The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b+Ly6C+Ly6G- monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b+Ly6C+Ly6G+ cells. The phenotype of the CD11b+Ly6C+Ly6G+ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b+Ly6C+Ly6G+ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b+Ly6C+Ly6G+ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G+ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b+Ly6C+Ly6G+ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction.

Background: Antigen-specific CD4 T cells are activated by small numbers of antigenic peptide-MHC class II (pMHC-II) complexes on dendritic cells (DCs). Results: Newly generated pMHC-II complexes are present in small clusters on the DC surface. Conclusion: pMHC-II clusters permit efficient T cell activation. Significance: The appearance of clustered pMHC-II reveals the organization of the T cell antigen receptor ligand on the DC surface.

IFN regulatory factor (IRF)3 plays a detrimental role in the cecal ligation and puncture (CLP) mouse model of sepsis. However, it is unclear which pathway activates IRF3 in this context. In this report, we investigate two pathways that activate IRF3: the Stimulator of Interferon Genes (STING) pathway (that senses cytosolic DNA) and the TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway (that senses dsRNA and LPS via Toll-like receptor 3 and 4). Initially, we examine the impact of these pathways using a severe CLP model (∼90% mortality). Both STING-KO and TRIF-KO mice are protected from severe sepsis, exhibiting reduced mortality, disease score, hypothermia, and inflammatory cytokines relative to WT counterparts. STING/TRIF-DKO mice exhibit a similar phenotype to each of the single KO strains, suggesting that these pathways have an interrelated function. Subsequently, we examine the impact of these pathways using a moderate CLP model incorporating clinical treatments (Lactated Ringer Solution and antibiotics, ∼36% mortality). In this case, STING-KO mice show a similar phenotype to WT counterparts, while TRIF-KO mice show improved disease score and hypothermia. During sepsis, innate immune receptors recognize bacterial ligands and host-derived danger signals, including cell-free DNA released into the circulation. We show that IRF3 is activated in cultured macrophages treated with bacteria derived from the mouse cecum, dependent on TRIF, and in macrophages treated with mouse genomic DNA/Lipofectamine 2000, dependent on STING. Together, our data demonstrate that both the STING and TRIF pathways can promote sepsis pathogenesis; however, their contribution depends on the severity of the disease model. We show that bacteria are abundant in the peritoneum following both severe and moderate CLP, while cell-free DNA is more highly elevated in the serum following severe CLP compared with sham and moderate CLP. Hence, the presence of bacteria and cell-free DNA may explain the variable phenotypes in our severe CLP model (dependent on TRIF and STING) versus our moderate CLP model (dependent on TRIF only).

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

Circadian rhythms coordinate an organism's activities and biological processes to the optimal time in the 24-h daylight cycle. We previously demonstrated that male C57BL/6 mice develop sepsis more rapidly when the disease is induced in the nighttime versus the daytime. In this report, we elucidate the mechanism of this diurnal difference. Sepsis was induced via cecal ligation and puncture (CLP) at zeitgeber time (ZT)-19 (2 am) or ZT-7 (2 pm). Like the males used in our prior study, female C57BL/6 mice had a worse outcome when CLP was induced at ZT-19 versus ZT-7, and these effects persisted when we pooled the data from both sexes. In contrast, mice with a mutated () gene had a similar outcome when CLP was induced at ZT-19 versus ZT-7. Bone marrow chimeras reconstituted with C57BL/6 immune cells exhibited a worse outcome when sepsis was induced at ZT-19 versus ZT-7, whereas chimeras with -mutated immune cells did not. Next, murine macrophages were subjected to serum shock to synchronize circadian rhythms and exposed to bacteria cultured from the mouse cecum at 4-h intervals for 48 h. We observed that IL-6 production oscillated with a 24-h period in C57BL/6 cells exposed to cecal bacteria. Interestingly, we observed a similar pattern when cells were exposed to the TLR2 agonist lipoteichoic acid. Furthermore, TLR2-knockout mice exhibited a similar sepsis phenotype when CLP was induced at ZT-19 versus ZT-7. Together, these data suggest that circadian rhythms in immune cells mediate diurnal variations in murine sepsis severity via a TLR2-dependent mechanism.

IFN regulatory factor 3 (IRF3) is a transcription factor that is activated by multiple pattern-recognition receptors. We demonstrated previously that IRF3 plays a detrimental role in a severe mouse model of sepsis, induced by cecal ligation and puncture. In this study, we found that IRF3-knockout (KO) mice were greatly protected from sepsis in a clinically relevant version of the cecal ligation and puncture model incorporating crystalloid fluids and antibiotics, exhibiting improved survival, reduced disease score, lower levels of serum cytokines, and improved phagocytic function relative to wild-type (WT) mice. Computational modeling revealed that the overall complexity of the systemic inflammatory/immune network was similar in IRF3-KO versus WT septic mice, although the tempo of connectivity differed. Furthermore, the mediators driving the network differed: TNF-a, IL-1b, and IL-6 predominated in WT mice, whereas MCP-1 and IL-6 predominated in IRF3-KO mice. Network analysis also suggested differential IL-6-related inflammatory programs in WT versus IRF3-KO mice. We created bone marrow chimeras to test the role of IRF3 within leukocytes versus stroma. Surprisingly, chimeras with IRF3-KO bone marrow showed little protection from sepsis, whereas chimeras with IRF3-KO stroma showed a substantial degree of protection. We found that WT and IRF3-KO macrophages had a similar capacity to produce IL-6 and phagocytose bacteria in vitro. Adoptive transfer experiments demonstrated that the genotype of the host environment affected the capacity of monocytes to produce IL-6 during sepsis. Thus, IRF3 acts principally within the stromal compartment to exacerbate sepsis pathogenesis via differential impacts on IL-6-related inflammatory programs.

No abstract

Antiviral CD8+ T cell recognition of MHC Class I-peptide complexes on the surface of professional APCs is a requisite step in an effective immune response following many potentially lethal infections. Although MHC Class I-peptide production is thought to be closely linked to the continued presence of virus, several studies have shown that the persistence of antigen presentation occurs for an extended period of time following the clearance of RNA viruses. However, the mechanism responsible for antigen presentation persistence following viral clearance was unknown until now. Here, we utilized a recombinant DNA virus expressing different forms of a model antigen to study the mechanism of prolonged antigen presentation in mice. We determined that the persistence of antigen presentation consists of three distinct mechanistic phases: ongoing viral replication, persistence of virally infected cells, and cross presentation of antigen. These data will allow manipulation of the form of antigen contained within viral vectors to produce the most effective and protective CD8+ T cell response to be generated following vaccination.