Sara L. Colpitts

The Nlrp3 Inflammasome Suppresses Colorectal Cancer Metastatic Growth in the Liver by Promoting Natural Killer Cell Tumoricidal Activity

The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.

Cutting Edge: The Role of IFN-α Receptor and MyD88 Signaling in Induction of IL-15 Expression In Vivo

IL-15 plays a multifaceted role in immune homeostasis, but the unreliability of IL-15 detection has stymied exploration of IL-15 regulation in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in innate cells including dendritic cells (DCs), macrophages, and monocytes. However, DC subsets expressed varying levels of EmGFP/IL-15 with CD8+ DCs constitutively expressing EmGFP/IL-15 and CD8− DCs expressing low EmGFP/IL-15 levels. Virus infection resulted in IL-15 upregulation in both subsets. By crossing the transgenic mice to mice deficient in specific elements of innate signaling, we found a cell-intrinsic dependency of DCs and Ly6C+ monocytes on IFN-α receptor expression for EmGFP/IL-15 upregulation after vesicular stomatitis virus infection. In contrast, myeloid cells did not require the expression of MyD88 to upregulate EmGFP/IL-15 expression. These findings provide evidence of previously unappreciated regulation of IL-15 expression in myeloid lineages during homeostasis and following infection.

IL-7 Receptor Expression Provides the Potential for Long-Term Survival of Both CD62Lhigh Central Memory T Cells and Th1 Effector Cells during Leishmania major Infection

Infection with the intracellular protozoan parasite Leishmania major induces a state of concomitant immunity wherein secondary immunity is dependent upon the persistence of the original pathogen. Our laboratory has described two populations of Leishmania-induced CD4+ T cells that contribute to immunity: CD62Lhigh central memory T (TCM) cells and CD62Llow effector T cells. To determine whether the prosurvival cytokine IL-7 contributes to maintaining these T cells, we examined expression of the IL7R on CD4+ T cells activated during L. major infection. We found that TCM cells present in chronically infected mice expressed high levels of the IL7R. However, in addition to the expression of the IL7R by TCM cells, CD62Llow cells responding to L. major infection expressed the IL7R. Additional experiments revealed that a large percentage of the IL7RhighCD62Llow cells were Th1 cells, based on transcription at the IFN-γ locus and up-regulation of the Th1-promoting transcription factor T-bet. The up-regulation of T-bet did not prevent IL7R expression by L. major-responding CD4+ T cells, nor did the absence of T-bet result in increased IL7R expression. Finally, blockade of IL7R signaling decreased the number of T-bet+CD4+ T cells, reduced IFN-γ production, and inhibited delayed-type hypersensitivity responses in immune mice challenged with L. major, indicating that IL7R signaling contributes to the maintenance of Th1 effector cells. Thus, both TCM and Th1 effector cells can express the IL7R during chronic L. major infection, which provides a potential means for their long-term survival in addition to the presence of persisting parasites.

Transcriptional Regulation of IL-15 Expression during Hematopoiesis

Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8+ and CD8−). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineage−Sca-1+c-Kit+) expressed high levels of IL-15, suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15+CD8+ and IL-15−/lowCD8− DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.

In vivo recognition of ovalbumin expressed by transgenic Leishmania is determined by its subcellular localization.

The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity.

The Early Generation of a Heterogeneous CD4+ T Cell Response to Leishmania major

CD4+ T cells are an essential component of both the primary and secondary immune response against the intracellular protozoan parasite Leishmania major. Our laboratory has previously shown that CD62Lhigh IL-7Rhigh central memory T (TCM) cells mediate protective immunity following secondary challenge. To determine when TCM cells develop, we examined the phenotype of Leishmania-specific CD4+ T cells in the first 2 wk following infection. As expected, we identified a population of CD4+ T cells present in the draining lymph node with the characteristics of effector T cells. However, in addition, a second population phenotypically resembling TCM cells emerged coincident with the effector population. These T cells, expressing CD62L, CCR7, and IL-7R, failed to produce IFN-γ, but had the capacity to give rise to IFN-γ–producing effector cells. Our studies also demonstrated that the degree of proliferation and the timing of lymph node entry impact TCM cell development. The early generation of TCM cells following L. major infection indicates that TCM cells may not only control secondary infections, but may also contribute to the control of the primary infection.

Early Effector CD8 T Cells Display Plasticity in Populating the Short- Lived Effector and Memory- Precursor Pools Following Bacterial or Viral Infection

Naïve antigen-specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations, which include memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). In the days before the peak of the T cell response, a third population called early effector cells (EECs) predominate the antigen-specific response. However, the contribution of the EEC population to the CD8 T cell differentiation program during an antimicrobial immune response is not well understood. To test if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EECs from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), where the relative frequency of each population is known to be different at the peak of the response. Sorted EECs transferred into uninfected hosts revealed that EECs were pre-programmed to differentiate based on early signals received from the distinct infectious environments. Surprisingly, when these same EECs were transferred early into mismatched infected hosts, the transferred EECs could be diverted from their original fate. These results delineate a model of differentiation where EECs are programmed to form MPECs or SLECs, but remain susceptible to additional inflammatory stimuli that can alter their fate.

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex.

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rβ/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of β2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of β2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.

Rapid αβ T-cell responses orchestrate innate immunity in response to Staphylococcal enterotoxin A

In the generation of a traditional immune response against invading pathogens, innate cells guide T cells by programming their differentiation. However, here we demonstrate that αβ T cells have an essential role in priming innate immunity in the lung after Staphylococcus aureus enterotoxin A (SEA) inhalation. We found that SEA induces waves of cellular activation, cytokine production, and migration into the lung tissue and airways. However, this innate response was completely inhibited in the absence of αβ T cells. Specifically, we found that interleukin (IL)-17A was required for the recruitment of neutrophils and monocytes into the lung. The cellular source of IL-17A was γδ T cells, which increased their IL-17A production following SEA but only in an αβ T-cell-dependent manner. Thus, rapid T-cell activation orchestrates innate immunity and may be a new point of therapeutic intervention for acute lung injury.

IL-15 receptor α signaling constrains the development of IL-17–producing γδ T cells

Significance γδ T cells producing IL-17 (γδ-17 cells) play an important role in promoting inflammation, but the mechanisms that regulate their development are still being explored. IL-15 and its receptor, IL-15Rα, function largely to promote the development and survival of lymphoid cells. Here we show that IL-15Rα signaling has an opposing effect on the γδ-17 population, such that a deficiency in IL-15Rα increases γδ-17 cells and their precursors. This work establishes a direct role for IL-15Rα signaling in vivo and raises questions about the role of IL-15 itself in this process. The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of IL-15 and its unique receptor, IL-15Rα, in the development of IL-17–producing γδ (γδ-17) T cells. Phenotypic analysis has shown that CD44high γδ-17 cells express IL-15Rα and the common gamma chain (CD132), yet lack the IL-2/15Rβ chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice, but not of IL-15 KO mice. The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. γδ-17 cells also were increased in the peripheral lymph nodes of transgenic knock-in mice, where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). Finally, an analysis of neonatal thymi revealed that the CD44lo/int precursors of γδ-17 cells, which also expressed IL-15Rα, were increased in newborn mice deficient in IL-15Rα signaling, but not in IL-15 itself. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny, with long-term effects on their peripheral homeostasis in the adult.