RORc expressing immune cells negatively regulate tertiary lymphoid structure formation and support their pro-tumorigenic functions
Tertiary lymphoid structures (TLSs) are formed in many cancer types and have been correlated with better prognosis and response to immunotherapy. In liver cancer, TLSs have been reported to be pro-tumorigenic as they harbor tumor progenitor cells and nurture their growth. The processes involved in TLS development and acquisition of pro- or anti-tumorigenic phenotype in cancer are largely unknown. RORc expressing immune cells have been previously implicated in TLS formation, however we find that they are not necessary for TLS neogenesis in the context of liver inflammation. On the contrary, RORc expressing cells negatively regulate TLS formation, since in their absence TLSs form in excess. CD4 cells are essential for liver TLS formation whereas B cells are required for TLS formation specifically in the absence of RORc expressing cells. Importantly, in chronically inflamed livers lacking RORc expressing cells, TLSs become anti-tumorigenic, resulting in reduced tumor load. Comparing liver pro- and anti-tumorigenic TLSs by transcriptional, proteomic and immunohistochemical analyses, revealed enrichment of exhausted CD8 cells that retained effector functions as well as germinal center B cells and plasma cells in anti-tumorigenic TLSs. Cell depletion experiments revealed a role mainly for B cells in limiting tumor development, possibly via tumor directed antibodies. Thus, RORc expressing cells negatively regulate B cell responses, and facilitate the pro-tumorigenic functions of hepatic TLSs. Overall design: The goal of this experiment was to analyze the composition and phenotype of immune cells from hepatic tertiary lymphoid structures (TLSs). Whole body RORc knock out mice with chronically inflammed livers were compared to controls.  The genotypes compared were: Alb-IKKß(EE)Hep;RORctm2Litt +/- (termed: IKK RORc Het or control) and Alb-IKKß(EE)Hep;RORctm2Litt -/- (termed: IKK RORc KO or Experiment). CD45+ immune cells were isolated from livers of 6 months old DEN-injected mice. Immune cells were isolated from 3 IKK RORc Het mice (cells were pooled after isolation), and from 2 IKK RORc KO mice.