High-dimensional analysis of effector CD4 T cell function following γHV68 infection in B6 and IL-10KO mice. High-dimensional analysis of effector CD4 T.

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High-dimensional analysis of effector CD4 T cell function following γHV68 infection in B6 and IL-10KO mice. High-dimensional analysis of effector CD4 T cell function following γHV68 infection in B6 and IL-10KO mice. Mass cytometric analysis of cells recovered from lungs of γHV68-infected B6 or IL-10KO mice harvested at 9 dpi, with cells subjected to pharmacologic stimulation for ICCS analysis using a 35-Ab panel (Table I). Files were normalized, with data gated on viable CD4 T cells, defined as 191Ir+193Ir+195Pt−152CD3ε+172CD4+174MHC II− events, where numbers indicate isotopic mass for each measured parameter. (A) CD4 T cells were imported into PhenoGraph and clustered on 33,022 total events (3669 events from each file) and 32 markers (excluding CD3, CD4, and MHC II; Table I), identifying 16 unique clusters portrayed on a tSNE plot. (B) All events from (A) are colored by CD44, IFN-γ, TNF-α, and IL-10 expression. (C) Events from (A) are colored based on their cytokine profile, stratified based on IFN-γ, TNF-α, and IL-10 expression. (D) Frequency of CD4 T cells in virally infected B6 and IL-10KO mice. (E) Comparison of CTLA-4, GITR, ICOS, Lag3, and PD-1 expression across CD4 T cells from B6 (top row) or IL-10KO (bottom row) mice, in which IFN-γ+ TNF-α+ CD4 T cells were identified by a green boundary line. Parameters with a different maximum scale value between B6 and IL-10KO are identified by italicized text and an asterisk. Data from virally infected lungs of B6 (n = 4) and IL-10KO (n = 5) mice harvested 9 dpi, with cells stimulated with PMA and ionomycin for 5 h prior to ICCS. Data for B6 mice were also included in Fig. 1, subjected to different clustering parameters (as outlined in Table I). Data show mean ± SEM with individual symbols denoting individual mice, with statistical analysis done by unpaired t test, corrected for multiple comparisons using the Holm-Sidak method. **p < 0.01, ***p < 0.001, ****p ≤ 0.0001. ns, not significant. Abigail K. Kimball et al. ImmunoHorizons 2019;3:94-109 Copyright © 2019 The Authors