Supplementary MaterialsSupplemental data jci-130-131696-s292. mainly toward the 3 end of the viral genome. Five novel MHC II tetramers were made using an immunodominant EFYQSTCSAVSKGYL (F-EFY) epitope restricted to HLA-DR4, -DR9, and -DR11 (combined allelic frequency: 15% in Europeans) and G-DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labeling revealed enrichment of resident memory TC-H 106 CD4+ T (Trm) cells in the lower airway; these Trm cells displayed progressive differentiation, downregulation of costimulatory molecules, and elevated CXCR3 expression as infection evolved. CONCLUSIONS Human infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of Trm recognizing novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific CD4+ cells, potentially accelerating the development of effective vaccines. TRIAL REGISTRATION ClinicalTrials.gov “type”:”clinical-trial”,”attrs”:”text”:”NCT02755948″,”term_id”:”NCT02755948″NCT02755948. FUNDING Medical Research Council, Wellcome Trust, National Institute for Health Research. = 0.0009; Supplemental Figure 1D). Our previous studies showed that preinfection nasal IgA levels correlate with protection from infection with RSV, but that systemic serum-neutralizing antibodies are clearly less protective in an TC-H 106 experimental challenge (32). These data were supported by similar (although nonsignificant) findings in this smaller cohort (Supplemental Figure 2). Open up in another home window Shape 1 Movement diagram outlining research participating and style topics.(A) Healthful adult volunteers (= 49) were enrolled and inoculated with RSV M37 for polyclonal Compact disc4+ T cell evaluation and epitope discovery. (B) Another cohort (= Il17a 8) was enrolled for tetramer evaluation of RSV-specific Compact disc4+ T cells. To monitor T cell proliferation and activation, whole bloodstream samples had been stained with antiCKi-67 and Compact disc38 for movement cytometric evaluation of Compact disc4+ T cells before inoculation (day time 0) and 3, 7, 10, 14, and 28 times after the problem (Shape 2A). In bloodstream, the frequency of activated CD4+ TC-H 106 T cells increased between 7 and 10 days after contamination, coinciding with viral clearance. Ki-67+CD38+CD4+ T cells peaked around day 10 (median, 1.33%; IQR, 1.87C1.08), after which they returned to baseline frequencies on disease resolution (median, 0.67%; IQR, 0.757C0.449; Physique 2B). Although the magnitude of the proliferative response was modest, activated and proliferating CD4+ T cells were significantly more frequent than in those challenged individuals who remained uninfected. Open in a separate window Physique 2 Enrichment of activated and regulatory CD4+ T cells in the lower airway during RSV contamination.(A) Whole blood (= 49) and BAL (= 24) samples were stained with anti-CD3, -CD4, -CD8, -CD38, and CKi-67 for analysis by flow cytometry. Plots are gated on CD3+CD4+ lymphocytes. One representative infected subject is shown for blood (upper panels) and BAL (lower panels). Median and individual data points of Ki-67+CD38+CD4+ T cells in the (B) blood and (C) BAL of infected (PCR+, red) or uninfected (PCRC, blue) volunteers are shown. Tests of the 5 TC-H 106 a priori hypotheses were conducted by Wilcoxons signed-rank test with Bonferroni-adjusted levels of 0.01 (** 0.001). (D) Frequencies of Ki-67+CD38+ cells on day 10 after contamination are compared between paired blood and BAL samples in infected individuals (= 12). Assessments of the 5 a priori hypotheses were conducted by Wilcoxons signed-rank test with Bonferroni-adjusted levels of 0.01 with no statistically significant differences seen. (E) Whole blood and BAL samples were stained with anti-CD3, -CD4, -FoxP3, and -CD25. One representative infected BAL sample is usually shown gated on CD3+CD4+ lymphocytes. (F) Mean and individual data points of FoxP3+CD25+CD4+ T cells in the blood and BAL of infected (PCR+, red circles) or uninfected (PCRC, blue squares) volunteers are shown. values for Wilcoxons signed-rank (intragroup) and Mann-Whitney assessments (intergroup) are shown. * 0.05. A subset of participants (= 24) underwent bronchoscopy with bronchoalveolar lavage (BAL) to sample the lower airway on days 0, 7 to 10, and 28 after inoculation; 12 of these individuals (50%) became infected following viral inoculation. Activation and Proliferation of CD4+ T cells in BAL was comparable to that in bloodstream, although there is significant variability between people (Body 2, A and C). Within people, activated Compact disc4+ T cells.