NF kappa B Regulator Bcl3 Controls Development and Function of Classical Dendritic Cells in Toxoplasma gondii

The atypical I{kappa}B family member Bcl3 associates with p50/NF-{kappa}B1 or p52/NF-{kappa}B2 homodimers in the nucleus, and positively or negatively modulates transcription in a context-dependent manner. In mice lacking Bcl3 globally or specifically in CD11c+ cells, Toxoplasma gondii infection is uniformly fatal and is associated with an impaired Th1 immune response. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, we investigated the role of Bcl3 specifically in cDCs in T. gondii infection in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. The conditional cDC Bcl3 KO was as susceptible to lethal T. gondii infection as the total Bcl3 KO and generated poor Th1 responses. Splenocyte single cell RNA seq in the model revealed defective Bcl3-dependent expression of genes involved in antigen processing. Consistent with this, soluble toxoplasma antigen presentation was impaired in Bcl3-deficient cDCs, and tetramer staining demonstrated defective T. gondii antigen-specific splenic CD4+ and CD8+ T cell responses in infected cDC Bcl3-/- mice. In vitro differentiation of bone marrow progenitors from wildtype and cDC Bcl3-/- mice using Flt3L, NOTCH and IFN-{gamma} stimulation recapitulated the defective Bcl3-dependent cDC antigen-presentation activity observed in vivo. Splenocyte single cell RNA seq also revealed the existence of a unique subpopulation of Zbtb46+LysM+ DC which exhibited Bcl3-dependent expansion after infection. We also detected cDCs coexpressing the monocytic markers CD64 and Ly6C (designated icDC1 and icDC2) mainly in infected spleen, which were less abundant in Bcl3flx/flx Zbtb46 cre mice. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infected mice, and shapes DC ontogeny. Author SummaryDendritic cells initiate immune responses against invading pathogens. As professional antigen presenting cells they process and present antigen via the major histocompatibility complex to T cells and thus activate them. Bcl3, an atypical member of the I{kappa}B family regulates the APC function of dendritic cells. In this study we show that expression of Bcl3 specifically in classical DCs is critical for host protection against a protozoan parasite, Toxoplasma gondii. Host protective proinflammatory mechanisms are compromised in mice deficient in Bcl3 in classical DCs leading to an elevated organ parasite load and eventually death of the infected animals. We also found the emergence of Bcl3-dependent hybrid DCs upon T. gondii infection, which have mixed phenotypic markers from DCs and monocytes. Antigen processing genes are significantly downregulated in Bcl3-deficient cDCs, which may account for defective cross presentation of T. gondii antigens. In an in vitro differentiation model, we showed that development of XCR1+cross presenting cDC1s is critically regulated by Bcl3. Overall, this study reveals the complexity of dendritic cell ontogeny and the role of Bcl3 in classical DC function in the context of Toxoplasma infection.