Sema3E Puts the Brakes on Neutrophils See article p.1023

Scoping Out Shrimp for Allergy Immunotherapy See article p.1034

IL-10R Signaling Sustains TR1 Function In Vivo See article p.1130

NKG2C/E Marks ThCTL See article p.1142

IL-15 Maintains NK Cells during Sepsis See article p.1320

Studies in humans and mice have shown that IL-10 is essential for maintaining intestinal homeostasis. Because they secrete high levels of IL-10, CD4+ T regulatory type 1 (TR1) cells are currently being examined in clinical trials for treatment of patients with inflammatory bowel disease (IBD). However, the factors and molecular signals sustaining TR1 cell activity are unknown and need to be elucidated to optimize the efficacy and safety of TR1 therapy. In this issue, Brockmann et al. (p. 1130) examined mouse and human cells to analyze the role of IL-10 signaling in the anti-inflammatory activity of TR1 cells. After confirming that pure populations of both in vivo- and in vitro-differentiated murine intestinal TR1 cells expressed functional IL-10 receptors (IL-10Rα), studies utilizing TR1 cells from mice in which IL-10 signaling is impaired (IL-10RImpaired) demonstrated that IL-10 signaling did not appear to be essential for TR1 differentiation or in vitro suppressive function. In a colitis model in which transfer of either naive CD4+Foxp3 T cells or CD4+IL-17+ effector cells into Rag−/− recipients resulted in severe colitis, disease development was prevented by the cotransfer of in vitro-differentiated TR1 cells from wild-type (WT) but not IL-10RImpaired animals, demonstrating that IL-10 signaling was required for TR1 cells to maintain their suppressive function in vivo. These studies also suggested that TR1 cells could not only suppress the development of pathogenic effector T cells, but could also control cells that were already differentiated. Studies of in vitro-differentiated WT and IL-10RImpaired TR1 cells showed that STAT3 and p38 MAP kinase activation were required for IL-27-driven differentiation of naive CD4+ T cells into TR1 cells and that IL-10 signaling was required for differentiated TR1 cells to maintain p38 MAP kinase activation and sustain production of IL-10, the cytokine that mediates the suppressive activity of these cells. Consistent with the observations in murine TR1 cells, blockade of IL-10R in purified circulating human TR1 cells significantly decreased IL-10 production and reactivation of in vitro-differentiated human TR1 cells in the presence of IL-10Rα blocking Ab resulted in reduced production of IL-10. Taken together, these data demonstrate that TR1 cells require IL-10 signaling to maintain regulatory function and indicate that while TR1-based T cell therapy may be a safe treatment for IBD, the importance of IL-10R signaling should be factored in to efforts to improve treatment efficacy.

To prevent the dangerous consequences of food allergy, allergen-specific immunotherapies need to be developed to impede IgE reactivity. In this issue, Mahajan et al. (p. 1034) sought to better understand human responses to tropomyosin (Pen a 1), the major allergen in shrimp allergy, with the goal of developing hypoallergenic forms of Pen a 1 for immunotherapy. To study human allergic responses in a controlled system, the authors engineered a rat basophilic cell line using CRISPR-Cas 9 gene editing technology to replace the endogenous FcεRI α subunit with the human version (hRBLrαKO cells). Use of these basophils revealed that the magnitude of degranulation in response to Pen a 1 stimulation was linked to allergen dose and valency and to the number of Fc receptors bound to Pen a 1–specific IgE on the cell surface, with measurable responses occurring even upon engagement of only a few hundred receptors. Mathematical modeling was used to predict FcεRI aggregation kinetics and provided a basis for the bell-shaped dose response curve observed upon Pen a 1 stimulation, and transmission electron microscopy was used to visualize the formation of FcεRI clusters following treatment of hRBLrαKO cells with Pen a 1. Degranulation of these cells persisted despite incubation of Pen a 1 with pepsin at concentrations mimicking gastric digestion, confirming the highly stable nature of this allergen. To develop less allergenic Pen a 1 fragments, the authors generated five overlapping polypeptides, each covering one major IgE binding region, and evaluated their secondary structures and binding to IgE from a shrimp-allergic individual. Treatment of IgE-sensitized hRBLrαKO cells with these polypeptides, which were predominantly monomeric, resulted in reduced secretory responses, relative to intact Pen a 1, and the polypeptides competitively inhibited Pen a 1-induced degranulation. Use of primary human basophils confirmed reduced histamine secretion following treatment with the truncated peptides versus intact Pen a 1, but donor-specific variations were observed in peptide reactivity. In characterizing the basophil response to Pen a 1 and demonstrating the potential for fragments of this allergen to reduce allergic responses, this study should provide a basis for the future development of treatments for shrimp allergy.

Interleukin-15 is essential for the maintenance and function of NK and memory CD8+ (mCD8+) T lymphocytes. Although NK cells, CD8+ T cells, and IL-15 have been shown to facilitate the pathology of septic shock, the factors underlying the mechanism by which this occurs remain unclear. In this issue, Guo at al. (p. 1320) investigate the effects of IL-15 on NK and mCD8+ T cells during acute sepsis. Compared with wild type (WT) controls, IL-15-deficient (IL-15 KO) mice demonstrated significant reductions in NK and mCD8+ T cells in the liver and spleen and showed increased survival during sepsis. Resistance to acute sepsis was associated with decreased plasma levels of proinflammatory cytokines such as IL-6, TNFα, IL-1β, IFN-γ, and IL-12 and a significant decrease in the peritoneal bacterial load. Treatment of IL-15 KO mice with an IL-15/IL-15Rα complex that acts as an IL-15 superagonist (IL-15 SA) 4 d prior to and during septic shock regenerated spleen and liver NK and mCD8+ T cell populations and re-established mortality of IL-15 KO mice during septic shock. Depletion of NK cells, but not CD8+ T cells, in IL-15 KO mice prior to IL-15 SA treatment partially reversed endotoxin-induced mortality, suggesting that IL-15 maintained NK but not CD8+ T cell populations in endotoxin-induced mortality. Consistent with these observations, neutralization of IL-15 in WT mice 4 d prior to endotoxin shock, but not 2 h prior, protected against septic shock and specifically depleted NK cells. Additionally, compared with control-treated animals, treatment of WT mice with IL-15 SA prior to endotoxin-induced shock worsened mortality, but this lethality was reversed in an IFN-γ knockout or by depleting NK cells, but not CD8+ T cells. Taken together, these data indicate that endogenous IL-15 contributes to the pathology of acute septic shock by maintaining NK cells, and to a lesser degree CD8+ T cells, which then facilitate systemic inflammation and organ injury through an as yet undefined mechanism that is likely dependent on IFN-γ production.

During infections, activated CD4+ T cells differentiate into various effector subsets, including CD4+ cytotoxic T lymphocytes (ThCTL) that mediate MHC class II–restricted cytotoxicity at sites of infection in multiple viral models. The lack of a signature cell surface marker to identify ThCTL has precluded elucidation of the mechanisms underlying the generation, function, and phenotype of ThCTL and, in this issue, Marshall et al. (p. 1142) demonstrate that expression of NKG2C/E identifies ThCTL in the lungs of influenza A virus (IAV)-infected mice. The authors identified as potential markers for ThCTL molecules that costained with Blimp-1, which was demonstrated to be required for the development, differentiation, and induction of effector CD4+ T cells in the lungs of IAV-infected mice. Knowing that human cytotoxic CD4+ T cells can express NKG2C, the authors demonstrated that the majority of Blimp-1+CD4+ effectors from IAV-infected animals also expressed NKG2X (X indicating any isoform) and that Blimp-1 deficiency was associated with a significant decrease in the expression of NKG2X when compared with IAV-infected wild-type controls. Furthermore, NKG2X+, but not NKG2X, CD4+ effector T cells from the lung were effective killers of MHC class II–expressing targets, confirming the ThCTL activity in this subpopulation of lung CD4+ effectors was associated with NKG2X expression. Further characterization of NKG2X expression on ThCTL from the lungs of IAV-infected animals revealed that ThCTL primarily expressed the activating isoforms C and E, which correlated with ThCTL function but were not directly involved in cytotoxicity. Phenotypic characterization of NKG2C/E+ cells indicated that these cells, which localized only to the site of IAV infection, were highly differentiated effectors that expressed high levels of P-selectin and T-bet and were readily stimulated to degranulate and to secrete IFN-γ. Finally, NKG2C/E+ ThCTL upregulated genes associated with cytotoxic function such as those encoding perforin and granzymes A, C, and F and downregulated the expression of genes associated with recirculation, such as Klf2, Ccr7, and S1p1r, and memory, such as Tcf7, Id3, and Il2. Taken together, these results demonstrate that NKG2C/E can be used to identify and characterize ThCTL and suggest that this marker will provide a valuable tool for future research examining the unique functions and potential therapeutic application of these cells.

Semaphorins, a family of axon guidance proteins, are also involved in immune regulation and modulation of cell migration in tissues outside of the nervous system; for example, semaphorin 3E (Sema3E) can control cellular trafficking in airway remodeling and atherosclerosis. Movassagh et al. (p. 1023) have now explored a role for Sema3E in neutrophil migration, first finding that PlexinD1, the receptor for Sema3E, was expressed at a high level on human neutrophils. Using a microfluidic device in addition to a standard transwell migration assay to measure chemotaxis, the authors found that Sema3E inhibited the trafficking of human neutrophils stimulated via CXCL8 but had little effect on homeostatic migration. The mechanism by which Sema3E inhibited human neutrophil migration appeared to involve F-actin depolymerization and downregulation of CXCL8-induced activity of the small GTPase Rac1. Extending these observations to a mouse model of allergic asthma, the authors first observed that, relative to wild-type (WT) mice, Sema3e−/− mice demonstrated enhanced accumulation of neutrophils in their lungs under steady-state conditions, and this difference was further augmented following intranasal challenge with house dust mite allergen (HDM). Expansion of neutrophils was also observed in the blood of Sema3e−/− mice, both at steady state and following induction of inflammation by i.p. injection of LPS. Suggesting therapeutic relevance for these data, treatment of HDM-sensitized WT mice with Sema3E-Fc prior to HDM challenge reduced neutrophil infiltration of the lung and airway inflammation relative to control Fc-treated animals. Sema3E thus appears to play an important role in the regulation of neutrophil migration, which has implications for the understanding and treatment of asthma and other diseases that involve neutrophil inflammation.