Journal: Science immunology

Article Title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4 + T cells to drive polyfunctionality and antitumor immunity

doi: 10.1126/sciimmunol.aba5962

Figure Lengend Snippet: The schema depicts the timeline of the experimental procedures. A20WT tumor cells were subcutaneously inoculated to mice. When tumor sizes reached 170 mm2, mice were randomly grouped and received either no treatment (No Tx), CTX only, CTX + control CD19CAR T cells, or CTX + CASTAT5 CD19CAR T cells. (A) Expression of CAR and CASTAT5 in transduced T cells. Total T cells purified from CD45.1 mice were transduced to express CD19CAR alone or co-express CD19CAR and CASTAT5. Representative dot plots show the levels of CD19CAR and CASTAT5 in transduced T cells. CD19CAR was measured by anti-Fab stain and CASTAT5 was evaluated by Thy1.1 expression. (B) Tumor growth curves of each mouse under each condition are shown. The numbers indicate the number of tumor-free mice among treated mice at the end point. (C) Relapsed tumors did not lose CD19 expression. Mice treated with CD19CAR T cells had initial tumor regression followed by relapse. Relapsed tumors were resected and processed into single cell suspension for evaluation of CD19 expression by flow-cytometry. Tumors from untreated mice were used for comparison. Representative dot plots shown represent the co-stain of CD19 and B220. (D) Frequencies of host B cells in blood. At the indicated time points, tail blood was collected and the presence of host B cells in blood was evaluated by CD19 and B220 co-stain. (E) Detection of CD19CAR viral vector in bone marrow by PCR. 30 days after T cell transfer, a cohort of mice were killed and bone marrow aspirates were collected. The presence of CD19CAR T cells was evaluated by PCR detection of the CD19CAR viral vector. (F) Mice cured by CTX+CASTAT5 CD19CAR T cells were resistant to A20WT re-challenge. Live A20WT tumor cells were subcutaneously injected to cured mice and a group of naive mice used as controls. Tumor sizes in mice 3 weeks after tumor inoculation are shown.

Article Snippet: CD26-CD19 cells were generated by transfecting cells with a vector encoding murine CD19 (pCMV3-mCD19, SinoBiological Inc.) using Lipofectamine 2000.

Techniques: Expressing, Purification, Staining, Flow Cytometry, Plasmid Preparation, Injection

Journal: Science immunology

Article Title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4 + T cells to drive polyfunctionality and antitumor immunity

doi: 10.1126/sciimmunol.aba5962

Figure Lengend Snippet: (A) Viral transduction efficiency in CD4+ and CD8+ T cells. CD4+ and CD8+ T cells were separately purified from CD45.1 mice. These cells were transduced to express CD19CAR alone or co-express CD19CAR and CASTAT5. Representative dot plots show the levels of CD19CAR and CASTAT5 in transduced T cells. CD19CAR was measured by anti-Fab stain and CASTAT5 was evaluated by Thy1.1 expression. The numbers in plots indicate the percentage of cells in the corresponding quadrant. (B) Experimental setup and treatment conditions. The schema depicts the experimental procedures. The different combinations of engineered CD4+ and CD8+ CD19CAR T cells used for adoptive transfer are outlined in the chart. (C) The frequencies of donor CD8+ T cells in recipient mice during the course of CD19CAR T cell therapy. At the indicated time points, tail blood was collected from mice and subjected to flow cytometry to detect the donor CD8+ T cells (CD45.1+CD8+). The frequencies of donor CD8+ T cells were plotted against time. The frequencies of host B cells (CD19+B220+) in blood were also evaluated by flow cytometry and summarized in (D). (E) Tumor growth curves of mice in each group. The numbers indicate the number of tumor-free mice among treated mice at the end point of experiment. (F and G) Phenotypic characterization of donor CD4+ and CD8+ CD19CAR T cells in selected groups. Ten days after T cell transfer, splenocytes isolated from a cohort of mice from the specified groups (3 mice/group) were subject to flow cytometry analysis. Splenocytes from groups V and VI were stimulated with PMA/ionomycin or A20 tumor cells for ICS to examine the cytokine profiles of control CD4+ and CASTAT5 CD4+ CD19CAR T cells (F). Numbers in representative dot plots indicate percentage of cells in the corresponding quadrant. Donor CD8+ T cells from groups I, IV and VI, which expressed CD19CAR alone, or co-expressed CASTAT5 and CD19CAR in the absence or presence of CASTAT5 CD4+ CD19CAR T cells, respectively, were examined for expression of the specified markers. The frequencies of cells positive for each of the markers and the mean fluorescence intensity (MFI) of these markers are summarized in bar graphs shown as mean ± s.d. of five samples per group. (G). n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: CD26-CD19 cells were generated by transfecting cells with a vector encoding murine CD19 (pCMV3-mCD19, SinoBiological Inc.) using Lipofectamine 2000.

Techniques: Transduction, Purification, Staining, Expressing, Adoptive Transfer Assay, Flow Cytometry, Isolation, Fluorescence