Circulating ß cell-specific CD8+ T cells restricted by high-risk HLA class I molecules show antigen experience in children with and at risk of type 1 diabetes.

In type 1 diabetes (T1D), autoreactive cytotoxic CD8+ T cells are implicated in the destruction of insulin-producing ß cells. The HLA-B*3906 and HLA-A*2402 class I genes confer increased risk and promote early disease onset, suggesting that CD8+ T cells that recognize peptides presented by these class I molecules on pancreatic ß cells play a pivotal role in the autoimmune response. We examined the frequency and phenotype of circulating preproinsulin (PPI)-specific and insulin B (InsB)-specific CD8+ T cells in HLA-B*3906+ children newly diagnosed with T1D and in high-risk HLA-A*2402+ children before the appearance of disease-specific autoantibodies and before diagnosis of T1D. Antigen-specific CD8+ T cells were detected using human leucocyte antigen (HLA) class I tetramers and flow cytometry was used to assess memory status. In HLA-B*3906+ children with T1D, we observed an increase in PPI5-12 -specific transitional memory CD8+ T cells compared to non-diabetic, age- and HLA-matched subjects. Furthermore, PPI5-12 -specific CD8+ T cells in HLA-B*3906+ children with T1D showed a significantly more antigen-experienced phenotype compared to polyclonal CD8+ T cells. In longitudinal samples from high-risk HLA-A*2402+ children, the percentage of terminal effector cells within the InsB15-24 -specific CD8+ T cells was increased before diagnosis relative to samples taken before the appearance of autoantibodies. This is the first study, to our knowledge, to report HLA-B*3906-restricted autoreactive CD8+ T cells in T1D. Collectively, our results provide evidence that ß cell-reactive CD8+ T cells restricted by disease-associated HLA class I molecules display an antigen-experienced phenotype and acquire enhanced effector function during the period leading to clinical diagnosis, implicating these cells in driving disease.

A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells.

CD8 T cells specific for islet autoantigens are major effectors of ß cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114-123 (VMNILLQYVV), with studies demonstrating both 114-123 and 114-122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) ß cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed. We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114-122 alone or both 114-122 and 114-123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114-123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201(+) human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114-123 as the predominant epitope in this region. These studies highlight the importance of understanding ß cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells.

Identification and characterisation of peptide binding motifs of six autoimmune disease-associated human leukocyte antigen-class I molecules including HLA-B*39:06.

Research on CD8 T cell-mediated inflammatory diseases requires a better understanding of target epitopes and the constraints placed upon these by major histocompatibility complex (MHC) class I binding restrictions, especially those that relate to predisposing alleles. We used linear trap quadrupole fourier transform (LTQ-FT) tandem mass spectrometry to identify naturally processed and presented peptides eluted from the MHC-negative myeloid leukaemia cell line K562 transfected with specific MHC class I genes. We provide information on the peptidome of HLA-B*39:06, which is associated with the autoimmune disease type 1 diabetes, and extend the analysis to include a further five human leukocyte antigen (HLA) alleles (HLA-A*02:01/-A*11:01/-A*24:02/-B*18:01/-B*38:01) studied under identical experimental conditions. We identified a total of 3095 individual peptides with a mascot score ≥40 (HLA-A*02:01 = 569 peptides, -A*11:01 = 904, A*24:02 = 257, -B*18:01 = 615, -B*38:01 = 453, -B*39:06 = 297). Peptides had a preferential length of nine amino acids and originated mainly from cytoplasmic or nuclear proteins. Eluted peptides revealed a strong binding motif with binding anchor positions at position 2 (P2) and the C-terminus (PΩ). Peptides eluted from HLA-A*02:01 showed a P2 preference for leucine (62% of total peptides have Leu at P2) and PΩ preference for valine (49%). Similar data are provided for HLA-A*11:01 (P2:Thr, 29%; PΩ:Lys, 49%), -A*24:02 (P2:Tyr, 78%; PΩ:Phe, 41%), -B*18:01 (P2:Glu, 77%; PΩ:Tyr, 32%), -B*38:01 (P2:His, 51%; PΩ:Leu, 45%) and -B*39:06 (P2:Arg/His, 24%; PΩ:Ala, 64%). This work thus gives an overview of the naturally processed and presented repertoire of several common and autoimmune disease-related HLA alleles, which may be useful in studying autoreactive CD8 T cell responses and the role of HLA in disease susceptibility.

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.