No abstract

Negative selection of self-reactive T-cells during thymic development, along with activation-induced cell death in peripheral lymphocytes, is designed to limit the expansion and persistence of autoreactive T-cells. Autoreactive T-cells are nevertheless present, both in patients with type 1 diabetes and in at-risk subjects. By using MHC class II tetramers to probe the T-cell receptor (TcR) specificity and avidity of GAD65 reactive T-cell clones isolated from patients with type 1 diabetes, we identified high-avidity CD4؉ T-cells in peripheral blood, coexisting with low-avidity cells directed to the same GAD65 epitope specificity. A variety of cytokine patterns was observed, even among T-cells with high MHC-peptide avidity, and the clones utilize a biased set of TcR genes that favor two combinations, V␣12-␤5.1 and V␣17-V␤4. Presence of these high-avidity TcRs indicates a failure to delete autoreactive T-cells that likely arise from oligoclonal expansion in response to autoantigen exposure during the progression of type 1 diabetes.

Self-reactive T cells populate the peripheral immune system, and likely form the reservoir from which autoreactive cells are derived. We analyzed a panel of self and non-self peptides presented by HLA-DR4, a class II molecule associated with autoimmunity, by immunization of mice transgenic for HLA-DR4. Significant structural avidity for T cell recognition, as measured by MHC class II tetramer binding to CD4 + T cells was only observed in mice immunized with the non-self antigens. T cell hybridomas were generated from mice immunized with the naturally processed self-peptide hGAD65 (552-572) and also from mice immunized with an influenza-derived non-self epitope (HA 306-318). T cells specific for the self peptide failed to bind tetramers and exhibited low functional avidity as measured by the peptide concentration required to reach half-maximum proliferation values. In contrast, T cells specific for the non-self HA (306-318) peptide exhibited high structural and functional avidity profiles. As recently described in studies of murine CD8 + T cell function, the predominance of low avidity recognition of self-peptide epitopes may be a characteristic feature of CD4 + T cells responding to autoantigens.

Major histocompatibility complex class II tetramer staining and activation analysis identified 2 distinct types of antigenspecific CD4 ؉ T cells in the peripheral blood of humans with type 1 (autoimmune) diabetes. T cells with low-avidity recognition of peptide-MHC ligands had low sensitivity to activation and inefficient activation-induced apoptosis. In contrast, high-avidity T cells were highly sensitive to antigen-induced cell death through apoptotic mechanisms, and both apoptosis-resistant high-and low-avidity T cells that survived prolonged tetramer treatment were rendered anergic to restimulation by antigen. In addition, however, apoptosis-resistant high-avidity T cells acquired regulatory features, being able to suppress both antigen-specific and nonspecific CD4 ؉ T-cell responses. Introduction␤-cell autoimmunity in type 1 diabetes is associated with the presence of both islet antigen (Ag)-specific T cells and autoantibodies, which form the basis for immune prediction and diseasemonitoring studies. 1,2 Glutamic acid decarboxylase 65 (GAD65) is one of the prevalent islet Ags, and CD4 ϩ T cells targeting its epitopes can be identified and characterized using major histocompatibility complex (MHC) class II tetramers (TMrs). 3 TMrs are soluble ligands for the MHC-restricted Ag specificity of the T-cell receptor (TCR), consisting of multimers of recombinant peptide-class II MHC molecules. Analogous to TCR-specific monoclonal antibodies (mAbs), TMrs are also endowed with signaling properties, being capable of activating target T cells and, on prolonged stimulation, of causing activation-induced cell death (AICD). 4 This latter outcome represents an appealing therapeutic approach to silence autoreactive T cells and restore tolerance in diabetes. 5 MHC-based reagents are therefore being investigated for both immune monitoring and intervention purposes. 6 In vivo preclinical models of immunomodulation by peptide-MHC reagents have been reported in a number of animal models of autoimmunity, including experimental allergic encephalomyelitis (EAE) 7 and experimental autoimmune myasthenia gravis, 8 and in transgenic models of autoimmune diabetes. 9,10 Mechanisms at play include AICD, 10 anergy, 8 T-helper 2 (Th2) immune deviation, 11 and induction of interleukin 10 (IL-10)-secreting cells. 9,10 One of the key determinants of differential outcomes for both autoimmune and normal T-cell responses is the functional T-cell avidity. The overall T-cell avidity is determined by a number of components, including the intrinsic TCR affinity for the peptide-MHC complex; the expression levels of TCR, CD4, and costimulatory molecules; differential membrane clustering of relevant receptors 12 ; and postreceptorial tunable signaling thresholds. 13 T-cell avidity provides a mechanism for sensing low-and high-density Ags, 12 for determining the hierarchy of epitope spreading, 14 for progression of the (auto)immune response, 15,16 and for eliminating high-avidity autoreactive T cells through AICD. 17 Sufficient T-cell avidity is also cr...