Single cell sequencing of the effects of blocking lymphotoxin alpha in the mesenteric lymph node and spleen

TNFDARE+/- mice develop TNF-driven transmural ileitis and mesenteric inflammation, and lose body mass, reminiscent of human Crohn’s disease. Here, we report that B cells regulate body mass and intestinal permeability during ileitis in a manner that is linked to lymphotoxin (LT)-a but not LTb, whereas B cell LTab2 promoted mesenteric TLS that suppressed lymphatic outflow. Using genetic approaches and neutralizing reagents, we connect LTa3, a ligand for TNFRs, expressed by activated follicular B cells to their appearance in draining lymph nodes and the generation or persistence of IgA+ ileal plasma cells. Some of the same B cells also expressed TNF. Opposite to LTa, expression of TNF by B cells was disadvantageous to the development of IgA+ ileal plasma cells, suggesting counterbalancing interplay between LTa3 and TNF in ileitis. These data highlight a multi-faceted role of B cells in ileitis that includes an unexpected role for B cell-derived LTa3 in the maintenance of body weight. We used single cell RNA sequencing to compare the immune cells that had trafficked from the ileum to either the mesenteric lymph node or the spleen under four conditions to understand how blocking lymphotoxin alpha homotrimer can alter the immune system. All mice had undergone a bone marrow transplant 30 weeks previously. Half had received WT bone marrow and half received bone marrow from TNFdARE mice, who have aberrantly stable tumor necrosis factor (TNF) mRNA and subsequently develop Crohn's disease-like ileal inflammation; both donors universally expressed the photoconvertible protein KikGR. A day prior to photoconversion, mice were dosed with either an anti-LTa3 antibody or an isotype control forming four groups: 1) WT mice given an isotype control, 2) WT mice given the anti-LTa3 antibody, 3) TNFdARE mice given an isotype control or 4) TNFdARE mice given the anti-LTa3 antibody. Surgery was performed on anesthetized mice to photoconvert haematopoietically derived cells in the ileum. The mesenteric lymph nodes and spleen were collected 20 hours later.