Elevated and cross-responsive CD1a-reactive T cells in bee and wasp venom allergic individuals.

The role of CD1a-reactive T cells in human allergic disease is unknown. We have previously shown that circulating CD1a-reactive T cells recognize neolipid antigens generated by bee and wasp venom phospholipase, and here tested the hypothesis that venom-responsive CD1a-reactive T cells associate with venom allergy. Circulating T cells from bee and wasp venom allergic individuals, before and during immunotherapy, were exposed to CD1a-transfected K562 cells in the presence of wasp or bee venom. T-cell response was evaluated based on IFNγ, GM-CSF, and IL-13 cytokine production. Venom allergic individuals showed significantly higher frequencies of IFN-γ, GM-CSF, and IL-13 producing CD1a-reactive T cells responsive to venom and venom-derived phospholipase than healthy individuals. Venom-responsive CD1a-reactive T cells were cross-responsive between wasp and bee suggesting shared pathways of allergenicity. Frequencies of CD1a-reactive T cells were initially induced during subcutaneous immunotherapy, peaking by weeks 5, but then reduced despite escalation of antigen dose. Our current understanding of venom allergy and immunotherapy is largely based on peptide and protein-specific T cell and antibody responses. Here, we show that lipid antigens and CD1a-reactive T cells associate with the allergic response. These data have implications for mechanisms of allergy and approaches to immunotherapy.

Effectiveness of SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on DMARDs: as determined by antibody and T cell responses.

OBJECTIVES: To assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on disease-modifying antirheumatic drugs (DMARDs). METHODS: This prospective study recruited 100 patients with RA on a variety of DMARDs for antibody and T cell analysis, pre-vaccination and 4 weeks post-vaccination. Positive antibody response was defined as sera IgG binding to ≥1 antigen. Those that remained seronegative after first vaccination were retested 4 weeks after second vaccination; and if still seronegative after vaccination three. A T cell response was defined an ELISpot count of ≥7 interferon (IFN)γ-positive cells when exposed to spike antigens. Type I IFN activity was determined using the luminex multiplex assay IFN score. RESULTS: After vaccine one, in patients without prior SARS-CoV-2 exposure, 37/83 (45%) developed vaccine-specific antibody responses, 44/83 (53%) vaccine-specific T cell responses and 64/83 (77%) developed either antibody or T cell responses. Reduced seroconversion was seen with abatacept, rituximab (RTX) and those on concomitant methotrexate (MTX) compared to 100% for healthy controls (p<0.001). Better seroconversion occurred with anti-tumour necrosis factor (TNF) versus RTX (p=0.012) and with age ≤50 (p=0.012). Pre-vaccine SARS-CoV-2 exposure was associated with higher quantitative seroconversion (≥3 antibodies) (p<0.001). In the subgroup of non-seroconverters, a second vaccination produced seroconversion in 54% (19/35), and after a third in 20% (2/10). IFN score analysis showed no change post-vaccine. CONCLUSION: Patients with RA on DMARDs have reduced vaccine responses, particularly on certain DMARDs, with improvement on subsequent vaccinations but with approximately 10% still seronegative after three doses.

A Personalized Rituximab Retreatment Approach Based on Clinical and B-Cell Biomarkers in ANCA-Associated Vasculitis.

Background: Time to relapse after rituximab for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is variable, and optimal retreatment strategy has remained unclear. In AAV following rituximab induction, the study objective was to evaluate clinical and B-cell predictors of relapse in order to develop a retreatment algorithm. Methods: A retrospective observational study was conducted in 70 rituximab-treated ANCA-associated vasculitis patients followed up for over 10 years. Complete response (CR) was defined as Birmingham Vasculitis Activity Score v3.0 = 0. Retreatment was given on clinical relapse, defined as new features or worsening of persistent disease (not by biomarker status). Peripheral B-cell subsets were measured using highly sensitive flow cytometry. Predictors were tested using multivariable Cox regression. Results: Median time to retreatment for cycles 1-5 were 84, 73, 67, 60, and 73 weeks. Over 467 patient-years follow-up, 158 relapses occurred in 60 patients; 16 (in 15 patients) were major (renal = 7, neurological = 4, ENT = 3, and respiratory = 2). The major-relapse rate was 3.4/100 patient-years. In multivariable analysis, concomitant immunosuppressant [HR, 0.48 (95% CI, 0.24-0.94)], achieving CR [0.24 (0.12-0.50)], and naïve B-cell repopulation at 6 months [0.43 (0.22-0.84)] were associated with longer time to relapse. Personalized retreatment using these three predictors in this cohort would have avoided an unnecessary fixed retreatment in 24% of patients. Area under the receiver operating characteristic for prediction of time to relapse was greater if guided by naïve B-cell repopulation than if previously evaluated ANCA and/or CD19+ cells return at 6 months had been used, 0.82 and 0.53, respectively. Conclusion: Our findings suggest that all patients should be coprescribed oral immunosuppressant. Those with incomplete response or with absent naïve B cells should be retreated at 6 months. Patients with complete response and naïve repopulation should not receive fixed retreatment. This algorithm could reduce unnecessary retreatment and warrant investigation in clinical trials.

Direct ex-vivo evaluation of pneumococcal specific T-cells in healthy adults.

Streptococcus pneumoniae is an encapsulated bacterium that causes significant global morbidity and mortality. The nasopharynxes of children are believed to be the natural reservoir of pneumococcus and by adulthood nasopharyngeal carriage is infrequent; such infrequency may be due to demonstrable pneumococcal specific T and B-cell responses. HLA Class 2 tetrameric complexes have been used to characterise antigen specific T-cell responses in a variety of models of infection. We therefore sought to determine the frequency and phenotype of pneumococcal specific T-cells, using a novel HLA-DRB1*1501 tetramer complex incorporating a recently defined T-cell epitope derived from the conserved pneumococcal serine/threonine kinase (StkP). We were able to detect direct ex-vivo StkP(446-60)-tetramer binding in HLA-DRB1*1501 adults. These StkP(446-60)-tetramer binding T-cells had increased CD38 expression and were enriched in CCR7- CD45RA+ expression indicating recent and on-going activation and differentiation. Furthermore, these StkP(446-60)-tetramer binding T-cells demonstrated rapid effector function by secreting interferon-gamma on stimulation with recombinant StkP. This is the first study to directly enumerate and characterise pneumococcal specific T-cells using HLA class 2 tetrameric complexes. We found that ex-vivo pneumococcal-specific T cells were detectable in healthy adults and that they were enriched with cell surface markers associated with recent antigen exposure and later stages of antigen-driven differentiation. It is likely that these activated pneumococcal specific T-cells reflect recent immunostimulatory pneumococcal exposure in the nasopharynx and it is possible that they may be preventing subsequent colonisation and disease.

Histone H3 serine 57 and lysine 56 interplay in transcription elongation and recovery from S-phase stress.

BACKGROUND: Acetylation of lysine 56 of histone H3 plays an important role in the DNA damage response and it has been postulated to play an as yet undefined role in transcription, both in yeast and in higher eukaryotes. Because phosphorylated human histone H3 serine 57 peptides have been detected by mass spectrometry we examined whether H3-S57 phosphorylation interplays with H3-K56 acetylation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To explore the physiological role of H3-S57, H3-K56 was mutated to mimic constitutively (un)acetylated forms of H3-K56 and these were combined with constitutively (un)phosphorylated mimics of H3-S57, in yeast. A phosphorylated serine mimic at position 57 lessened sensitivities to a DNA replication fork inhibitor and to a transcription elongation inhibitor that were caused by an acetylated lysine mimic at position 56, while the same substitution exacerbated sensitivities due to mimicking a constitutive non-acetylated lysine at position 56. Strikingly, opposite results were obtained in the context of a serine to alanine substitution at position 57 of histone H3. CONCLUSIONS/SIGNIFICANCE: The phenotypes elicited and the context-dependent interplay of the H3-K56 and -S57 point mutations that mimic their respective modification states suggest that serine 57 phosphorylation promotes a nucleosomal transaction when lysine 56 is acetylated. We speculate that histone H3-S57 couples H3-K56 acetylation to histone quaternary structures involving arginine 40 on histone H4 helix 1.

Tracking antigen-specific T-cells during clinical tolerance induction in humans.

Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen specific T-cell responses during immunotherapy can provide novel insights into mechanisms of tolerance induction in humans and identify new potential treatment targets.

Rapid effector function of circulating CD4+ T cells specific for immunodominant regions of the conserved serine/threonine kinase found in Streptococcus pneumoniae (StkP) in healthy adults.

Streptococcus pneumoniae is an encapsulated bacterium that causes significant global morbidity and mortality. There is emerging evidence that T cells contribute to the immunity that protects humans from S. pneumoniae-associated disease. However, no T-cell epitopes have been identified as yet in this bacterium and there are no data that address the functional nature of T cells specific for pneumococcal-derived epitopes. We sought to define T-cell epitopes in the conserved serine/threonine kinase, found in S. pneumoniae (StkP) and to investigate specific interferon γ (IFN-γ) production resulting from such T-cell activation in healthy donors. We were able to detect the activation of T cells in response to pneumococcal whole-cell antigen or StkP-derived peptides in all 15 individuals. We found that the majority of the T-cell responses were directed against the extracellular, penicillin-binding protein and serine/threonine kinase-associated domains. We proceeded to characterize the immunodominant epitope in detail and observed HLA-DRB1(*) 1501 restriction. This is the first study that has identified T-cell responses to peptides derived from a protein from S. pneumoniae and has shown that in healthy adults, specific T cells have rapid IFN-γ production compatible with effector cell differentiation. The use of such T-cell epitopes will aid in the future monitoring of T-cell responses to both S. pneumoniae infection and vaccination in humans.