No abstract

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8 T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-␥) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B ؉ CD8 ؉ T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.

Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in anti-viral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. Here we demonstrate that not only ECTV but also vaccinia virus and Lymphocytic Choriomeningitis virus induce CD4-CTL, but that the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that Major Histocompatibility Complex Class II molecules on CD11c + cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that anti-viral CD4-CTL and non-cytolytic T Helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment; and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors suggesting that further post-transcriptional regulation is required for CD4-CTL differentiation. Finally, using CRISPR-Cas9 deletion of Runx3 in CD4 T cells, we demonstrate that the development of CD4-CTL but not of classical Th1 CD4 T cells requires Runx3 following ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of post-transcriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTL) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTL require sustained antigen presentation and are induced by CD11c-expressing antigen presenting cells. Moreover, we show that CD4-CTL are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTL upregulate protein levels of the transcription factors ThPOK, Runx3 and GATA-3 post-transcriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents CD4-CTL but not of classical Th1 cells. These results advance our knowledge of how CD4-CTL are induced during viral infection.