Journal: bioRxiv

Article Title: Influence of age on functional memory T cell diversity

doi: 10.1101/2021.05.29.446296

Figure Lengend Snippet: (A) Transcription Factor-Regulatory Element-Target Gene (TF-RE-TG) networks in CD73 − (left) or CD73 + cells (right) were modeled as described in Suppl. Figure 3. Red and yellow nodes represent transcriptional factors (TF) or chromatin regulators (CR); the green nodes represent their target genes (TG) that are differentially expressed in CD73 + and CD73 − memory T cells. The size of TF nodes corresponds to the number of TF connections. ( B-D ): 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 was assessed. Results are compared by two-tailed paired t-test. N.S: not significant.

Article Snippet: To overexpress RUNX2 in human T cells, we purchased commercial RUNX2/GFP lentivector (Origene, Cat#: RC212884L4).

Techniques: Isolation, Infection, shRNA, Cell Culture, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Influence of age on functional memory T cell diversity

doi: 10.1101/2021.05.29.446296

Figure Lengend Snippet: (A and B) Freshly isolated memory T cells were activated in vitro by anti-CD3/CD28 Dynabeads for 4 days followed by culture with TGFβ /IL-15 for 3 days. CD4 (A) and CD8 (B) T cells were analyzed by flow cytometry for the T RM -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 T RM development conditions for 7 days. GFP + c ells were gated and analyzed for CD69 and CD103 expression. (G) Expression profile of 16 of 19 T RM 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 (<35y, red symbol) and older (>65y, black symbol) individuals were differentiated under 4 days of Dynabeads stimulation and 3 days of TGFβ treatment. Expression of CD73, CD69, CXCR6 and CD103 were analyzed by flow cytometry; results are summarized as box plots (H,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 ttest. One-way ANOVA was used for multi-group comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: To overexpress RUNX2 in human T cells, we purchased commercial RUNX2/GFP lentivector (Origene, Cat#: RC212884L4).

Techniques: Isolation, In Vitro, Flow Cytometry, Infection, shRNA, Expressing, Two Tailed Test