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.

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.

High levels of type 2 cytokine-producing cells in chronic fatigue syndrome.

The aetiology of chronic fatigue syndrome (CFS) is not known. However, it has been suggested that CFS may be associated with underlying immune activation resulting in a Th2-type response. We measured intracellular production of interferon (IFN)-gamma and interleukin (IL)-2; type 1 cytokines), IL-4 (type 2) and IL-10 (regulatory) by both polyclonally stimulated and non-stimulated CD4 and CD8 lymphocytes from patients with CFS and control subjects by flow cytometry. After polyclonal activation we found evidence of a significant bias towards Th2- and Tc2-type immune responses in CFS compared to controls. In contrast, levels of IFN-gamma, IL-2 and IL-10-producing cells were similar in both study groups. Non-stimulated cultures revealed significantly higher levels of T cells producing IFN-gamma or IL-4 in CFS patients. Concluding, we show evidence for an effector memory cell bias towards type 2 responsiveness in patients with CFS, as well as ongoing type 0 immune activation in unstimulated cultures of peripheral blood cells.

Predictors of fatigue following the onset of infectious mononucleosis.

BACKGROUND: Infectious mononucleosis (IM) is a risk factor for chronic fatigue. Reduced activity is the most consistent factor found to be associated with poor outcome following the onset of infectious mononucleosis. However, little is known about the biological mechanisms involved in the pathogenesis of chronic fatigue following IM and no study, so far, has examined the relation between certain illness beliefs and poor outcome. This study explored immunological, endocrine, behavioural and cognitive responses to the acute illness and assessed which components of these groups of risk factors predicted a chronic course. METHOD: Using a prospective cohort design, 71 primary care patients with IM were enrolled onto the study and interviewed. Their recovery was explored by postal questionnaire up to 1 year later. RESULTS: In the univariate analysis, increased baseline levels of immune activation were associated with fatigue at baseline and 3 months. Cortisol levels were not associated with fatigue at any point. Using multivariate models of clinical and psychosocial baseline factors, severity of symptoms and illness perceptions were found to predict fatigue 3 months later. At 6 months, fatigue was best predicted by female gender and illness perceptions, and at 12 months by female gender and a symptoms-disability factor. CONCLUSIONS: In the multivariate analysis no factors were found to predict poor outcome at all time-points. Instead the pattern of predictors changed over time, partly but not completely consistent with our a priori predictions. Larger studies are needed to explore further the predictive nature of biopsychosocial factors in the pathogenesis of chronic fatigue related to IM. The psycho-behavioural predictors found in this study are amenable to intervention. Such interventions should be tested in randomized controlled trials.

Antinuclear autoantibodies (ANA) in Gulf War-related illness and chronic fatigue syndrome (CFS) patients.

It is established that veterans of the 1991 Gulf War have an increased frequency of experiencing multiple symptoms. The underlying mechanism of these ailments is unclear, although they do not correspond to any clearly defined syndrome. The most common symptoms overlap with those of chronic fatigue syndrome (CFS). CFS was recently associated with a novel subtype of antinuclear autoantibody (ANA) that reacts with nuclear envelope (NE) antigens. NE autoantibodies are not known to be linked with any distinct clinical condition, but have been observed in patients with unusual mixed chronic autoimmune disorders and connective tissue diseases. In this study we examined whether NE ANAs are a feature of patients with CFS and symptomatic Gulf War veterans (sGWV). We studied the prevalence of ANA in 130 sGWV, 90 well Gulf War veterans (wGWV), 128 symptomatic Bosnia and Era veterans (sBEV), 100 CFS patients, and 111 healthy control subjects matching for age and sex. We found no significant difference in the prevalence of ANAs between any of the groups. None of the patients/or veterans we studied had ANA of the NE type. Our results show that multisymptom illness due to CFS or related to Gulf War service is not associated with antinuclear autoimmunity.