PMC

CD127 expression inversely correlates with T-bet expression in CD8⁺ T-cells. (A) Gating strategy for identifying CD8⁺ or CD4⁺ CD127⁺ T-cell memory subsets. Flow cytometry data shown were gated as follows: singlets, lymphocytes, Aqua Blue⁻ (live cells), CD14⁻CD16⁻CD19⁻, CD3⁺, CD8⁺ or CD4⁺, CD127⁺. A representative donor is shown. (B) The frequency of CD127⁺ CD8⁺ (black), or CD4⁺ (white) T-cells expressing T-bet or Eomes is shown. **p < 0.004.

Resting CD4⁺ T_reg cells express low levels of T-bet. (A) Gating strategy for identifying CD4⁺ and CD8⁺ T_reg T-cells. Flow cytometry data shown were gated as follows: singlets, lymphocytes, Aqua Blue⁻ (live cells), CD14⁻CD16⁻CD19⁻, CD3⁺, CD8⁺ or CD4⁺, and CD25⁺ FoxP3⁺. A representative donor is shown. (B) The frequency of CD8⁺ and CD4⁺ T_reg T-cells is shown. (C) The frequency of T-bet⁺ CD8⁺ and CD4⁺ T_reg T-cells is shown. (D) Graphical representation of the mean frequency of T-bet^hi (gray) and T-bet^lo (white) CD8⁺ and CD4⁺ T_reg T-cells is shown. The box and whisker graphs display 25–75% (box) and 10–90% (whisker). The line in the box represents the median value. *p < 0.04, **p < 0.004.

T-bet expression in antigen-experienced B-cells. (A) T-bet gating strategy for B-cell populations is shown. Transitional, naïve, memory B-cells, and plasmablasts populations are depicted as a contour plot overlaying a density plot of total B-cells. T-bet⁺ events are gated from a representative donor. (B) The frequency of T-bet⁺ B-cells within B-cell subpopulations is shown. Each symbol represents an individual subject. Statistical differences of interest, as measured using non-parametric Wilcoxon matched, paired two-tailed t tests, are described in the text. *p < 0.04. (C) Histograms depicting T-bet expression levels in B-cells and NK cells from a representative donor. Histograms represent the following subsets: naïve B-cells (thick black line), memory B-cells (thin black line), plasmablasts (shaded gray), CD56^bright NK cells (gray line), and CD56^dim NK cells (shaded black).

Co-expression of T-bet and Eomes in γδ T-cells and CD4⁻CD8⁻ iNKT cells. (A,B) Gating strategies for identifying (A) γδ T-cells and (B) iNKT cells. γδ T-cells and iNKT cells were gated as follows: singlets, lymphocytes, Aqua Blue⁻ (live cells), CD14⁻CD16⁻CD19⁻, CD3⁺, γδ TCR⁺ (γδ T-cells) or Vα24αJα18⁺ (iNKT cells). T-bet and Eomes expression within these cells from a representative donor is shown. (C) The frequency of T-bet expression in total γδ T-cells and iNKT cells is shown. Each symbol represents an individual donor. (D) Graphical representation of the mean frequency of T-bet^hi (gray) and T-bet^lo (white) expression in these populations is shown. The box and whisker graphs display 25–75% (box), 10–90% (whisker), and the median value (line). (E) The frequency of Eomes⁺ γδ and iNKT cells is shown for each cell subset. (F) Median fluorescence intensity (MFI) is shown for Eomes⁺ cells within each cell subset. (G) The frequency of T-bet and Eomes co-expression within each cell population is shown. *p < 0.04.

Distinct T-bet and Eomes expression patterns between CD56^bright and CD56^dim NK cells. (A) Gating strategy for identifying NK cell subsets. NK subpopulations were gated as follows: singlets, lymphocytes, Aqua Blue⁻ (live cells), and CD14⁻CD19⁻CD3⁻. Mature NK cell populations, CD56^bright (CD56^hi CD16⁻) and CD56^dim (CD56^lo CD16⁺), from a representative donor are gated. T-bet and Eomes expression in CD56^bright cells (red) and CD56^dim cells (blue) are plotted. (B) The frequency of T-bet expression in CD56^bright and CD56^dim NK cells is shown. Each symbol represents and individual donor. (C) Box and whisker graphical representation of the frequency of T-bet^hi (gray) and T-bet^lo (white) expression for each NK population is shown. The box and whisker graphs display 25–75% (box), 10–90% (whisker), and the median value (line). (D) The frequency of Eomes-expressing NK cells is shown. (E) Eomes MFI from Eomes⁺ cells from each NK population is shown. (F) Frequency of T-bet and Eomes co-expression within each subset. *p < 0.04.

T-bet and Eomes expression correlates with T_EM and effector CD8⁺ T-cells. (A) Gating strategy for identifying CD8⁺ T-cell subsets. Flow cytometry data shown were gated as follows: singlets, lymphocytes, Aqua Blue⁻ (live cells), CD14⁻CD16⁻CD19⁻, CD3⁺, CD4⁻, CD8⁺. Boolean gating of CD27, CCR7, and CD45RO was used to define CD8⁺ subsets from eight normal donors. Effector CD8⁺ T-cells from a representative donor are shown. (B) The frequency of T-bet⁺ CD8⁺ T-cells within naïve (CCR7⁺CD45RO⁻CD27⁺), T_CM (CCR7⁺CD45RO⁺CD27⁺), T_EM (CCR7⁻CD45RO⁺CD27⁻), and effector (CD27⁻CD45RO⁻CCR7⁻) cells is shown. Each symbol represents an individual donor. (C) Graphical representation of the mean frequency of T-bet^hi (gray) and T-bet^lo (white) CD8⁺ T-cells is shown for each memory subset. The box and whisker graphs display 25–75% (box) and 10–90% (whisker). The line in the box represents the median value. (D) The frequency of CD8⁺ T-cells expressing Eomes is shown for each subset. (E) Median fluorescence intensity (MFI) is shown for Eomes within each subpopulation. (F) Co-expression of T-bet and Eomes within each memory subset is shown. *p < 0.04, **p < 0.004.

T-bet and Eomes expression associates with T_EM and effector CD4⁺ T-cell populations. (A) Gating strategy for identifying CD4⁺ T-cell subsets. Flow cytometry data shown were gated as follows: singlets, lymphocytes, Aqua Blue⁻ (live cells), CD14⁻CD16⁻CD19⁻, CD3⁺, CD8⁻, CD4⁺. Boolean gating of CD27, CCR7, and CD45RO was used to define CD4⁺ subsets from eight normal donors. Effector CD4⁺ T-cells from a representative donor are shown. (B) The frequency of T-bet⁺ CD4⁺ T-cells within naïve (CCR7⁺CD45RO⁻CD27⁺), T_CM (CCR7⁺CD45RO ⁺CD27⁺), T_EM (CCR7⁻CD45RO⁺CD27⁻), and effector (CD27⁻CD45RO⁻CCR7⁻) cells is shown. Each symbol represents an individual donor. (C) Graphical representation of the mean frequency of T-bet^hi (gray) and T-bet^lo (white) CD4⁺ T-cells is shown for each subset. The box and whisker graphs display 25–75% (box) and 10–90% (whisker). The line in the box represents the median value. (D) The frequency of CD4⁺ T-cells expressing Eomes is shown for each subset. (E) Median fluorescence intensity (MFI) is shown for Eomes within each subpopulation. (F) Co-expression of T-bet and Eomes within each memory subset is shown. *p < 0.04, **p < 0.004.