Correction: γδ T cell IFNγ production is directly subverted by Yersinia pseudotuberculosis outer protein YopJ in mice and humans.

[This corrects the article DOI: 10.1371/journal.ppat.1010103.].

γδ T cell IFNγ production is directly subverted by Yersinia pseudotuberculosis outer protein YopJ in mice and humans.

Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity.

Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4.

Intestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed that microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is a ligand for PXR in vivo, and IPA downregulated enterocyte TNF-α while it upregulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2(-/-)) mice showed a distinctly "leaky" gut physiology coupled with upregulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2(-/-)Tlr4(-/-) mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway that involves luminal sensing and signaling by TLR4.

Cutting Edge: Batf3 Expression by CD8 T Cells Critically Regulates the Development of Memory Populations.

The basic leucine zipper transcription factor ATF-like 3 (BATF3) is required for the development of conventional type 1 dendritic cells that are essential for cross-presentation and CD8 T cell-mediated immunity against intracellular pathogens and tumors. However, whether BATF3 intrinsically regulates CD8 T cell responses is not well studied. In this article, we report a role for cell-intrinsic Batf3 expression in regulating the establishment of circulating and resident memory T cells after foodborne Listeria monocytogenes infection of mice. Consistent with other studies, Batf3 expression by CD8 T cells was dispensable for the primary response. However, Batf3 -/- T cells underwent increased apoptosis during contraction to contribute to a substantially reduced memory population. Batf3 -/- memory cells had an impaired ability to mount a robust recall response but remained functional. These findings reveal a cell-intrinsic role of Batf3 in regulating CD8 T cell memory development.

The accumulation of Vγ4 T cells with aging is associated with an increased adaptive Vγ4 T cell response after foodborne Listeria monocytogenes infection of mice.

BACKGROUND: It is generally accepted that aging has detrimental effects on conventional T cell responses to systemic infections. However, most pathogens naturally invade the body through mucosal barriers. Although mucosal sites are highly enriched in unconventional immune sentinels like γδ T cells, little is currently known about the impact of aging on unconventional mucosal T cell responses. We previously established that foodborne infection with a mouse-adapted internalin A mutant Listeria monocytogenes (Lm) generates an adaptive intestinal memory CD44hi CD27neg Vγ4 T cells capable of co-producing IL-17A and IFNγ. Therefore, we used this model to evaluate the impact of aging on adaptive Vγ4 T cell responses elicited by foodborne infection. RESULTS: Foodborne Lm infection of female Balb/c and C57BL/6 mice led to an increased adaptive CD44hi CD27neg Vγ4 T cell response associated with aging. Moreover, Lm-elicited CD44hi CD27neg Vγ4 T cells maintained diverse functional subsets despite some alterations favoring IL-17A production as mice aged. In contrast to the documented susceptibility of aged mice to intravenous Lm infection, mice contained bacteria after foodborne Lm infection suggesting that elevated bacterial burden was not a major factor driving the increased adaptive CD44hi CD27neg Vγ4 T cell response associated with mouse age. However, CD44hi CD27neg Vγ4 T cells accumulated in naïve mice as they aged suggesting that an increased precursor frequency contributes to the robust Lm-elicited mucosal response observed. Body mass did not appear to have a strong positive association with CD44hi CD27neg Vγ4 T cells within age groups. Although an increased adaptive CD44hi CD27neg Vγ4 T cell response may contribute to foodborne Lm resistance of C57BL/6 mice aged 19 or more months, neither anti-TCRδ or anti-IL-17A treatment impacted Lm colonization after primary infection. These results suggest that γδTCR signaling and IL-17A are dispensable for protection after primary foodborne Lm infection consistent with the role of conventional T cells during the early innate immune response to Lm. CONCLUSIONS: Lm-elicited adaptive Vγ4 T cells appear resistant to immunosenescence and memory Vγ4 T cells could be utilized to provide protective immune functions during enteric infection of aged hosts. As such, oral immunization might offer an efficient therapeutic approach to generate unconventional memory T cells in the elderly.

Precursor Abundance Influences Divergent Antigen-Specific CD8+ T Cell Responses after Yersinia pseudotuberculosis Foodborne Infection.

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 epitope was inserted into YopE in Y. pseudotuberculosis and antigen-specific CD8+ T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE69-77 elicited significantly larger CD8+ T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen, and liver between 7 and 30 days postinfection, despite residing in the same protein and having an affinity for H-2Kb similar to that of OVA257-264. YopE-specific CD8+ T cell precursors were ∼4.6 times as abundant as OVA-specific precursors in the MLNs, spleens, and other lymph nodes of naive mice, explaining the dominance of YopE69-77 over OVA257-264 at early infection times. However, other factors contributed to this dominance, as the ratio of YopE-specific to OVA-specific CD8+ T cells increased between 7 and 30 days postinfection. We also compared the YopE-specific and OVA-specific CD8+ T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short-lived effectors, while higher percentages of OVA-specific cells were memory precursor effectors at day 30 postinfection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8+ T cells generated in response to the protective YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.

Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.

Bioactive sphingolipids are modulators of immune processes and their metabolism is often dysregulated in ulcerative colitis, a major category of inflammatory bowel disease (IBD). While multiple axes of sphingolipid metabolism have been investigated to delineate mechanisms regulating ulcerative colitis, the role of acid ceramidase (AC) in intestinal inflammation is yet to be characterized. Here we demonstrate that AC expression is elevated selectively in the inflammatory infiltrate in human and murine colitis. To probe for mechanistic insight into how AC up-regulation can impact intestinal inflammation, we investigated the selective loss of AC expression in the myeloid population. Using a model of intestinal epithelial injury, we demonstrate that myeloid AC conditional knockout mice exhibit impairment of neutrophil recruitment to the colon mucosa as a result of defective cytokine and chemokine production. Furthermore, the loss of myeloid AC protects from tumor incidence in colitis-associated cancer (CAC) and inhibits the expansion of neutrophils and granulocytic myeloid-derived suppressor cells in the tumor microenvironment. Collectively, our results demonstrate a tissue-specific role for AC in regulating neutrophilic inflammation and cytokine production. We demonstrate novel mechanisms of how granulocytes are recruited to the colon that may have therapeutic potential in intestinal inflammation, IBD, and CAC.-Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., Obeid, L. M. Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.

Retinoic acid signaling during priming licenses intestinal CD103+ CD8 TRM cell differentiation.

CD8 tissue-resident memory T (TRM) cells provide frontline protection at barrier tissues; however, mechanisms regulating TRM cell development are not completely understood. Priming dictates the migration of effector T cells to the tissue, while factors in the tissue induce in situ TRM cell differentiation. Whether priming also regulates in situ TRM cell differentiation uncoupled from migration is unclear. Here, we demonstrate that T cell priming in the mesenteric lymph nodes (MLN) regulates CD103+ TRM cell differentiation in the intestine. In contrast, T cells primed in the spleen were impaired in the ability to differentiate into CD103+ TRM cells after entry into the intestine. MLN priming initiated a CD103+ TRM cell gene signature and licensed rapid CD103+ TRM cell differentiation in response to factors in the intestine. Licensing was regulated by retinoic acid signaling and primarily driven by factors other than CCR9 expression and CCR9-mediated gut homing. Thus, the MLN is specialized to promote intestinal CD103+ CD8 TRM cell development by licensing in situ differentiation.

The use of foodborne infection to evaluate bacterial pathogenesis and host response.

Foodborne bacterial infections are a major cause of gastrointestinal illness. Murine models have been widely used to interrogate bacterial pathogenesis and host response to better understand the pathogens that cause gastrointestinal disease. Humans are usually exposed to these pathogens through consumption of contaminated food products. However, most murine models of foodborne infection rely on oral gavage to deliver pathogens directly into the stomach. While expedient, the gavage procedure may lead to microabrasions in the esophagus that allow direct access of the pathogen to the blood, which can alter bacterial pathogenesis and the host response under study. In this chapter, the alternative approach of foodborne infection through the consumption of inoculated food is described using the human pathogen Listeria monocytogenes (Lm). A detailed protocol of this methodology is provided with details of assessing bacterial burden and the host immune response. Translation of these methods to other foodborne pathogens will allow a more accurate assessment of bacterial pathogenesis and host immunity in more physiologic murine models.

A blend of broadly-reactive and pathogen-selected Vγ4 Vδ1 T cell receptors confer broad bacterial reactivity of resident memory γδ T cells.

Although murine γδ T cells are largely considered innate immune cells, they have recently been reported to form long-lived memory populations. Much remains unknown about the biology and specificity of memory γδ T cells. Here, we interrogated intestinal memory Vγ4 Vδ1 T cells generated after foodborne Listeria monocytogenes (Lm) infection to uncover an unanticipated complexity in the specificity of these cells. Deep TCR sequencing revealed that a subset of non-canonical Vδ1 clones are selected by Lm infection, consistent with antigen-specific clonal expansion. Ex vivo stimulations and in vivo heterologous challenge infections with diverse pathogenic bacteria revealed that Lm-elicited memory Vγ4 Vδ1 T cells are broadly reactive. The Vγ4 Vδ1 T cell recall response to Lm, Salmonella enterica serovar Typhimurium (STm) and Citrobacter rodentium was largely mediated by the γδTCR as internalizing the γδTCR prevented T cell expansion. Both broadly-reactive canonical and pathogen-selected non-canonical Vδ1 clones contributed to memory responses to Lm and STm. Interestingly, some non-canonical γδ T cell clones selected by Lm infection also responded after STm infection, suggesting some level of cross-reactivity. These findings underscore the promiscuous nature of memory γδ T cells and suggest that pathogen-elicited memory γδ T cells are potential targets for broad-spectrum anti-infective vaccines.

Autographa californica multicapsid nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH.

The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

TGF-ß: Many Paths to CD103+ CD8 T Cell Residency.

CD8 tissue-resident memory T (TRM) cells primarily reside in nonlymphoid tissues without recirculating and provide front-line protective immunity against infections and cancers. CD8 TRM cells can be generally divided into CD69+ CD103- TRM cells (referred to as CD103- TRM cells) and CD69+ CD103+ TRM cells (referred to as CD103+ TRM cells). TGF-ß plays a critical role in the development and maintenance of CD103+ CD8 TRM cells. In this review, we summarize the current understanding of tissue-specific activation of TGF-ß mediated by integrins and how it contributes to CD103+ CD8 TRM cell development and maintenance. Furthermore, we discuss the underlying mechanisms utilized by TGF-ß to regulate the development and maintenance of CD103+ CD8 TRM cells. Overall, this review highlights the importance of TGF-ß in regulating this unique subset of memory CD8 T cells that may shed light on improving vaccine design to target this population.

FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages.

FTY720 is a treatment for relapsing remitting multiple sclerosis (MS). It is an analog of sphingosine-1-phosphate (S1P) and targets S1P receptors 1, 3, 4, and 5. Recent reports indicate an association between long-term exposure to FTY720 and cases of cryptococcal infection. Here, we studied the effect of FTY720 and its derivative, BAF312, which only target S1P receptors 1 and 5, in a mouse model of cryptococcal infection. We found that treatment with FTY720, but not with BAF312, led to decreased survival and increased organ burden in mouse cryptococcal granulomas. Both FTY720 and BAF312 caused a profound CD4+ and CD8+ T cell depletion in blood and lungs but only treatment with FTY720 led to cryptococcal reactivation. Treatment with FTY720, but not with BAF312, was associated with disorganization of macrophages and with M2 polarization at the granuloma site. In a cell system, FTY720 decreased phagocytosis and production of reactive oxygen species by macrophages, a phenotype recapitulated in the S1pr3-/- knockout macrophages. Our results suggest that FTY720 reactivates cryptococcosis from the granuloma through a S1P receptor 3-mediated mechanism and support the rationale for development of more-specific receptor modulators for therapeutic use of MS.

Mucosal CD8 T Cell Responses Are Shaped by Batf3-DC After Foodborne Listeria monocytogenes Infection.

While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlAMLm). InlAMLm trafficked intracellularly from the intestines to the MLN and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this, InlAMLm initially disseminated from the gut to the MLN normally in Batf3-/- mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlAMLm. At this time Batf3-/- mice displayed reduced InlAMLm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3-/- mice also exhibited profound defects in the induction and gut-homing of InlAMLm-specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3-/- mice, indicating a critical role for Batf3 in generating anti-InlAMLm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlAMLm infection and in driving the establishment of intestinal Lm-specific effector T cells.

Isolating Lymphocytes from the Mouse Small Intestinal Immune System.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Listeria Monocytogenes: A Model Pathogen Continues to Refine Our Knowledge of the CD8 T Cell Response.

Listeria monocytogenes (Lm) infection induces robust CD8 T cell responses, which play a critical role in resolving Lm during primary infection and provide protective immunity to re-infections. Comprehensive studies have been conducted to delineate the CD8 T cell response after Lm infection. In this review, the generation of the CD8 T cell response to Lm infection will be discussed. The role of dendritic cell subsets in acquiring and presenting Lm antigens to CD8 T cells and the events that occur during T cell priming and activation will be addressed. CD8 T cell expansion, differentiation and contraction as well as the signals that regulate these processes during Lm infection will be explored. Finally, the formation of memory CD8 T cell subsets in the circulation and in the intestine will be analyzed. Recently, the study of CD8 T cell responses to Lm infection has begun to shift focus from the intravenous infection model to a natural oral infection model as the humanized mouse and murinized Lm have become readily available. Recent findings in the generation of CD8 T cell responses to oral infection using murinized Lm will be explored throughout the review. Finally, CD8 T cell-mediated protective immunity against Lm infection and the use of Lm as a vaccine vector for cancer immunotherapy will be highlighted. Overall, this review will provide detailed knowledge on the biology of CD8 T cell responses after Lm infection that may shed light on improving rational vaccine design.

Combining Adoptive Cell Therapy with Cytomegalovirus-Based Vaccine Is Protective against Solid Skin Tumors.

Despite many years of research, cancer vaccines have largely been ineffective in the treatment of established cancers. Many barriers to immune-mediated destruction of malignant cells exist, and these likely limit the efficacy of cancer vaccines. In this study, we sought to enhance the efficacy of a cytomegalovirus (CMV)-based vaccine targeting melanoma by combining vaccination with other forms of immunotherapy. Adoptive cell therapy in humans and in animal models has been shown to be effective for tumor regression. Thus, in this study, we assessed whether CMV-based vaccines in combination with adoptively transferred antitumor T cells could provide greater antitumor protection than either therapy alone. Our results show that adoptive cell therapy greatly enhanced the antitumor effects of CMV-based vaccines targeting the foreign model antigen, OVA, or the melanoma differentiation antigen, gp100. Combination adoptive cell therapy and vaccination induced the upregulation of the inhibitory ligands, PD-L1, and Qa-1b, on B16 tumor cells. This expression paralleled the infiltration of tumors by vaccine-stimulated T cells which also expressed high levels of the receptors PD-1 and NKG2A/C/E, suggesting a potential mechanism of tumor immune evasion. Surprisingly, therapeutic blockade of the PD-1/PD-L1 and NKG2A/Qa-1b axes did not delay tumor growth following vaccination, suggesting that the presence of inhibitory ligands within malignant tissue may not be an effective biomarker for successful combination therapy with CMV-based vaccines. Overall, our studies show that therapeutic CMV-based vaccines in combination with adoptive T cell transfer alone are effective for tumor rejection.

Reviving virus based cancer vaccines by using cytomegalovirus vectors expressing modified tumor antigens.

Cancer vaccines that have utilized various immunization strategies to induce antitumor immunity have largely failed in clinical settings. We have recently developed a cancer vaccine using a cytomegalovirus (CMV) based vector that expressed a modified melanoma antigen that elicited a robust antitumor CD8+ T cell response and tumor rejection.

Pregnane X Receptor Regulates Pathogen-Induced Inflammation and Host Defense against an Intracellular Bacterial Infection through Toll-like Receptor 4.

The nuclear pregnane X receptor (PXR) plays a central role in regulating xenobiotic metabolism. We now report a novel role for PXR as a critical negative regulator of innate immunity after infection. Pxr(-/-) mice exhibited remarkably elevated pro-inflammatory cytokine and chemokine production following infection with Listeria monocytogenes (Lm). Despite the more robust innate immune response, Pxr(-/-) mice were highly susceptible to Lm infection. Surprisingly, disruption of the Toll-like receptor 4 (TLR4) but not TLR2 signaling restored the inflammation to normal levels and the ability to clear Lm in Pxr(-/-) mice. Mechanistically, the heightened inflammation in Pxr(-/-) mice resulted in the death of inflammatory monocytes that led to the enhanced susceptibility to Lm infection. These data demonstrated that PXR regulated pathogen-induced inflammation and host defense against Lm infection through modulating the TLR4 pathway. In summary, we discovered an apical role for PXR in regulating innate immunity. In addition, we uncovered a remarkable negative impact of the TLR4 pathway in controlling the quality of the inflammatory response and host defense against a gram-positive bacterial infection.

Cytomegalovirus-Based Vaccine Expressing a Modified Tumor Antigen Induces Potent Tumor-Specific CD8(+) T-cell Response and Protects Mice from Melanoma.

The presence of tumor-infiltrating CD8(+) T cells is associated with tumor regression and better prognosis. Cytomegalovirus (CMV) infection elicits a robust and long-lasting CD8(+) T-cell response, which makes CMV a potentially promising vaccine vector against cancer. In the current study, we used recombinant murine CMV (MCMV) strains as prophylactic and therapeutic vaccines in an aggressive B16 lung metastatic melanoma model. Immunization with MCMV-expressing ovalbumin (OVA) induced a potent OVA-specific CD8(+) T-cell response and was effective in protecting mice from OVA-expressing B16 melanoma in an antigen-dependent manner. We engineered MCMV to express a modified B16 melanoma antigen gp100 (MCMV-gp100KGP). Immunization with MCMV-gp100KGP was highly effective in overcoming immune tolerance to self-antigen and induced a strong, long-lasting gp100-specific CD8(+) T-cell response even in the presence of preexisting anti-CMV immunity. Furthermore, both prophylactic and therapeutic vaccinations of mice with MCMV-gp100KGP effectively protected mice from highly aggressive lung B16-F10 melanoma, and the protection was mediated by gp100-specific CD8(+) T cells. We showed that MCMV is a superior vaccine vector compared with a commonly used vesicular stomatitis virus vector. Collectively, our studies demonstrate that CMV is a promising vaccine vector to prevent and treat tumors.