Fig 2: CD40L induces drug resistance in MCL cell lines.a, b Viability assays were performed after prestimulation of REC-1 and MAVER-1 cells with CD40L (100 ng/ml, 18 h) or H2O as a control and additional treatment with sotrastaurin (a, 3 µM) or ibrutinib (b, 400 nM) for 48 h. Viability was determined with the MTT assay. c REC-1 cells were treated with various drugs (Akti-1/2, 1 µM; NVP-BEZ 235, 25 nM; AZD8330, 500 nM; ERK inhibitor II, 10 µM; 2-Bromoaldisine, 1 µM) or DMSO for 20 min before stimulation with CD40L (100 ng/ml, 18 h) or H2O as a control and subsequent treatment with sotrastaurin (3 µM), ibrutinib (400 nM) or DMSO for additional 48 h. Viability was determined with the MTT assay (*p < 0.001; **p < 0.0001; ***p < 0.00001)

Fig 3: CD40L activates alternative NFκB signaling independently of the BCR pathwayThe MCL cell lines REC-1 a and MAVER-1 b were preincubated with CD40L (100 ng/ml) or H2O for 18 h and subsequently treated with either sotrastaurin (STN, 3 μM), ibrutinib (Ibru, 400 nM) or DMSO as a control for an additional 24 h. Cytoplasmic and nuclear protein extracts were analyzed by Western blot for the indicated proteins; “sh.” stands for a short and “l.” for a long exposure time for RelB detection. c L-929 cells were transfected with a CD40L expression plasmid or a control plasmid and co-cultured with REC-1 and MAVER-1 cells for 18 h. Subsequently, the cells were treated with sotrastaurin (3 µM), ibrutinib (400 nM) or DMSO for an additional 24 h, and whole cell lysates were analyzed by Western blot

Fig 4: Cangrelor regulates platelet aggregation and neutrophil recruitment. A Platelet surface expression CD40L. B Western blots of platelet lysates CD40L. C The number of neutrophils in BALF. D The levels of edema formation in the lung. Results are presented as means ± SD (n = 8). **p < 0.01, *** p < 0.001 compared to the sham control; ##P < 0.01, ###P < 0.001 compared to CLP

Fig 5: Juxtacrine signals influence the tSV output of TH synapses.a–k Normalized Synaptic Transfer (NST%) of various vesicular proteins in response to BSLBs presenting different juxtacrine signals. Please refer to Supplementary Fig. 1a for a representative gating strategy showing the identification of single BSLBs and T cells by flow cytometry. a TCR, b CD2, c CD28, d CD63, e CD81, f BST2, and g CD40L vesicular transfer to control BSLBs (dark green circles) and BSLBs presenting 100 molec./µm2 of each juxtacrine signals CD80/CD86/ICOSL (brown circles), CD58 (dark brown circles), PD-L1/PD-L2 (light brown circles), HIV-1 gp120 (light gray circles), and OX40L/4-1BBL (black circles). h–i Representative half-overlaid histograms showing CD40L transfer to BSLB with various juxtacrine signals and in the presence of either 200 (h) or 2000 (i) molec./µm2 of a-CD3e-Fab. j NST% of CD4 to control and HIV-1 gp120-presenting BSLBs. k Representative histograms showing the loss of CD4 on TH (bottom left panels and blue histograms). Data represents means ± SEM of n = 5 (a–g, j) biologically independent samples across three independent experiments. Normality was determined using Shapiro–Wilk test and statistical significance was determined by two-tailed Multiple t test with Holm–Šídák correction (a = 0.05) for the multiple comparisons of tSV transfer between control BSLBs and BSLBs decorated with different juxtacrine signals (a–g, j). Adjusted P values are shown next to each significant comparison.