Characterization of the role of viral sensing and migratory capacity in regulatory T cells after neurotropic viral infection

Abstract

Regulatory T cells (Tregs) are recognized as suppressors of autoimmunity, yet Tregs also play an important although variable role in the development of an immune response to infection. The mechanism of Treg sensing of infection and migration to immune activation sites during viral infections is not yet clearly defined. Furthermore, given the rapid spread of flaviviruses such as West Nile virus (WNV) and Zika virus, it is critical that we develop a thorough and complete understanding of the key mediators of an effective anti-flaviviral response. One key family of viral sensors, the RIG-I-like receptors, are known to signal through the adaptor protein mitochondrial antiviral-signaling protein or MAVS. MAVS-null mouse models experimentally infected with WNV display signs of grossly dysregulated immunity, elevated viral titers, and death that correlate with a failure of Treg expansion following infection. Thus, we sought to determine if intrinsic MAVS signaling is required for participation of Tregs in anti- WNV immunity. Despite evidence of increased Treg cell division indicated by Ki67 expression, FoxP3 expression was down-regulated after WNV infection in MAVS-deficient mice. Additionally, we observed the successful generation of an effective anti-WNV immune response in both the secondary lymphoid organs as well as the central nervous system when Tregs lacked MAVS, thereby demonstrating that Treg detection of the presence of WNV through the RIG-I like receptor/MAVS signaling pathway is not required for the generation of effective immunity. Together, these data suggest that while MAVS signaling has a considerable impact on Treg identity, this effect is not mediated by intrinsic MAVS signaling but rather is likely an effect of the overproduction of pro-inflammatory cytokines generated in MAVS- deficient mice after WNV infection. We also investigated the effect of Treg migratory marker expression both at steady state and on the development of an effective WNV-directed immune response. We utilized an ITGb1Flox x FoxP3Cre conditional knockout mouse (CKO) model to generate Tregs that lack the ability to express β1-integrin, a transmembrane protein reported to be necessary for migration to sites of inflammation and the central nervous system. Through flow cytometric analyses, we found that wild type (WT) Treg expression of β1-integrin correlated with an activated, migratory phenotype as indicated by increased surface expression of CD44, CXCR3, and CCR5. In addition, we found that when the balance of β1-integrin +/- Tregs is altered, the expression of suppressive proteins such as CD73 and CTLA4 is significantly altered. Analysis of the CD8+ T cell compartment within the CKO revealed that a greater percentage of CD8+ T cells displayed signs of activation including increased CD44, CXCR3 and Ly6C expression at steady state as compared to WT controls. When these mice were infected with WNV, CKOs and WT controls generated similar frequencies of WNV-specific CD8+ T cells in response, both at peak of the infection in the spleen and when assessed at an early memory time point. However after a second exposure to WNV, expansion of CKO-derived WNV-specific CD8+ T cells was greatly diminished despite significant proliferation of non-WNV-specific CKO-derived CD8+ T cells in the same mouse. In sum, these data suggest that Treg migratory capability is critical for the maintenance and development of a functional CD8+ T cell response both at steady state and after an infectious challenge. Through our investigations of Treg function, both in viral sensing and migratory function, we sought to further the understanding of Tregs as key players in responding to infection and in maintaining homeostasis. Although MAVS signaling was found to be dispensable for Treg function during WNV infection, this finding extends previous work and answers a lingering question in the field. In addition, our work on β1-integrin on Tregs suggests a potential novel function for an integrin that was previously attributed a minor role in migration. As such, the following report is intended to document these findings and place them within the context of current immunological knowledge.