Figure 1. Multiple human circulating memory T cell subsets can generate T_RM in vitro.

(A) The gating strategy used for sorting of human peripheral blood CD45RA⁻ memory T cells into purified populations of CCR7⁻L-selectin⁻ effector memory T cells (T_EM), CCR7⁺L-selectin⁻ migratory memory T cells (T_MM) and CCR7⁺L-selectin⁺ central memory T cells (T_CM) is shown. (B) Experimental strategy for the study of T_RM generation in vitro. Sorted purified T cell subsets from human blood were stimulated for 24 hours with an anti-CD3/CD2/CD28 beads, cultured on monolayers of keratinocytes, fibroblasts or on tissue culture plastic and analyzed by flow cytometry. (C) Seven days of culture on keratinocyte monolayers generated single positive CD69⁺CD103⁻ and double positive CD69⁺CD103⁺ T cells (left panel), similar in phenotype to those observed in healthy human skin (right panel). (D, E) Time course of CD69 and CD103 acquisition in vitro after culture on keratinocyte monolayers. A representative culture is shown; similar results were obtained in a total of four donors. (F-I) All memory subsets tested gave rise to CD69⁺ T cells, some of which also upregulated CD103. Results for CD4 (top) and CD8 (bottom) T cells are shown after culture on tissue culture plastic (plastic), keratinocyte monolayers (ker) or fibroblast monolayers (fib). The mean and SEM of at least six donors are shown. (J, K) Culture on keratinocyte monolayers generated increased numbers of CD4 (J) and CD8 (K) T cells expressing T_RM markers. The mean and SEM of a minimum of four donors are shown. (L) CD4⁺ and CD8⁺ T cells were equally efficient at up regulating CD69 (left panel) but CD8⁺ T cells were more efficient at generating double positive CD69⁺CD103⁺ T cells. The mean and SEM of at least four donors cultured on keratinocyte monolayers are shown. (M,N) Naïve T cells did not upregulate T_RM markers in vitro. CD4⁺ (M) and CD8⁺ (N) CD45RO⁻CD45RA⁺ naïve T cells were bead stimulated and cultured on keratinocyte monolayers then analyzed by flow cytometry. Similar results were obtained in a total of three naïve donors. Naïve T cells produced significantly fewer CD69⁺ CD103⁺ T cells (right panels); the mean and SEM of three naïve donors and six memory donors are shown. Significance was determined by a one-way ANOVA and Tukey’s post-hoc test for multiple comparisons (F-K, M,N) or by two-tailed T-tests (L).