Fig 1: IDO1-shRNA treatment decreases the expression of inhibitory receptors of CD4+/CD8+ T cells in vivo. Lymphocytes from lymph nodes and spleen of LLC-bearing tumor mice were collected on day 21 injection of LLC cells. CD4+ and CD8+ T cells were stained with anti-PD-1 and anti-BTLA and analyzed by flow cytometry. Expression of PD-1 and BTLA on CD4+ T cells in the (A) lymph nodes and (B) spleen of tumor-bearing mice without treatment, or treated with scrambled-shRNA or IDO1-shRNA. (C) Expression of PD-1 and BTLA on CD8+ T cells from the lymph nodes of tumor-bearing mice without treatment or treated with scrambled-shRNA or IDO1-shRNA. Statistical analysis of the expression of PD1 and BTLA on CD4+ (right panel in A and B) and CD8+ (right panels in C) was performed from three independent experiments. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; IDO1, indoleamine 2,3-dioxygenase 1; LN, lymph node; PD-1, programmed death-1; sh, short hairpin; SP, spleen.
Fig 2: Tumors increase inhibitory receptor expression in both CD4+ and CD8+ T cells in vivo. Tumor-bearing mice were inoculated subcutaneously with LLC cells, and lymphocytes were collected for analysis after 21 days. Mice without tumors were used as the control. (A) Flow cytometry analysis of CD4+/CD8+ lymphocytes from lymph nodes of CD3+ T cells in mice without tumors and mice with tumors. (B) Flow cytometry analysis of CD4+/CD8+ lymphocytes from the spleen of CD3+ T cells in mice without tumors and mice with tumors. (C and D) Percentage of PD1 and BTLA in CD4+/CD8+ T cells from the lymph nodes and the spleen in mice without tumors vs. mice with tumors. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; LN, lymph node; PD-1, programmed death-1; SP, spleen.
Fig 3: IDO1-siRNA in LLC cells inhibits T-cell exhaustion in vitro. (A and B) LLC cells were transfected with IDO1-siRNA or GL2-siRNA and co-cultured with lymphocytes isolated from spleen. Lymphocytes were collected and (A) PD-1 and (B) BTLA were detected by flow cytometry. n=3. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; GL2, glabra 2; IDO1, indoleamine 2,3-dioxygenase 1; PD-1, programmed death-1; siRNA, small interfering RNA; control, untransfected cells.
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