Journal: bioRxiv
Article Title: Enhancing the iNKT cell immunotherapy platform by combining optimised CAR endodomains with novel iNKT engagers
doi: 10.1101/2025.10.23.683869
Figure Lengend Snippet: Higher proliferative potential and avidity of CD28z CAR-iNKT A. Structure of CARs with different co-stimulatory molecules sharing the same BCMA scFV-CD8spacer-CD8aTM design. B. Left: iNKT frequency in PBMC before and after iNKT microbead-based selection as identified after staining with the shown Abs and flow-cytometry. Right: cumulative data from eight different healthy donors. C. High transduction efficiency for all 5 CARs is shown in flow-cytometry histogram (left) and cumulative data for different donors (right; n=5). CAR staining was performed with anti-linker mAb. D. Surface expression of BCMA and CD1d detected by flow cytometry in MM1.S and H929 cells (left) and cytotoxic activity of BCMA CAR iNKT cells with different co-stimulatory molecules against MM1.S and H929 cells as compared to non-transduced (NT) iNKT cells are shown (representative of n=5 different donors). E. Immune synapse formation of BCMA CAR iNKT cells conjugated to MM1.S target cells. 3D reconstruction of z-stacks is shown and used to quantitate the distance of the centrosome (in green) to the synapse; distance < 3 µM = polarised centrosome (n=replicates of 3 different donors). F. Proliferative capacity of CD28z CAR-iNKT cells as compared to all other CAR- and NT iNKT cells in short-term assay in the presence of IL15 alone assessed by real-time IncuCyte®imaging (n=3 donors). G. Absolute numbers and fold expansion of CD28z CAR iNKT cells after stimulation with alphaGalCer-loaded C1R-CD1d cells as compared to all other CAR iNKT cells (n=3 donors). H. Left: CD28z CAR iNKT cell binding capacity on MM1.S myeloma cells under increasing levels of acoustic force as compared to all other CARs and NT iNKT cells; right: CAR-iNKT binding at rForce 1000pN (data representative of n=2 donors, datapoint shown for individual run). I. Avidity of CAR-iNKT cells against MM1.S myeloma cells as assessed under shear stress by flow chamber assay. From left: Representative image of cell binding at 24 dyne/cm , cell binding efficiency at different shear tress levels, cumulative data for cell binding efficiency at 24dyne/cm (data representative of n=4 donors). ns-not significant, * p<0.05, ** p<0.01, **** p< 0.0001.
Article Snippet: iNKT cells were isolated from healthy donor-derived peripheral blood mononuclear cells (PBMCs) sourced from NHS Blood and Transplant (NCI licence no. NCI2758) using anti-iNKT microbeads (cat no. 130-094-842; Miltenyi Biotec) followed by stimulation with irradiated PBMCs and anti-CD3 and anti-CD28 antibodies (cat no. 130-091-441; Miltenyi Biotec) and cultured in R10 media (RPMI 1640 supplemented with 10% fetal bovine serum and 1% Pen/strep, 4mM L-Glutamine, 1% non-essential amino acids, 1mM sodium pyruvate, 15mM Hepes buffer, 0.05mM 2-Mercaptoethanol) supplemented with 150U IL-15 (cat. no 130-095- 764; Miltenyi Biotec) and expanded as described , .
Techniques: Selection, Staining, Flow Cytometry, Transduction, Expressing, Activity Assay, Imaging, Binding Assay, Shear, Boyden Chamber Assay
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