Extended Data Fig. 2: Phenotypic analyses of pMIG- and BATF-transduced CAR TILs. | Nature Immunology

Extended Data Fig. 2: Phenotypic analyses of pMIG- and BATF-transduced CAR TILs.

From: BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells

Extended Data Fig. 2

a, Retroviral transduction efficiencies for CAR and MSCV-IRES-eGFP retroviral expression plasmids, assessed as expression of Thy1.1 and GFP respectively. FMO, fluorescence-minus-one control. b, Histograms showing BATF (left), JUN (middle), and MAFF (right) expression after retroviral transduction of CD8+ T cells with the corresponding retroviral expression plasmids or pMIG empty-vector control, assessed by flow cytometry with antibodies to the endogenous proteins. c, Histograms showing CAR expression (assessed by staining for the Myc tag) in pMIG- (grey), BATF- (red), JUN- (sky blue) and MAFF- (orange) transduced CAR T cells. d-e, Replicate tumor growth experiments using B16F0-hCD19 (d) and MC38-hCD19 (e) tumor cells. 1×105 tumor cells were injected subcutaneously into the left flank of C57BL/6 mice at day 0 (D0) in 100 μl phosphate-buffered saline (PBS); 3×106 control pMIG-, JUN-, MAFF-, or BATF-transduced CAR T cells were adoptively transferred by retro-orbital injection at day 7. Tumor sizes were measured by caliper. f, Histograms showing expression of the indicated markers by each group of CAR TILs, assessed by flow cytometry. g, Left panels, Histograms showing expression of CD44, CD62L, CD127 and KLRG1. Middle panels, Overlaid contour plots of CD44 and CD62L (top) and CD127 and KLRG1 (bottom) in pMIG- (grey) and BATF- (red) transduced CAR TILs. Right panel, expression of the markers quantitated as MFI. h, Left panels, Histograms showing expression of TNF, IFNγ, granzyme B, and CD107a after resting in T cell media or after stimulation with PMA and ionomycin for 4 h. Right panel, expression of the markers quantitated as MFI. i, pMIG (n = 6) and BATF (n = 6). Quantitation of TCF1+ and TCF1– CAR TILs. Each circle in g, h, and i represents one mouse, and the bar graphs represent the mean ± standard error of mean (s.e.m.). Data in d-i were obtained from two independent biological experiments. Data in g, h, and i were analyzed by two-tailed unpaired Student’s t-test.

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