2 research outputs found

    Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells

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    T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR

    Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA?Sequencing

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    Objective:To obtain the comprehensive transcriptome profile of human citrulline?specific B cells from patients with rheumatoid arthritis (RA). Methods:Citrulline? and hemagglutinin?specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA?sequencing, and expression of key protein molecules was evaluated by aptamer?based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. Results:Citrulline?specific B cells, in comparison to citrulline?negative B cells, from patients with RA differentially expressed the interleukin?15 receptor ? (IL?15R?) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositive RA patients, the expression of IL?15R? protein was enriched in citrulline?specific B cells from the patients’ peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast?like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts. Conclusion:To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases