Fig. 2

Effects of generalized immunosuppression on T splenocyte populations. a The figure illustrates the gating strategies employed to identify the distribution of T lymphocytes in the spleen. Debris was excluded, and lymphocytes included, using a forward scatter area (FSC-A) versus side scatter area (SSC-A) gate. Single cells (singlets) were then selected on a FSC-A versus FSC-W plot. This population was then analyzed in a FL1/SSC plot in order to quantify the percentage of CD3-APC positive cells. An FL2/FL1 plot was used to quantify the percentage of CD4-FITC and CD3-APC positive cells in the total live cell population. Similarly, an FL3/FL1 plot was used to quantify the percentage of CD8-PE and CD3-APC positive cells in the total live cell population. To determine Foxp3+CD25+ Tregs, CD3+CD4+ T cells were further gated on the SSC (FL2) for CD25-PE-Cy7 and FSC (FL1) for Foxp3-PerCP-Cy5.5. The cell population at Q2 are the % Foxp3+CD25+ /CD4+ Tregs. For each sample, 100,000 events were acquired. b Analysis of splenic T lymphocytes revealed that 3xTg-AD males treated with CY exhibited a significant increase in spleen-derived CD3⁺ T cells compared to vehicle-treated littermates (p < .001). This effect occurred independent of sex, as CY also normalized low CD3⁺ T cells in vehicle-treated 3xTg-AD females (p < .001). The loss of CD3⁺ T cells was more pronounced in 3xTg-AD males than females (p < .001), while a similar sex difference was not seen in WT controls. c CY partially restored low CD3⁺CD4⁺ T cell counts apparent in vehicle-treated 3xTg-AD males (p < .001) and females (p < .001). d CY also normalized low CD3⁺CD8⁺ T cells in the 3xTg-AD substrain (p < .001) such that levels were comparable to WT controls. e In addition to mitigating the loss of T effector cells, CY treatment abated the rise in the proportion of Foxp3+CD25+/CD4+ Tregs apparent in vehicle-treated 3xTg-AD males (p < .001) and females (p < .001). WT Veh males (n = 5–8), WT CY males (n = 5–11), 3xTg-AD Veh males (n = 5–7), 3xTg-AD CY males (n = 5–10), WT Veh females (n = 5–8), WT CY females (n = 5–10), 3xTg-AD Veh females (n = 5–6), 3xTg-AD CY females (n = 5–6). Overall group comparisons were carried out using three-way ANOVA (Genotype × Treatment × Sex) followed by post hoc t tests. Error bars = SEM, *p ≤ .05, **p < .01, ***p < .001