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.
Fig 2: CD73+ and CD73- memory T cells are governed by distinct transcription factor networks(A) Transcription factor-regulatory element-target gene (TF-RE-TG) networks in CD73- (left) or CD73+ cells (right) were modeled as described in Figure S3. Red and yellow nodes represent transcriptional factors (TFs) or chromatin regulators (CRs); the green nodes represent their target genes (TGs) that are differentially expressed in CD73+ and CD73- memory T cells. The size of TF nodes corresponds to the number of TF connections.(B–E) Freshly isolated human total T cells were activated and infected with GFP+ lentivirus containing RUNX2 shRNA (B) RUNX2 cDNA (C), RUNX3 shRNA (D), and RUNX3 cDNA (E) respectively. TR30021, pCDH, and Lenti-Control served as respective controls. Transduced cells were cultured for 7 days, before CD73 expression in gated GFP+ cells were assessed.Results are compared by two-tailed paired t test. N.S., not significant. See also Figure S5.
Supplier Page from OriGene Technologies for RUNX2 Human shRNA Plasmid Kit (Locus ID 860)