LNSC regulation of immunity and infection

PROJECT SUMMARY Lymph nodes (LNs) are essential for the induction of adaptive immune responses. The organization and environment of LNs is coordinated by lymph node stromal cells (LNSCs), which include heterogenous populations of fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs). These cells provide a physical scaffold for immune cell migration and produce numerous signals to regulate migration, adhesion, localization, function, and survival of hematopoietic cells. Moreover, we discovered that foreign protein antigens are acquired and retained by LN LECs which enhances CD8+ T cell memory and protection against rechallenge, a process we termed antigen archiving. LNs also limit pathogen dissemination via the capture of viral particles by macrophages lining the subcapsular sinus. Remarkably, for several mosquito-transmitted RNA alphaviruses, including chikungunya virus (CHIKV), we found instead that this function is mediated by the scavenger receptor MARCO expressed by distinct subsets of LECs. Although LEC-mediated capture of lymph-borne alphavirus particles limits viral dissemination, this viral capture is associated with profound LN inflammation, interferon responses, and disorganization that impairs germinal center formation and the capacity of LECs to acquire and archive foreign antigen. The primary goals of this collaborative and interdisciplinary project are to define mechanistically how discrete LNSCs regulate viral dissemination to distal tissues and contribute to the development of antiviral immune responses. In addition, we will determine how alterations to specific LNSCs during viral infection influence protective immunity from both previous and subsequent antigenic challenges. A better understanding of the role of distinct LNSCs in the development of immune responses, and how viral infection alters these responses, could identify new ways to rationally tune immune responses in health and disease. In Specific Aim 1, we will use mice with genetic deficiencies in key CHIKV cell entry molecules, the type I IFN receptor, or Irf5 in specific LECs or FRCs, and single-cell RNA analyses, to define how viral interactions with discrete LNSCs, and LNSC responses to viral infection, limit viral spread in the LN and to distal sites. In addition, we will determine how viral interactions with discrete LNSCs regulate the development of antiviral B and T cell responses including the activation and differentiation of B cells, the avidity, neutralization capacity, and durability of the polyclonal anti-viral Ab response, and the priming, proliferation, and differentiation of virus- specific T cells. In Specific Aim 2, we will define how virus-specific interactions with LECs, MARCO, and virus- specific inflammatory responses impact antigen trafficking, acquisition, and archiving in vivo and in human LECs and LN explants in vitro. We also will elucidate how viral targeting of LNSCs alters responses to previously archived antigen and archiving of exogenous antigen post-infection. These innovative molecular, genetic, immunological, and in vivo approaches will provide new mechanistic insight into how LNSCs respond to viral infection and regulate immunity.