Extended Data Fig. 6: Inhibition of TNF-α signaling and the ROCK1/2 pathway quells TRM-driven intestinal inflammation.
From: Human organoids with an autologous tissue-resident immune compartment
a, Line graphs quantifying expression of predicted TNF-α targets associated with T cell activation and cytotoxicity, 48 h after treatment with 5 ng/ml EpCAM TCB in the presence of the TNF-α-neutralizing antibody Adalimumab or an isotype control. Left graph displays expression in CD4+ T cells and the right graph displays expression in CD8+ T cells. Two-tailed paired T-test. Data derived from three biological replicates. b, Luminex quantification of CCL2 chemokine in the supernatant of IIO cultures, 48 h after treatment with 5 ng/ml EpCAM TCB in the presence of the TNF-α-neutralizing antibody Adalimumab or an isotype control. Two-tailed paired T-test. Data derived from three biological replicates. c, Luminex quantification of TNF-α in the supernatants of IIO cultures, 24 h and 48 h after treatment with either 5 ng/ml EpCAM TCB or a non-targeting control. Data derived from three biological replicates. Mean and SD. d, Bihourly quantification of caspase 3/7 signal in organoid cultures treated with increasing concentrations of recombinant TNF-α cytokine for 40 h. TRM-containing IIO cultures treated with 5 ng/ml EpCAM TCB served as a positive control for caspase 3/7 induction. Technical triplicates representative of 2 independent biological replicates, mean and SD. e, CellProfiler assessment of TRM migration speed during 1 h live-imaging of IIO cultures, either in the presence of 10 µM ROCKi or vehicle control. Each dot represents a migrating cell tracked for more than 20 frames. Violin plots are a collation of duplicate videos from one IIO culture, n = 471 for vehicle and 546 for ROCKi treated. Similar results were observed in 3 biologically-independent IIO cultures. Bar shows mean. Two-tailed unpaired Mann-Whitney test. f, Line graphs quantifying expression of CD25, GzmB and perforin in CD4+ T cells (left graph) CD8+ T cells (right graph) isolated from IIO co-cultures 72 h after treatment with 5 ng/ml EpCAM-targeting T-cell bispecific antibodies in the presence of 10 µM ROCKi or vehicle control, as determined via flow cytometry. Two-tailed paired T-test, 3 biological replicates. g, Expression of intracellular TNF-α in CD4+ T cells (left graph) CD8+ T cells (right graph) isolated from IIO co-cultures 72 h after treatment with 5 ng/ml EpCAM-targeting T-cell bispecific antibodies in the presence of 10 µM ROCKi or vehicle control. Two-tailed paired T-test, 3 biological replicates, mean and SD h, Viability of T cells within IIO co-cultures 72 h after treatment with 5 ng/ml EpCAM-targeting T-cell bispecific antibodies in the presence of 10 µM ROCKi or vehicle control, as determined via flow cytometry. Cells were first gated based on CD3 expression and defined as viable via the live/dead dye Efluor780. n = 3 biological replicates, mean and SD.
