGYY15 treatment reverses overt hyperglycemia in 80% of new onset diabetic mice with significant improvement in β-islet insulin granule score. Structure-activity relationship studies demonstrate that the 15-mer peptide is optimal for efficacy and that five specific amino acids are critical in its activity. KGYY15 is well tolerated and functions to control the culprit Th40 effector T cells that are causative in T1D. The peptide sequence is 80% homologous to the human sequence suggesting an important candidate for translational studies.
CD40 plays a prominent role in autoimmunity. We described its expression on CD4 cells termed CD4CD40 cells. Diabetogenic T cell clones are CD40+ while non-diabetogenic clones are CD40-. CD4CD40 cells expand in number in NOD mice developing progressive insulitis and hyperglycemia and are necessary for adoptive transfer of T1D to NOD.scid recipients. CD4CD40 cells are found in human subjects, significantly (p< 10-7) increased in T1D, including new onset and long term (> 40 yrs) T1D subjects. CD4CD40 cells are expanded regardless of HLA haplotype but occur at normal levels in non-diabetics even those carrying predictive HLAs. CD4CD40 cells are significantly increased in diagnosed pre-diabetic subjects suggesting a biomarker for T1D diagnosis. CD4CD40 cells produce predominantly Th1 cytokines but also produce IL-17 suggesting an intermediary between Th1 and Th17 cells. Blocking CD40 - CD154 with anti-CD154 antibody prevents diabetes and contains CD4CD40 cell expansions. A 15 amino acid peptide derived from the CD154 protein sequence binds directly to CD4CD40 cells and prevents hyperglycemia onset. Furthermore the peptide reverses hyperglycemia in new onset T1D mice. This peptide constricts CD4CD40 cell numbers similarly to anti-CD154 treatment but does not disrupt immune homeostasis. Peptide treated mice have less islet infiltrates and greater insulin production than controls. Peptide treated mice also have increases in FoxP3+ cells, suggesting a tolerogenic mechanism.
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