Anna Skowera

A single autoimmune T cell receptor recognizes more than a million different peptides

Background: How does a limited pool of <108 T cell receptors (TCRs) provide immunity to >1015 antigens? Results: A single TCR can respond to >one million different decamer peptides. Conclusion: This unprecedented level of receptor promiscuity explains how the naïve TCR repertoire achieves effective immunity. Significance: TCR degeneracy has enormous potential to be the root cause of autoimmune disease. The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <108 human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>1015 distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8+ T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type “index” preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >108 peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8+ T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.

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)‐γ 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‐γ, 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‐γ 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.

Chronic fatigue syndrome: an update focusing on phenomenology and pathophysiology

Purpose of review Chronic fatigue syndrome is a controversial condition especially concerning its clinical definition and aetiopathogenesis. Most recent research progress has been made in phenomenology and pathophysiology and we focused our review on these two areas. Recent findings The phenomenology research supports the notion of a discrete fatigue syndrome which can be distinguished from depression and anxiety. The current case definition, however, may need an improvement based on empirical data. Recent advances in understanding the pathophysiology of chronic fatigue syndrome continue to demonstrate the involvement of the central nervous system. Hyperserotonergic state and hypoactivity of the hypothalamic–pituitary–adrenal axis constitute other findings, but the question of whether these alterations are a cause or consequence of chronic fatigue syndrome still remains unanswered. Immune system involvement in the pathogenesis seems certain but the findings on the specific mechanisms are still inconsistent. Genetic studies provide some evidence of the syndrome being a partly genetic condition, but environmental effects seem to be still predominant and identification of specific genes is still at a very early stage. Summary The recent findings suggest that further research is needed in improving the current case definition; investigating overlaps and boundaries among various functional somatic syndromes; answering the question of whether the pathophysiologic findings are a cause or consequence; and elucidating the involvement of the central nervous system, immune system and genetic factors.

Cellular Immune Activation in Gulf War Veterans

The etiology and pathology of illnesses related to the first Persian Gulf War are unclear. Among the constellation of symptoms noted in sick veterans, some, such as skin rashes, musculoskeletal pains, and neuropsychiatric problems, have been proposed to reflect an underlying immune dysfunction. In this study we explored the hypothesis that sickness following deployment to the Gulf in 1991 is associated with altered immune function, and we examine possible associated exposures. In particular, we focused on peripheral blood Th1/Th2 balance by measuring intracellular production of IFN-γ, IL-2 (Th1), IL-4 (Th2), and IL-10 by CD4 T cells, using a nested case control study design within a large epidemiological survey. We compared symptomatic Gulf War veterans (sGWV) with well GWVs (wGWV), and a second control group of symptomatic veterans who served in Bosnia or were nondeployed military personnel of the same era. We found evidence for an altered immune status in sGWV in comparison to the other study groups. In particular, ongoing Th1-type immune activation was associated with multisymptom illness in GWVs, with sick veterans having significantly elevated levels of IFN-γ and IL-2 producing CD4+ cells in the absence of in vitro stimulation compared with wGWVs (P = 0.01 and P =0.001). In vitro polyclonal activation revealed significantly elevated levels of IL-10 producing memory CD4 cells in sGWVs (P <0.001), but other cytokines were normal. In terms of possible exposures that might influence immune function, we found a trend for reduced levels of IFN-γ producing cells after polyclonal activation with increasing numbers of vaccines administered (P <0.05) but no changes in other cytokines. These data show that multisymptom illness in Gulf War veterans is characterized by ongoing Th1-type immune activation and a biased generation of memory cells secreting the suppressor cytokine, IL-10.

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.

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.

Analysis of Anthrax and Plague Biowarfare Vaccine Interactions with Human Monocyte-Derived Dendritic Cells

The anti-biowarfare anthrax and plague vaccines require repeated dosing to achieve adequate protection. To test the hypothesis that this limited immunogenicity results from the nature of vaccine interactions with the host innate immune system, we investigated molecular and cellular interactions between vaccines, dendritic cells (DCs), and T cells and explored the potential for adjuvants (pertussis) to boost induction of host immunity. Human monocyte-derived DCs were matured in the presence of vaccines and analyzed for their ability to induce Th1/Th2 development from naive T cells, expression of cell surface maturation/costimulation molecules, and cytokine production. The vaccines showed different behavior patterns. Although the plague vaccine is equivalent to control maturation factors in maturation and stimulation of DCs and induces strong MLR and Th outgrowth, the anthrax vaccine is a poor inducer of DC maturation, as indicated by low levels of HLA-DR, CD86, and CD83 induction and minimal proinflammatory cytokine production. Interestingly, however, anthrax vaccine-treated DCs stimulate Th1 and Th2 outgrowth and a limited MLR response. There was no sustained negative modulatory effects of the anthrax vaccine on DCs, and its limited stimulatory effects could be overridden by coculture with pertussis. These results were supported by analysis of anthrax vaccine recall responses in subjects vaccinated using pertussis as an adjuvant, who demonstrate anthrax-specific effector T cell responses. These data show that the anthrax vaccine is a suboptimal DC stimulus that may in part explain the observation that it requires repeated administration in vivo and offer a rational basis for the use of complementary DC-maturing adjuvants in combined immunotherapy.