Figure 3. CX₃CR1 and CD62L expression identifies four distinct memory CD8⁺ T-cell populations.

(a–f) Naive CD45.1⁺ OT-I ^CX3CR1-GFP T cells (5 × 10²) were adoptively transferred into CD45.2 mice subsequently infected with AdOVA. (a) At 60 d.p.i., CX3CR1 expression was determined in CD45.1⁺ OT-I-derived KLRG1⁺ T cells, CD62L^lowCD127⁺CD44⁺ memory T cells (TEM) and CD62L^hiCD127⁺CD44⁺ memory T cells (TCM) isolated from the spleen. (b) Representative analysis of CD62L and GFP (CX₃CR1) expression in CD127⁺ CD44⁺ memory OT-I^CX3CR1-GFP T cells from a. (c,d) Four populations of CD127⁺ CD44⁺ memory OT-I^CX3CR1-GFP T cells after AdOVA infection separated by CD62L and GFP (CX₃CR1) expression levels were (c) analysed for GzmB expression or (d) sorted and analysed for IL-2 expression after PMA/ionomycine stimulation for 5 h. Unstimulated T cells from each population (empty circles) served as control. n=3 or 4 for each group; *P<0.05, **P<0.01, ANOVA. (e,f) Adoptive transfer of identical numbers (2 × 10³ cells) of FACSorted CD45.1⁺ cells from the four populations of memory T cells into CD45.2⁺ mice (n=4) that were subsequently infected with AdOVA. (e) At 8 d.p.i., total numbers of CD45.1⁺ T cells and (f) frequencies of CX₃CR1⁺ cells among CD45.1⁺ T-cell progeny was determined in the spleen. *P<0.05, ***P<0.001, ANOVA. (g) Representative analysis showing CD62L and CX₃CR1 expression in human CD45RO⁺CD3⁺CD8⁺ PBMCs. (h,i) Flow cytometric determination of expression of GzmB in the four cell populations stratified by CD62L and CX₃CR1 expression. ***P<0.001, ANOVA. (j) IL-2 expression in FACSorted cells from these four T-cell populations subjected to PMA/ionomycine stimulation for 5 h. **P<0.01 and ***P<0.001, ANOVA. Fluorescence-activated cell sorting (FACS) plots are representative for three (a–c) or five (g,h) independent experiments. Data from one of three independent experiments are show in c–f each dot represents T cells from one mouse. (i,j) Pooled data for T cells from n=6 individual donors.