Viral infections often gain access to the body of their host by exploiting areas of natural vulnerability, such as the semipermeable surfaces of mucosal tissues which are adapted for adsorption of nutrients and other diffusible molecules. Once the microbes have crossed the epithelial barrier, they can disperse to other tissues where eradication may not be possible. The best opportunity for successful immune intervention is immediately after infection while the pathogen is confined to a localized area of the body. Cytotoxic T lymphocytes (CTL) which reside at the site where the infection begins can make an important contribution to immunity by reducing early dissemination of the infection. Because the lungs provide easy access points for many pathogens to enter the body, they require protection from many complementary mechanisms, including pathogen-specific cytotoxic T cells. In this study we show that an enduring response to pathogen-derived peptide antigens facilitates sustained surveillance of the lungs by pathogen-specific CTL during the recovery from influenza virus infection. Our studies show that these processed peptide antigens reinforce expression of two homing receptors (CD69 and CD103) which help recently activated virus-specific CTL colonize the lungs during a mild inflammatory response. We suggest that this requirement for prolonged antigen presentation to reinforce local CTL responses in the lungs explains why protective cellular immunity quickly declines following influenza virus infection and other viral infections that enter the body via mucosal tissues.

Pulmonary influenza infection causes prolonged lymph node hypertrophy while processed viral antigens continue to be presented to virus-specific CD8 T cells. We show that naïve, but not central/memory, nucleoprotein (NP)-specific CD8 T cells recognized antigenbearing CD11b + DC in the draining lymph nodes more than 30 days after infection. After these late transfers, the naïve CD8 T cells underwent an abortive proliferative response in the mediastinal lymph node (MLN), where large clusters of partially activated cells remained in the paracortex until at least a week after transfer. A majority of the endogenous NP-specific CD8 T cells that were in the MLN between 30 and 50 days after infection also showed signs of a continuing response to antigen stimulation. A high frequency of endogenous NP-specific CD8 T cells in the MLN indicates that late antigen presentation may help shape the epitope dominance hierarchy during reinfection.

CD134 and CD137 primed CD8 T cells mount powerful effector responses upon recall, but even without recall these dual costimulated T cells respond to signal 3 cytokines like IL-12. We searched for alternative signal 3 receptor pathways and found the IL-1 family member IL-36R. While IL-36 alone did not stimulate effector CD8 T cells, in combination with IL-12, or more surprisingly IL-2, induced striking and rapid TCR-independent IFN-γ synthesis. To understand how signal 3 responses functioned in dual costimulated T cells we showed that IL-2 induced IL-36R gene expression in a JAK/STAT dependent manner. These data help delineate a sequential stimulation process where IL-2 conditioning must precede IL-36 for IFN-γ synthesis. Importantly, this responsive state was transient, and functioned only in effector T cells capable of aerobic glycolysis. Specifically, as the effector T cells metabolized glucose and consumed O2, they also retained potential to respond through IL-36R. This suggests that T cells use innate receptor pathways like the IL-36R-axis when programmed for aerobic glycolysis. To explore a function for IL-36R in vivo we showed that dual costimulation therapy reduced B16 melanoma tumor growth while increasing IL-36R gene expression. In sum, cytokine therapy to eliminate tumors may target effector T cells, even outside of TCR specificity, as long as the effectors are in the correct metabolic state.

The cytokine gamma interferon (IFN-γ), with antimicrobial and immunoregulatory functions, can be produced by T cells following stimulation through their T cell receptors (TCRs) for antigen. The innate cytokines type 1 IFNs and interleukin-12 (IL-12) can also stimulate IFN-γ production by natural killer (NK) but not naive T cells. High basal expression of signal transducer and activator of transcription 4 (STAT4), used by type 1 IFN and IL-12 to induce IFN-γ as well as CD25, contributes to the NK cell responses. During acute viral infections, antigen-specific CD8 T cells are stimulated to express elevated STAT4 and respond to the innate factors with IFN-γ production. Little is known about the requirements for cytokine compared to TCR stimulation. Primary infections of mice with lymphocytic choriomeningitis virus (LCMV) demonstrated that although the elicited antigen-specific CD8 T cells acquired STAT4-dependent innate cytokine responsiveness for IFN-γ and CD25 induction ex vivo, TCR stimulation induced these through STAT4-independent pathways. During secondary infections, LCMV-immune CD8 T cells had STAT4-dependent IFN-γ expression at times of innate cytokine induction but subsequently expanded through STAT4-independent pathways. At times of innate cytokine responses during infection with the antigen-distinct murine cytomegalovirus virus (MCMV), NK and LCMV-immune CD8 T cells both had activation of pSTAT4 and IFN-γ. The T cell IFN-γ response was STAT4 and IL-12 dependent, but antigen-dependent expansion was absent. By dissecting requirements for STAT4 and antigen, this work provides novel insights into the endogenous regulation of cytokine and proliferative responses and demonstrates conditioning of innate immunity by experience.