Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) Luciferase-based cytotoxicity assay to validate the impact of selected HITs on FluTC-mediated MO3.13-A2-Luc killing. MO3.13-A2-Luc cells were transfected with IRG-specific pool of 30 siRNA (siTOOLs) and co-cultured with FluTC as described in . Graph indicates cytotoxicity/viability ratio normalized to Scr control. (B-C) Luminex-based cytokine analysis of the FluTC – MO3.13-A2-Luc cell co-cultures. MO3.13-A2-Luc cells transfected either with Scr siRNA or IRG-specific siRNA pool and (B) co-cultured with FluTC for 24 h or (C) cultured in CM. (B) MCP-1 and (C) IL-8 levels were depicted. (B) Each line represents an independent experiment; values indicate the average of (B) triplicates or (C) duplicates. (A) Representative data of at least 2 independent experiments with triplicates per sample. (A-C) Graphs show mean +/- SD. P-values were calculated using (A) two-tailed student’s t-test (B) Ratio-paired t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001. (D) Cytokine analysis to determine IFNγ, TNFα, FasL and TRAIL secretion by anti-CD3/CD28 activated FluTC. Values represent the mean ± SD of triplicates.

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Luciferase, Cytotoxicity Assay, Transfection, Cell Culture, Control, Luminex, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) FACS analysis to determine the surface expression of IFNG-R1, TNF-R1, TNF-R2, FAS, TRAIL-R1, and TRAIL-R2 in MO3.13-A2-Luc cells. Grey histograms represent the isotype control. (B) Impact of IRG knockdown on TRAIL-R2 surface expression in MO3.13-A2-Luc cells. Left panel: Representative overlay of histograms to compare TRAIL-R2 expression on IRG +/- MO3.13-A2-Luc cells. Right panel: Compiled data of mean fluorescence intensity (MFI) for TRAIL-R2 expression. (C) Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze TRAIL-induced apoptosis over 48 hours in IRG +/- MO3.13-A2-Luc cells. Incucyte® Cytotox-Red Dye was added to the transfected MO3.13 oligodendrocytes together with TRAIL treatment as an indicator of apoptosis. The graph shows total red object integrated intensity per well (RCU x µm²/Image). (D) Western blot analysis of total/cleaved caspase-3/8/9, TRAF2, phosho(p)/total TAK1, MKK4, JNK, NF-κB, pLKB1 and Bcl2 in IRG +/- MO3.13-A2-Luc cells upon 4 h treatment with TRAIL. Experiment was run in two separate blots each including Scr samples. Each HIT was shown in comparison to Scr sample analyzed in the same blot (Blot1: Scr, siSTK11, siKCNH8 and siABCA2; Blot2: Scr, siSLC1A3 and siCHRNA1). Representative data of (A & D) two, (B-C) three independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001).

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Expressing, Control, Knockdown, Fluorescence, Cytotoxicity Assay, Transfection, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Impact of SIK3 on TRAIL-mediated MO3.13-A2-Luc killing was determined by (A) Luciferase-based cytotoxicity assay as described in supp and (B) real-time cytotoxicity assay as described in . Representative data of (A) three (B) two independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01).

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Luciferase, Cytotoxicity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: Summary of molecular pathways involved in the downstream signaling of STK11/LKB1, KCNH8, ABCA2, SLC1A3/EAAT1 and/or CHRNA1/AChR-mediated immune resistance. Activation of caspase-3/-8/-9, MKK4, JNK1, NF-κB and expression of TRAF2, TRAIL-R2, IL-8 was experimentally depicted in this study, whereas the potential involvement of the other molecular components was deducted from the previous studies as described and referred in the results and discussion sections above. Colors of the lines depicting the regulatory networks are matched with colors of the IRG icons. Activating and inhibitory effects are indicated by arrows and blocked lines respectively. The figure was generated using biorender.com (Created in BioRender. Beckhove, P. (2026) https://BioRender.com/2adzain )

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Activation Assay, Expressing, Generated