RNAseq Transcriptomic Analysis of Polysaccharide A (PSA)-responding CD4+ T cells in Mice

Polysaccharide A (PSA), a capsular carbohydrate from the commensal gut bacteria Bacteroides fragilis, has been shown to possess both potent T cell-dependent pro- and anti-inflammatory properties. PSA is able to induce abscess and adhesion formation in sepsis models, but can also inhibit asthma, inflammatory bowel disease (IBD) and experimental autoimmune encephalomyelitis (EAE) through MHCII-dependent activation of CD4+ T cells. Yet, despite decades of study, the ability of PSA to balance both these pro- and anti-inflammatory responses remains poorly understood. Here we utilized an unbiased systems immunology approach consisting of RNAseq transcriptomics, high-throughput flow cytometry and Luminex analysis to characterize the full impact of PSA-mediated stimulation of CD4+ T cells. We found that exposure to PSA resulted in the upregulation and secretion of IFN?, TNFa, IL-6 and CXCL10, consistent with an interferon responsive gene (IRG) signature. Importantly, PSA stimulation also led to expression of immune checkpoint markers Lag3, Tim3 and especially PD1, which were also enriched and sustained in the gut associated lymphoid tissue of PSA-exposed mice. Taken together, PSA responding cells display an unusual mixture of pro-inflammatory cytokines and anti-inflammatory surface receptors, consistent with the ability to both cause and inhibit inflammatory disease. Overall design: Splenic CD4+ T cells were stimulated with splenic antigen presenting cells (MHCII+) and 50 ug/ml purified Polysaccharide A (PSA) derived from the commensal bacterium Bacteroides fragilis for either zero or 7 days. CD4+ T cells were isolated after activation using magnetic beads and used as a source of mRNA for deep sequencing. There are three replicates in each group (zero stimulation, 7 days stimulation).