Fig 1: Divergent roles for NLRP3 in CD4+ and CD8+ T cells. a, b Extracellular and intracellular C5aR1, C5aR2, C5 and C5a in resting human CD8+ T cells. Freshly purified CD8+ T cells were evaluated for extra- and intracellular C5aR1 (green histograms), C5aR2 (red histograms), C5 and C5a expression by FACS (a) or confocal microscopy (b, scale bar = 7 µm). c Levels of C5aR1, C5aR2, and C5 expression in resting CD4+ and CD8+ T cells and C5a generation in activated CD8+ T cells (n = 3). d Effect of CD46 co-stimulation on NLRP3 expression in T cells. T cells from healthy donors were left non-activated (NA) or stimulated with the depicted antibodies and NLRP3 expression measured 60 h post activation (n = 4). Upper panels show a representative cytometry plot of NLRP3 expression and lower panels the corresponding quantifications (gray histograms, isotype controls; black histograms, non-activated cells; green, blue, or red histograms, CD3, CD3 + CD28, or CD3 + CD46 activation, respectively; gating strategy for Fig. 4a–d is shown in Supplementary Fig. 7b). e, f Effect of CD46 co-stimulation on IL1B gene transcription at 6 h post activation ((e) n = 2)) and IL-1ß secretion ((f) n = 4)) in CD4+ and CD8+ T cells at 60 h post activation (for an uncropped image of panel (e), see Supplementary Fig. 8). g Impact of MCC950 on IL-1ß and IFN-? production from CD46-activated T cells. T cells were CD3 + CD46-activated with or without MCC950 (10 µM) and cytokine production determined (60 h) (n = 4). h, i Effects of MCC950 on active caspase-1 generation and granzyme B expression in CTLs. CTLs were treated as in (g) and active caspase-1 ((h) n = 6 and granzyme B measured (i, n = 3). j TCR and CD46 activation fail to induce IL-18 in human CTLs (72 h post activation, n = 4). Error bars denote mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant; ctrl, control. Statistical analyses were performed using One-way ANOVA with Tukey Multiple Comparison test or the Paired Student’s t-test where appropriate