Longitudinal changes of T-cell functions in spleen as a representative organ in aging Eµ-TCL1 and WT mice and after AT. Fresh splenocytes from 3-, 6-, and 12-month-old WT and Eµ-TCL1 mice and from mice with full CLL after AT were stimulated for 6 hours with phorbol 12-myristate 13-acetate/ionomycin ± CD107a antibody in the presence of brefeldin/monensin for the last 5 hours of culture. Cells were then harvested, surface stained, fixed, permeabilized, and stained with antibodies against IL-2, IL-4, IFN-γ, and GrB. Unstimulated cells were used as controls and also stained with anti-ki-67 to assess proliferation. (A) Heat map summary of P values describing statistical differences in functional T-cell subsets. Groups were compared with the relative percentage of functional subsets in 3-month-old mice. Mann-Whitney test was used for nonnormally distributed data and unpaired t test for normally distributed data, as determined by Shapiro-Wilk normality test. Blue, relative expansion; red, relative loss of cell subsets; nd, no data; ns, nonsignificant. *P < .05, **P < .001, ***P < .0001. Percentages of (B) CD3⁺CD4⁺ and (C) CD3⁺CD8⁺ viable cells positive for IL-2, IL-4, and IFN-γ after mitogenic stimulation. Cells were gated based on unstimulated cell populations (supplemental Figure 1C). (D) Effector cell cytotoxicity was assessed by CD107a localization to the cell surface upon mitogenic stimulation. Naive/Ag-experienced viable CD3⁺CD8⁺ cytotoxic effector T-cell subsets were discriminated by expression of CD44. Unstimulated cells were used as controls. T-cell function was compared by calculating ratios of CD44⁺CD107a⁺ vs CD44⁺CD107a⁻ cells out of all CD3⁺CD8⁺ T cells to describe enrichment (increased ratio) or loss (decreased ratio) of effector cells within the CD44⁺ population (supplemental Figure 1D). Longitudinal changes of intracellular (E) GrB and (F) IFN-γ were described in a similar fashion. (G) To assess changes in the ability to form immune synapse, splenic T cells were mixed with 7-amino-4-chloromethylcoumarin–labeled, super Ag-pulsed healthy B cells at a 1:1 ratio, centrifuged onto polylysine-coated microscope slides, and F-actin was stained with rhodamine-phalloidin. Synapse formation between T and B cells was quantified by confocal laser-scanning microscopy using AxioVision image analysis software. The synapse area is depicted as median area of T-cell F-actin immune synapses (μm²) value. (H) Ex vivo proliferation of unstimulated CD3⁺CD8⁺ T cells was assessed by intranuclear ki-67 based on FMO controls and is given as a ratio among CD44⁺ cells as described in panel D. (I) In vivo proliferation of CD8⁺ and CD4⁺ T cells was assessed after injection with 100 µg/g body weight EdU 20 hours before euthanasia as percentage of EdU⁺ cells after gating on CD5⁺ CD19⁻ cells (ie, T cells). All graphs depict median ± interquartile range and combine data from all cohorts of mice at each age group.