Activation of insulin-reactive CD8 T-cells for development of autoimmune diabetes.

OBJECTIVE: We have previously reported a highly diabetogenic CD8 T-cell clone, G9C8, in the nonobese diabetic (NOD) mouse, specific to low-avidity insulin peptide B15-23, and cells responsive to this antigen are among the earliest islet infiltrates. We aimed to study the selection, activation, and development of the diabetogenic capacity of these insulin-reactive T-cells. RESEARCH DESIGN AND METHODS: We generated a T-cell receptor (TCR) transgenic mouse expressing the cloned TCR Valpha18/Vbeta6 receptor of the G9C8 insulin-reactive CD8 T-cell clone. The mice were crossed to TCRCalpha-/- mice so that the majority of the T-cells expressed the clonotypic TCR, and the phenotype and function of the cells was investigated. RESULTS: There was good selection of CD8 T-cells with a predominance of CD8 single-positive thymocytes, in spite of thymic insulin expression. Peripheral lymph node T-cells had a naïve phenotype (CD44lo, CD62Lhi) and proliferated to insulin B15-23 peptide and to insulin. These cells produced interferon-gamma and tumor necrosis factor-alpha in response to insulin peptide and were cytotoxic to insulin peptide-coated targets. In vivo, the TCR transgenic mice developed insulitis but not spontaneous diabetes. However, the mice developed diabetes on immunization, and the activated transgenic T-cells were able to transfer diabetes to immunodeficient NOD.scid mice. CONCLUSIONS: Autoimmune CD8 T-cells responding to a low-affinity insulin B-chain peptide escape from thymic negative selection and require activation in vivo to cause diabetes.

Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands.

Insulin-reactive CD8 T cells are amongst the earliest islet-infiltrating CD8 T cells in NOD mice. Cloned insulin B15-23-reactive cells (designated G9C8), restricted by H-2K(d), are highly diabetogenic. We used altered peptide ligands (APL) substituted at TCR contact sites, positions (p)6 and 8, to investigate G9C8 T cell function and correlated this with structure. Cytotoxicity and IFN-gamma production assays revealed that p6G and p8R could not be replaced by any naturally occurring amino acid without abrogating recognition and functional response by the G9C8 clone. When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide. The antagonist activity in cytotoxicity and IFN-gamma production assays can be correlated with conformational changes induced by different structures of the MHC-peptide complexes, shown by molecular modeling. We conclude that p6 and p8 of the insulin B15-23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide. This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin.

CD86 has sustained costimulatory effects on CD8 T cells.

CD80 and CD86 both costimulate T cell activation. Their individual effects in vivo are difficult to study as they are coordinately up-regulated on APCs. We have studied mice expressing rat insulin promoter (RIP)-CD80 and RIP-CD86 on the NOD and NOD.scid genetic background to generate in vivo models, using diabetes as a readout for cytotoxic T cell activation. Accelerated spontaneous diabetes onset was observed in NOD-RIP-CD80 mice and the transfer of diabetes from 6-wk-old NOD mice to NOD.scid-RIP-CD80 mice was greater compared with NOD-RIP-CD86 and NOD.scid-RIP-CD86 mice, respectively. However, the secondary in vivo response was maintained if T cells were activated through CD86 costimulation compared with CD80. This was demonstrated by greater ability to cause recurrent diabetes in NOD-RIP-CD86 diabetic mice transplanted with 6-wk-old NOD islets and adoptively transferred diabetes from diabetic NOD-RIP-CD86 mice to NOD.scid mice. In vitro, CD80 costimulation enhanced cytotoxicity, proliferation, and cytokine secretion in activated CD8 T cells compared with CD86 costimulation. We demonstrated increased CTLA-4 and programmed death-1 inhibitory molecule expression following costimulation by both CD80 and CD86 (CD80 > CD86). Furthermore, T cells stimulated by CD80 were more susceptible to inhibition by CD4(+)CD25(+) T cells. Overall, while CD86 does not stimulate an initial response as strongly as CD80, there is greater sustained activity that is seen even in the absence of continued costimulation. These functions have implications for the engineered use of costimulatory molecules in altering immune responses in a therapeutic setting.

The influence of the major histocompatibility complex on development of autoimmune diabetes in RIP-B7.1 mice.

The most important genetic susceptibility factor for type 1 diabetes is encoded in the major histocompatibility complex (MHC). The nonobese diabetic (NOD) mouse, which develops spontaneous diabetes, expresses H-2g7 comprising the MHC class I molecules Kd and Db and the MHC class II molecule I-Ag7. However, neither B6.H-2g7 mice, in which H-2g7 is expressed on the C57BL/6 genetic background, nor the nonobese resistant (NOR) mouse, in which H-2g7 is expressed on a genetic background that is 88% similar to NOD mice, develop diabetes. Immune tolerance can be broken in these diabetes-resistant mice expressing H-2g7 if the costimulatory molecule B7.1 is present on the islet beta cells. This does not occur if only single MHC class I components of the H-2g7 haplotype are present, such as Kd in BALB/c mice or Db in C57BL/6 mice, both of which develop only a low level of diabetes when B7.1 is expressed. The presence of I-Ag7 leads to the development of an autoimmune T-cell repertoire, and local costimulation of CD8 T-cells precipitates aggressive diabetes. This implies that a major role of the MHC class II molecules in diabetes is the development of an autoreactive T-cell repertoire.