The goal of this project is to identify potential mechanisms by which CD44 acts as an immune checkpoint inhibitor in the setting of chronic virus infection and whether CD44 represents a prospective therapeutic target to reverse T cell dysfunction. Our studies using the LCMV (lymphocytic choriomeningitis virus) Cl13 chronic infection model show that Cd44 deficiency supports viral clearance rather than chronic infection. Virus- specific CD8 and CD4 T cells became multifunctional as measured by cytokine production, and downregulated PD-1 as well as several other inhibitory receptors. The effective anti-viral response was accompanied by 30% mortality and immune pathology, underscoring that a key brake on the immune response had been released. Cd44-deficiency exclusively in T cells or in hematopoietic cells did not prevent chronic Cl13 infection or T cell exhaustion. Notably, however, Cd44-deficiency only in non-hematopoietic cells promoted T cell responses that led to viral clearance. This outcome was not due to differences in the infectivity of Cl13 in WT vs Cd44-/- mice. Thus, our findings reveal an unrecognized, novel immune inhibitory function of CD44 that is independent of its direct roles in regulating T cells. To initiate studies of non hematopoietic cells in CD44- dependent immune regulation of the response to Cl13, we first focused on fibroblastic reticular cells (FRCs) since these cells are potent regulators of T cell immune responses and contribute to the immune suppression in the Cl13 model. We found that Cd44-/- FRCs had lower expression of Nos2 than WT FRCs after infection, and that independent of infection, Cd44-/- FRCs produced less NO than WT FRCs when stimulated by IFN-? and TNF?, indicating an FRC intrinsic regulatory role of CD44. Cd44-deficiency was also linked to dramatic downregulation Trim11, an E3-ubiquitin ligase that can block IFN-? production, which we found to be produced in higher levels by Cd44-/- FRCs. We did not find differences in expression of PD-L1, IL-7, or Ccl19/21 that could impair T cell responses. From these results, we hypothesize that during chronic infection, CD44 expression by FRCs promotes NO production and limits IFN-? production thereby contributing to inhibition of T cell responses, promoting T cell exhaustion, and supporting viral persistence. However, FRCs produce other inhibitory mediators which we have not yet tested for CD44-dependent regulation that could also impact the anti-viral response. Based on the differences WT and Cd44-/- FRCs we have identified to date, our goal is to determine whether CD44 expression by these cells plays a fundamental role or ancillary role in the regulating T cell responses, to identify the ligands of CD44 that regulate FRC suppressive function, and to identify signaling mechanisms that regulate FRC responses.
Many viruses and cancers survive by inhibiting the immune responses of T lymphocytes. We discovered a new molecular pathway involving CD44 that regulates immune responses but functions in cells that do not themselves mediate immune responses. This application proposes studies to better understand this pathway and determine it potential as a therapeutic target for treatment of chronic virus infections and cancer.