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. 2022 Jan 14;219(2):e20210815. doi: 10.1084/jem.20210815

Figure 6.

Figure 6.

Exogenous C1q is anti-apoptotic, and human apCAF C1q increases viability of intratumoral CD4 T cells. (A) Immunofluorescence for C1qbp shows surface expression in human intratumoral CD4 T cells. Samples were analyzed by confocal microscopy with a 40× objective. Bars, 40 µm. MHCII+ CAFs, MHCII− CAFs, and intratumoral CD4+ T cells were FACS sorted from human lung tumors and analyzed for C1q by qPCR (n = 4 patients). Pt, patient. (B) CD4 T cells and CAFs were sorted from the same tumor fragment and co-cultured with aC1qbp-blocking antibody or isotype (ISO) control. Absolute (Abs.) numbers of live T cells were assessed by FACS with counting beads (n = 5 patients). (C) Peripheral blood (PB) CD4 T cells from a healthy donor were sorted and activated with aCD3/aCD2/aCD28 beads before undergoing serum starvation with or without purified C1q. Representative FACS plots of two experiments. (D) As in C, plus aC1qbp-blocking antibody or isotype control. Cumulative data of cell viability measured by Annexin V//dead stain by FACS (n = 6 donors). Representative FACS plots of caspase-3 activity. (E) CD4 T cells, total CAFs, and MHCII− CAFs were sorted from the same tumor fragment. T-CAFs were co-cultured with aC1qbp-blocking antibody or isotype control. T cell apoptosis was assessed using Annexin V and PI staining. Representative FACS plots and cumulative data are shown (n = 3 patients). (A–E) *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Error bars, mean ± SEM; one–tailed unpaired or paired t test. FSC-A, forward scatter-A; PBMC, peripheral blood mononuclear cell.