In This Issue

Numerous studies have demonstrated modulation of immune responses to pathogens by sex steroid hormones, but sex-dependent effects on susceptibility to Staphylococcus aureus skin and soft tissue infection (SSTI) have not been investigated. Analysis of health records of patients receiving treatment for S. aureus SSTI by Castleman et al. (p. 657) revealed an increased incidence of infection in males compared with females. Consistent with these data in humans, female mice s.c. infected with a highly virulent strain of S. aureus (LAC) showed significantly reduced dermonecrosis, reduced local levels of IL-1β, TNF-α, IL-6, and CXCL1, and increased bacterial clearance when compared with LAC-infected males. This innate resistance was dependent on estrogen, as LAC-infected ovariectomized mice displayed increased dermonecrosis and local inflammatory cytokine levels, which were restored by short-term administration of estrogen to levels similar to those observed in sham-operated controls. Sex bias was largely driven by differential responses to the virulence factor α-hemolysin (Hla); local inflammatory cytokines and bacterial clearance increased in female mice infected with an Hla-deficient strain of S. aureus when compared with LAC-infected females. Furthermore, transcription of nlrp3 and Il-1β were significantly reduced at the site of LAC infection in females relative to infected males, suggesting that sex bias observed in S. aureus–infected animals may be attributed to sex-dependent regulation of Hla-driven NLRP3 inflammasome activation. Sex-based differences were also observed following injection of Hla alone. Finally, neutrophils from females demonstrated significantly elevated S. aureus bactericidal activity in vivo and ex vivo, indicating an additional sex-dependent factor that may contribute to the innate sex bias in S. aureus SSTI. Taken together, these data suggest that estrogen-dependent differences in both neutrophil bactericidal activity and innate responsiveness to Hla play important roles in limiting SSTI in females, further highlighting the contributions of gender to infectious disease susceptibility and severity.

Low-affinity Fc γ receptor type IIIA (FcγIIIA or CD16a), which is encoded by the gene FCGR3A, is expressed during the final stage of NK cell maturation and is critical for Ab-dependent cell-mediated cytotoxicity by NK cells. Although the function of CD16a in NK cells has been well characterized, much less is known about epigenetic mechanisms controlling transcriptional and posttranscriptional regulation of CD16 expression. Victor et al. (p. 565) compared DNA methylation and expression patterns of FCGR3A and FCGR3B (which is expressed in neutrophils and has a genomic sequence nearly identical to that of FCGR3A) in purified CD16a⁺ and CD16a⁻ NK cells. They found greatly diminished DNA methylation in the FCGR3A medial promoter-1 (Pmed1-A) of CD16a⁺ relative to CD16a⁻ NK cells. Assays in which plasmid luciferase expression in transfected NK or myeloid cells was driven by the variant promoters of FCGR3A and FCGR3B showed that he Pmed1-A region of FCGR3A was only activated in NK cells. This activity was abolished following in vitro methylation of Pmed1-A sequences, indicating that the activity of FCGR3A is cell type specific and methylation dependent. Alignment of Pmed1 sequences of FCGR3A and FCGR3B showed that FCGR3A has more CpG dinucleotides and mutations in these CpG motifs abolished the activity of Pmed1-A in NK cells. Additionally, the authors identified three potential microRNAs that may regulate the activity of FCGR3A. Luciferase assays revealed that miR-218 expression negatively regulated the activity of FCGR3A. To validate these observations in human cells, miR-218 was overexpressed in primary human NK cells, resulting in decreased CD16a surface expression when compared with control cells. Collectively, these results describe two epigenetic mechanisms regulating CD16a expression in primary human NK cells; DNA methylation and CpG motifs in Pmed1-A provide cell-specific transcriptional regulation, while miR-218 provides an additional layer of posttranscriptional regulation during the final stage of the NK cell development. As NK-mediated cytotoxicity is key for mAb therapy, this study may provide direction for improving the use of engineered NK cells.

Toll-like receptor agonists promote antitumor responses through induction of cytokine production by T cells and activation of dendritic cells. Although TLR2 stimulation is known to induce clonal expansion of memory CD8⁺ T cells, the role of TLR2 in T_h1 lineage development remains unclear. Ibrahim et al. (p. 847) examined the effects of the TLR2 agonist Pam₃Cys₄ in an EG.7 tumor model and found that CD4⁺ T cells carrying Pam₃Cys₄ cargo are resistant to the regulatory effects of TGF-β on T_h1 development. Naive CD45.1⁺ OVA-specific OT2 cells with or without Pam₃Cys₄ cargo were transferred into CD45.2⁺ C57BL/6 wild-type or TLR2^−/− hosts bearing established TGF-β and OVA-expressing EG.7 tumors. OT2 cells carrying Pam₃Cys₄ cargo (OT2^Pam3) cells had significantly elevated gene transcripts associated with T_h1 lineage development, including T-bet and its target genes. OT2 cells homed to tumor-draining lymph nodes (TDLN) independent of Pam₃Cys₄ sequestration, but elevated numbers of IFN-γ⁺ OT2 and IFN-γ⁺ CD8⁺ T cells accumulated in TDLN of hosts that received OT2^Pam3 cells. Moreover, OT2^Pam3 tumor-infiltrating cells expressed IFN-γ, regardless of their expression of programmed cell death protein-1 (PD-1) and the T cell Ig mucin-3 (TIM-3). However, Ifng, Prf1 Gzmb1, and Fasl mRNA levels were decreased in the PD-1⁺ fraction, suggesting a loss of T_h1 effector phenotype. OT2^Pam3 cell-treated hosts rejected tumors in the setting of PD/PD-L1 blockade, which had only a slight effect on tumor growth in untreated and OT2-treated recipients. The authors also examined the effects of TGF-β1 on regulatory T cell (Treg) development. TGF-β1 treatment led to upregulation of Foxp3 in OT2 cells grown in culture, but CD4⁺ T^Pam3 cells were resistant to TGF-β1−induced Treg development and displayed attenuated TGF-β receptor signaling. Finally, examination of the effects of Pam₃Cys₄ cargo on Akt activation in wild-type and TLR2 CD4⁺ T cells following TCR engagement revealed that the TLR2–MyD88–PI3K pathway antagonizes TGF-β–mediated suppression of T_h1 development. Taken together, these data elucidate the role of TLR2 in regulation of TGF-β–mediated suppression of T_h1 cell development and tumor immune evasion, suggesting that the sequestration of TLR ligands by T cells may enhance adoptive T cell immunotherapies.

Sphingosine-1 phosphate (S1P) is a lipid mediator that plays important roles in diverse biological processes, including the regulation of murine lymphocyte trafficking. However, few studies have addressed the role of S1P receptors (S1PRs) in primary human lymphocytes and, thus, Drouillard et al. (p. 551) examined migration of human T cell subsets in response to S1P gradients. In Transwell migration assays, naive blood T cells did not respond to S1P, whereas naive tonsil CD4⁺ and CD8⁺ T cells migrated in response to an S1P gradient. Migration of CD8⁺ central and effector memory T cells (TCM and TEM, respectively) was inhibited by S1P. TCM cells displayed an intermediate migratory response, suggesting that different S1PRs mediate migration in T cell subsets. S1PR1, S1PR2, and S1PR4 were expressed at high levels in human tonsil T cell subsets, whereas S1PR5 was expressed at low levels in CD8⁺ TEM cells. S1PR1 expression was downregulated during T cell differentiation, but S1PR2 and S1PR4 were constitutively expressed. S1PR1 inhibitors FTY720 and SEW2871 inhibited tonsil naive T cell migration, whereas the S1PR4-specific inhibitor CYM50358 did not. TCM, TEM, tonsil resident memory T cell (TRM), and naive T cell migration were restored in the presence of S1PR2 inhibitor JTE-013. These data suggest that the relative expression levels of S1PR1 and S1PR2 in different T cell subsets impact the cells’ migratory behavior. Specifically, S1P inhibited spontaneous migration of CD69⁺CD103⁺, but not CD69⁺CD103⁻ TRM cells. S1PR1 was downregulated in activated T cells and undetectable in TRM cells, but S1PR2 levels were comparable to that of other T cell subsets. Finally, migration of naive CD4⁺ and CD8⁺ T cells in response to CXCL12 was enhanced in the presence of S1P. In contrast, CXCL12-induced migration of TEM and TCM cells was inhibited by S1P, an effect reversed by JTE-013 treatment. Taken together, these data demonstrate that human T cell subsets display opposing migration patterns in response to S1P, a carefully regulated response that is mediated by differential S1PR expression. Thus, S1PR1 and S1PR2 have opposing function and lymphocyte responses to S1P are modulated during T cell recirculation and tissue residency, which allows fine-tuning of T cell migration.

Necroptosis is a form of inflammatory cell death triggered by TNF and initiated by receptor-interacting protein kinase 1 (RIPK1). To examine the role of RIPK1 in dendritic cells (DCs), O’Donnell et al. (p. 737) generated mice carrying DC-specific deletion of RIPK1 (Ripk1^{DC KO}) by crossing mice containing a floxed allele of Ripk1 to CD11cCre transgenic mice. In vitro, RIPK1-deficient DCs showed increased sensitivity to necroptosis, whereas in vivo, Ripk1^{DC KO} mice had increased numbers of conventional DCs in the bone marrow, but not in the spleen. Moreover, Ripk1^{DC KO} mice developed splenomegaly and lymphadenopathy by 16 wk, with evidence of fibrosis in the spleen, skin, and lungs. Ripk1^{DC KO} mice exhibited inflammation, with increased numbers of splenic neutrophils and inflammatory monocytes. Significant increases in serum TNF-α, IFN-γ, and anti-nuclear autoantibodies (ANAs) were also observed in Ripk1^{DC KO} mice when compared with CD11cCre control mice. Administration of LPS precipitated death by endotoxic shock, with 100% Ripk1^{DC KO} mortality within 30 h compared with 60% mortality of control mice by 70 h. Ripk1^{DC KO} mice crossed with mice expressing kinase-inactive RIPK1, or those harboring a deletion of either Ripk3 or mixed lineage kinase domain-like (Mlkl), a pseudo-kinase activated by RIPK1 signaling, displayed an attenuated inflammatory and autoimmune phenotype, as evidenced by reduced spleen fibrosis, diminished splenomegaly, and decreased splenic inflammatory cell infiltration. Ripk1^{DC KO} mice lacking TNFR1 or IFN-γ exhibited splenic fibrosis, but decreased lymphadenopathy in the absence of TNFR1, demonstrating that the lymphadenopathy observed in Ripk1^{DC KO} mice is partially dependent on TNF-α. Similarly, mice lacking MyD88 displayed a decrease in inflammatory phenotype, suggesting that the inflammation observed in Ripk1^{DC KO} mice is exacerbated by TLR signaling. Finally, in the absence of type I IFNR, inflammation was unaffected, but ANA development was abrogated, implicating type I IFN signaling in autoimmunity but not inflammation. Thus, RIPK1 mediates DC necroptosis, revealing a role of necroptosis in autoimmunity and fibrosis, even in the absence of microbial products. These studies demonstrate that chronic necroptosis promotes generation of autoreactive B cells and ANA production.