Longitudinal analysis of mucosa-associated invariant T cells in sepsis reveals their early numerical decline with prognostic implications and a progressive loss of antimicrobial functions.

Sepsis-elicited immunosuppression elevates the risk of secondary infections. We used a clinically relevant mouse model and serial peripheral blood samples from patients to assess the antimicrobial activities of mucosa-associated invariant T (MAIT) cells in sepsis. Hepatic and splenic MAIT cells from B6-MAITCAST mice displayed increased CD69 expression and a robust interferon-γ (IFNγ) production capacity shortly after sublethal cecal ligation and puncture, but not at a late timepoint. Peripheral blood MAIT cell frequencies were reduced in septic patients at the time of intensive care unit (ICU) admission, and more dramatically so among nonsurvivors, suggesting the predictive usefulness of early MAIT cell enumeration. In addition, at ICU admission, MAIT cells from sepsis survivors launched stronger IFNγ responses to several bacterial species compared with those from patients who subsequently died of sepsis. Of note, while low human leukocyte antigen (HLA)-DR+ monocyte frequencies, widely regarded as a surrogate indicator of sepsis-induced immunosuppression, were gradually corrected, the numerical insufficiency of MAIT cells was not resolved over time, and their CD69 expression continued to decline. MAIT cell responses to bacterial pathogens, a major histocompatibility complex-related protein 1 (MR1) ligand, and interleukin (IL)-12 and IL-18 were also progressively lost during sepsis and did not recover by the time of ICU/hospital discharge. We propose that MAIT cell dysfunctions contribute to post-sepsis immunosuppression.

MAIT cells accumulate in ovarian cancer-elicited ascites where they retain their capacity to respond to MR1 ligands and cytokine cues.

The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.

Physical restraint mouse models to assess immune responses under stress with or without habituation.

Physical confinement, or restraint, is a psychological stressor used in rodent studies. A single restraint episode elevates blood corticosterone levels, a hallmark of stress responses. Repeated restraint results in habituation (or desensitization), whereas chronic exposure to unpredictable stressors fails to induce habituation. Here, we provide our protocols and guidelines in using three mouse restraint models, namely prolonged restraint stress, repeated restraint stress, and chronic variable stress, to examine immunological homeostasis/competence, or lack thereof, under stress with or without habituation. For complete information on the generation and use of these protocols, please refer to Rudak et al. (2021).

In Vivo Cytotoxicity by α-GalCer-transactivated NK Cells.

Invariant natural killer T (iNKT) cells are innate-like, lipid-reactive T lymphocytes known for their potent immunomodulatory properties. In addition to expressing and utilizing cytolytic effector molecules of their own against certain target cells, iNKT cells can be stimulated with α-galactosylceramide (α-GalCer) to augment the cytotoxic capacity of natural killer (NK) cells. Herein, we describe a flow cytometry-based in vivo killing assay that enables examination of α-GalCer-promoted cytotoxicity against ß2 microglobulin knockout (ß2M-/-) target cells, which mimic tumor and virus-infected cells displaying little to no MHC class I molecules on their surface. Using an anti-asialo GM1 antibody, which depletes NK cells but not iNKT cells, we confirmed that the increased clearance of ß2M-/- cells in α-GalCer-primed recipients was mediated by NK cells. The protocol detailed here can be leveraged to assess the functional fitness of iNKT cells and their crosstalk with NK cells and to further our understanding of α-GalCer-promoted cytotoxicity in preclinical immunotherapeutic applications.

Chronic stress physically spares but functionally impairs innate-like invariant T cells.

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

Measles Virus Infects and Programs MAIT Cells for Apoptosis.

Measles virus (MeV) binds, infects, and kills CD150+ memory T cells, leading to immune amnesia. Whether MeV targets innate, memory-like T cells is unknown. We demonstrate that human peripheral blood and hepatic mucosa-associated invariant T (MAIT) cells and invariant natural killer T cells express surprisingly high levels of CD150, more than other lymphocyte subsets. Furthermore, exposing MAIT cells to MeV results in their efficient infection and rapid apoptosis. This constitutes the first report of direct MAIT cell infection by a viral pathogen. Given MAIT cells' antimicrobial properties, their elimination by MeV may contribute to measles-induced immunosuppression and heightened vulnerability to unrelated infections.

Glycolipid Stimulation of Invariant NKT Cells Expands a Unique Tissue-Resident Population of Precursors to Mature NK Cells Endowed with Oncolytic and Antimetastatic Properties.

Invariant NKT (iNKT) cells are innate-like T lymphocytes that recognize and respond to glycolipid Ags such as α-galactosylceramide (α-GalCer). This unique property has been exploited in clinical trials for multiple malignancies. While investigating mouse iNKT cell responses to α-GalCer in vivo, we found a dramatically enlarged tissue-resident population surprisingly coexpressing select dendritic cell, NK cell, and B cell markers. Further phenotypic and functional analyses revealed the identity of this B220+CD11c+MHC class II+NK1.1+ population as precursors to mature NK (pre-mNK) cells, which also expressed high levels of proliferation and tissue retention markers but diminished sphingosine-1-phosphate receptor 1, a receptor that facilitates tissue trafficking. Accordingly, FTY720, a sphingosine-1-phosphate receptor 1 antagonist, failed to prevent pre-mNK cells' intrahepatic accumulation. We found iNKT cell-driven expansion of pre-mNK cells to be dependent on IL-12 and IL-18. Although α-GalCer-transactivated pre-mNK cells lost their capacity to process a model tumor Ag, they selectively expressed granzyme A and directly lysed YAC-1 thymoma cells through granule exocytosis. They also contributed to ß2 microglobulin-deficient target cell destruction in vivo. Therefore, α-GalCer treatment skewed pre-mNK cell responses away from an APC-like phenotype and toward killer cell-like functions. Finally, the ability of α-GalCer to reduce the pulmonary metastatic burden of B16-F10 mouse melanoma was partially reversed by in vivo depletion of pre-mNK cells. To our knowledge, our findings shed new light on iNKT cells' mechanism of action and glycolipid-based immunotherapies. Therefore, we introduce pre-mNK cells as a novel downstream effector cell type whose anticancer properties may have been overlooked in previous investigations.

Stress-elicited glucocorticoid receptor signaling upregulates TIGIT in innate-like invariant T lymphocytes.

Stress is known to impede certain host defense mechanisms, including those governed by conventional T lymphocytes. However, whether innate-like T lymphocytes, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, are impacted by stress is unclear. Herein, we report that prolonged psychological stress caused by physical confinement results in robust upregulation of T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), an immune checkpoint receptor that controls antitumor and antiviral immune responses. Elevated TIGIT expression was found not only on NK and conventional T cells, but also on iNKT and MAIT cells. Stress-provoked TIGIT upregulation was reversed through treatment with the glucocorticoid receptor (GR) antagonist RU486, but not with 6-hydroxydopamine that induces chemical sympathectomy. A Cre/Lox gene targeting model in which GR was ablated in cells expressing Lck under its proximal promoter revealed that TIGIT upregulation in stressed animals stems from direct GR signaling in T and iNKT cells. In fact, long-term oral administration of exogenous corticosterone (CS) to wild-type C57BL/6 (B6) mice was sufficient to increase TIGIT expression levels on T and iNKT cells. In vitro treatment with CS also potently and selectively upregulated TIGIT, but not CTLA-4 or LAG-3, on mouse iNKT and MAIT hybridomas. These results were recapitulated using primary hepatic iNKT and MAIT cells from wild-type B6 and B6.MAITCAST mice, respectively. Subjecting B6.MAITCAST mice to physical restraint also raised the frequency of TIGIT+ cells among hepatic MAIT cells in a GR-dependent manner. Finally, we found that TIGIT is similarly upregulated in a chronic variable stress model in which animals are exposed to unpredictable heterotypic stressors without developing habituation. Taken together, our findings link, for the first time to our knowledge, GR signaling to TIGIT expression. We propose that glucocorticoid hormones dampen immune responses, in part, by enhancing TIGIT expression across multiple critical subsets of effector lymphocytes, including innate-like T cells. Therefore, TIGIT may constitute an attractive target in immune-enhancing interventions for sustained physiological stress.

Therapeutic control of leishmaniasis by inhibitors of the mammalian target of rapamycin.

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species.

MAIT cell-mediated cytotoxicity: Roles in host defense and therapeutic potentials in infectious diseases and cancer.

Mucosa-associated invariant T (MAIT) cells are unconventional, innate-like T lymphocytes that sense the presence of MHC-related protein 1 (MR1)-restricted ligands and select inflammatory cues. Consequently, they release potent immunomodulatory mediators, including IFN-γ, TNF-α, and/or IL-17. MAIT cells can also be viewed as killer cells. They display several NK cell-associated receptors, carry granules containing cytotoxic effector molecules, and swiftly upregulate perforin and granzymes upon activation. Accordingly, MAIT cells are capable of lysing MR1-expressing cells infected with a variety of pathogenic bacteria in in vitro settings and may also mount cytotoxic responses during microbial infections in vivo. Of note, MAIT cell hyperactivation during certain infections may impede their ability to elicit inflammatory and/or cytotoxic responses to secondary stimuli. In addition, MAIT cells isolated from within and from the margin of tumor masses exhibit diminished functions. We propose that MAIT cell-mediated cytotoxicity can be induced, bolstered, or restored to assist in clearing infections and potentially in reducing tumor loads. In this review, we discuss our current understanding of MAIT cells' lytic functions and highlight the pressing questions that need to be addressed in future investigations. We also offer a picture, however hypothetical at this point, of how harnessing the full cytotoxic potentials of MAIT cells may be a valuable approach in the immunotherapy of infectious and malignant diseases.

Mucosa-associated invariant T cells in malignancies: a faithful friend or formidable foe?

Mucosa-associated invariant T (MAIT) cells are a subset of innate-like T lymphocytes known for their ability to respond to MHC-related protein 1 (MR1)-restricted stimuli and select cytokine signals. They are abundant in humans and especially enriched in mucosal layers, common sites of neoplastic transformation. MAIT cells have been found within primary and metastatic tumors. However, whether they promote malignancy or contribute to anticancer immunity is unclear. On the one hand, MAIT cells produce IL-17A in certain locations and under certain circumstances, which could in turn facilitate neoangiogenesis, intratumoral accumulation of immunosuppressive cell populations, and cancer progression. On the other hand, they can express a potent arsenal of cytotoxic effector molecules, NKG2D and IFN-γ, all of which have established roles in cancer immune surveillance. In this review, we highlight MAIT cells' characteristics as they might pertain to cancer initiation, progression, or control. We discuss recent findings, including our own, that link MAIT cells to cancer, with a focus on colorectal carcinoma, as well as some of the outstanding questions in this active area of research. Finally, we provide a hypothetical picture in which MAIT cells constitute attractive targets in cancer immunotherapy.

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vß-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

Invariant Natural Killer T Cells Are Pathogenic in the HLA-DR4-Transgenic Humanized Mouse Model of Toxic Shock Syndrome and Can Be Targeted to Reduce Morbidity.

During toxic shock syndrome (TSS), bacterial superantigens trigger a polyclonal T -cell response leading to a potentially catastrophic "cytokine storm". Whether innate-like invariant natural killer T (iNKT) cells, with remarkable immunomodulatory properties, participate in TSS is unclear. Using genetic and cell depletion approaches, we generated iNKT cell-deficient, superantigen-sensitive HLA-DR4-transgenic (DR4tg) mice, which were compared with their iNKT-sufficient counterparts for responsiveness to staphylococcal enterotoxin B (SEB). Both approaches indicate that iNKT cells are pathogenic in TSS. Importantly, treating DR4tg mice with a TH2-polarizing glycolipid agonist of iNKT cells reduced SEB-inflicted morbidity/mortality. Therefore, iNKT cells may constitute an attractive therapeutic target in superantigen-mediated illnesses.