Regulatory and Effector CD4 T Cells in Nonobese Diabetic Mice Recognize Overlapping Determinants on Glutamic Acid Decarboxylase and Use DistinctVβGenes

Abstract

The 524–543 region of glutamic acid decarboxylase (GAD65), GAD65(524–543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524–543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-Ag7-restricted determinants present in the GAD65(524–543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR Vβ gene use. CD4+ T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530–543 (p530); however, T cells to the overlapping determinant 524–538 (p524) dominate the response only after immunization with GAD65(524–543). All p530-responsive T cells used the Vβ4 gene, whereas the Vβ12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524–543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGFβ upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524–543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.

N‐terminal flanking residues of a diabetes‐associated GAD65 determinant are necessary for activation of antigen‐specific T cells in diabetes‐resistant mice

“…The sequence of p524-543 is SRLSKVAPVIKARMMEYGTT, and the only acidic residue, E539, is unlikely to function as a P9 anchor with the bulky R536 in P6. We previously set forth a hypothesis of two-registers to explain the distinction between the N-and C-terminal peptides, p524-538 and p530-543 [23]. Peptide p524-538 could bind to I-A g7 using I533 and R536 as anchors for the P6 and P9 pockets, whereas for the peptide p530-543, M537 and Y540 were likely to be anchors binding to the P6 and P9 pockets, respectively.…”

“…We previously showed that T cells recognizing the N-terminal p524 peptide use BV12 TCR very frequently and T cells specific for the C-terminal p530 peptide are largely BV4 [23]. In fact, we found that three out of the five p524-538-specific hybridoma clones that we generated, including 524.59.3 and 524.169.17, used BV12 TCR, whereas, all four p530-543-specific clones, including 530.45.19 and 530.97.3, were BV4.…”

“…Interestingly, this 20mer peptide appeared to be comprised of two distinct determinants: an N-terminal moiety (p524-538 (p524)) and a C-terminal moiety (p530-543 (p530)). Importantly, two distinct T cell populations could be induced by these two overlapping peptides, and it appeared that they had opposing functions: the p530-specific T cells are invasive and may be pathogenic [21,22], whereas the p524-specific T cells are regulatory in nature and can prevent diabetes by adoptive transfer [23]. In this study, we mapped the fine specificity of clones specific for either p524 or p530 to define the minimal antigenic cores of these two determinants, and found that p543 contains multiple epitopes containing a central overlapping core sequence, p530-539, with different lengths of flanking residues at their N or C termini.…”

“…In addition, our recent studies show that processing within the long p524-543 peptide differs between NOD and NOR mice (K. P. Jensen et al, manuscript submitted). Therefore, endogenous priming to GAD65 in NOD preferentially activates C terminus-reactive T cells.The NOD T cell repertoire is highly focused and frequently uses BV4 TCRWe previously showed that T cells recognizing the N-terminal p524 peptide use BV12 TCR very frequently and T cells specific for the C-terminal p530 peptide are largely BV4 [23]. In fact, we found that three out of the five p524-538-specific hybridoma clones that we generated, including 524.59.3 and 524.169.17, used BV12 TCR, whereas, all four p530-543-specific clones, including 530.45.19 and 530.97.3, were BV4.…”

Anatomy of T cell autoimmunity to myelin oligodendrocyte glycoprotein (MOG): Prime role of MOG44F in selection and control of MOG‐reactive T cells in H‐2b mice

“…However, unlike MOGreactive T cell clones, not all of the PLP-reactive clones needed the same nonameric core epitope for their stimulation [35]. In other studies describing T cells reactive against MBP89-101 in SJL/J mice [36,37], against antigens/epitopes in rheumatoid arthritis (such as collagen II [38][39][40]) or against antigens/epitopes in type I diabetes (such as insulin [41,42] or glutamic acid decarboxylase 65 [43,44]), both restricted and highly diverse TCR repertoires have been reported to be associated with reactivity to the disease-relevant antigen/epitope. However, in the studies where diverse TCR expression for recognition of the epitopic region was demonstrated, the nonameric core sequence essential for stimulation of the heterogeneous T cells was not assigned [40,41].…”