Fig 1: CD73+ memory T cells are prone to differentiate into cells with a TRM phenotype(A and B) Freshly isolated memory T cells were activated in vitro by anti-CD3/CD28 Dynabeads for 4 days followed by culture with TGF-ß/IL-15 for 3 days. CD4 (A) and CD8 (B) T cells were analyzed by flow cytometry for the TRM-associated markers CD69, CXCR6, and CD103 in CD73+ and CD73- cells.(C–F) Freshly isolated human total T cells were activated and infected by GFP+ lentivirus containing RUNX2 shRNA (C, TR30021 as a control), RUNX2 cDNA (D, Lenti-Control as a control), RUNX3 shRNA (E, TR30021 as a control) or RUNX3 cDNA (F, pCDH as a control) and differentiated under TRM development conditions for 7 days. GFP+ cells were gated and analyzed for CD69 and CD103 expression.(G) Expression profile of 16 of 19 TRM core genes in the CXCR6+CD69+ and the CXCR6-CD69- CD4 T cell subsets that have the highest and the lowest CD73 expression, respectively. The remaining three genes (CX3CR1, S1PR5, and CRTAM) were undetectable and are not shown. qPCR results are shown as 2(-delta Ct) *10 -5.(H–K) Freshly isolated memory CD4 (H/J) and CD8 (I/K) T cells from young (<35 years, red symbol) and older (>65 years, black symbol) individuals were differentiated under 4 days of Dynabeads stimulation and 3 days of TGF-ß treatment. Expression of CD73, CD69, CXCR6, and CD103 were analyzed by flow cytometry; results are summarized as boxplots (H and I). Frequencies of CD73+ cells correlated with those of CD69+CXCR6+ cells for CD4 T cells (J) and CD103+ cells for CD8 T cells (K) as determined by Pearson’s correlation analysis.Data were compared by two-tailed paired or unpaired t test. One-way ANOVA was used for multi-group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figures S6 and S7.