B Cells Are Not Required for Sarcoidosis Granuloma Formation in Vitro

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Abstract RATIONALE: Sarcoidosis is an inflammatory disorder characterized by non-caseating granulomas. Granulomas develop through the complex interplay of several immune cells. The role each cell type has in the formation and persistence of non-caseating granulomas remains poorly understood. Refractory cases of sarcoidosis may improve with rituximab treatment, which depletes B-cells. However, the role of B cells in non-caseating, sarcoidosis granuloma formation is unclear. We utilized an in vitro granuloma model to study the impact of B cell depletion on granuloma formation in sarcoidosis. We hypothesized that B cells are required for granuloma formation in sarcoidosis. METHODS: We obtained peripheral blood mononuclear cells (PBMCs) from people with sarcoidosis (n=19), not currently on therapy, and we either depleted their samples of CD19+ cells (B cells) with anti-CD19 antibodies or left them un-depleted (whole). Both populations of cells were exposed to either uncoated or purified protein (PPD)-coated, DAPI florescent beads. In vitro granuloma formation was evaluated by counting the number and measuring the size of granulomas formed on days 4 and 7 with an epifluorescence microscope. Secreted cytokines and RNA were collected on day 7. RESULTS: Approximately ∼32% (n=6) of donors with sarcoidosis produced in vitro granulomas in the presence of PPD-coated beads. By day 7, there was no difference between the number of granulomas formed in whole PBMC or CD19 depleted samples, however the granuloma size formed in CD19 depleted samples (PPD-uncoated) were larger in the presence of PPD-coated beads compared to respective controls. Absolute granuloma size with PPD-coated beads on day 7 between groups were no different (p=0.6). A subset of donors had increased granuloma size when CD19 cells were depleted (n=3), while others had decreased granuloma size (n=3). CONCLUSION: Our preliminary data suggests that B cells are not required for in vitro granuloma formation in sarcoidosis. While granuloma size is increased in the absence of CD19 cells, this difference between the two groups is lost when comparing absolute granuloma size because a subset of donors show increased in vitro granuloma formation, in the absence of CD19 cells, and others have decreased formation. B-cells are known to have both stimulating and inhibiting forms in granuloma formation; therefore this finding suggests that CD19 depletion is either removing B cells that are stimulatory or inhibitory to granulomatous inflammation. Identifying this subset in sarcoidosis would determine who might benefit most from specific therapies like rituximab and improve outcomes in these severe forms of sarcoidosis.

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