A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Maternal-Fetal Immunologic Response to SARS-CoV-2 Infection in a Symptomatic Vulnerable Population: A Prospective Cohort.

BACKGROUND: Coronavirus disease 2019 (COVID-19) disproportionally affects pregnant women and their newborn; however, little is known about variables that modulate maternal-fetal immune response to infection. METHODS: We prospectively studied socioeconomic, biologic, and clinical factors affecting humoral immunity in 87 unvaccinated pregnant women hospitalized in Buenos Aires for symptoms consistent with COVID-19. RESULTS: The number of days between symptom onset and childbirth predicted maternal and newborn virus spike protein receptor binding domain (RBD)-specific immunoglobulin G (IgG). These findings suggest newborns may benefit less when mothers deliver soon after COVID-19 infection. Similarly, a longer time between symptom onset and birth predicted higher in utero transfer of maternal IgG and its concentration in cord blood. Older gestational age at birth was associated with lower maternal to cord blood IgG ratio. Of women with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, 87% developed RBD-specific IgA responses in breast milk within 96 hours of childbirth. IgA was not significantly associated with time from infection but correlated with maternal serum IgG and placental transfer. CONCLUSIONS: These results demonstrate the combined role of biologic, clinical, and socioeconomic variables associated with maternal RBD-specific antibodies and supports early vaccination strategies for COVID-19 in socioeconomically vulnerable pregnant women. CLINICAL TRIALS REGISTRATION: NCT04362956.

The Murine Neonatal Fc Receptor Is Required for Transport of Immunization-Induced C. difficile-Specific IgG to the Gut and Protection against Disease but Does Not Affect Disease Susceptibility.

The pathology associated with Clostridioides difficile disease is caused in large part by TcdB, an intracellular bacterial toxin that inactivates small GTPases. Despite C. difficile causing enteric disease, antitoxin IgG is a clear correlate of protection against infection-associated pathology. Immunization with TcdB-based immunogens or passive transfer of monoclonal antibodies specific for the TcdB carboxy-terminal domain (CTD) confers protection following C. difficile infection. Whether the mechanism by which circulating IgG is delivered to the gut depends on specific receptor-mediated transport or is solely reflective of infection-induced damage to the gut remains unclear. Here, we tested the hypothesis that neonatal Fc receptor (FcRn) is required for the delivery of systemic TcdB-specific IgG to the gut and protection against C. difficile-associated pathology. FcRn-expressing mice and FcRn-deficient littermates were immunized subcutaneously with Alhydrogel adjuvant-adsorbed CTD before challenge with live C. difficile spores. FcRn was required for the delivery of systemic TcdB-specific IgG to the gut and for vaccine-induced protection against C. difficile-associated disease. The lack of FcRn expression had minimal effects on the composition of the gut microbiome and did not affect susceptibility to C. difficile infection in nonimmunized mice. In further experiments, intraperitoneal injection of immune sera in FcRn-deficient mice led to the transport of protective IgG to the gut independently of infection, confirming a reported method of bypassing the FcRn. Our results reveal an FcRn-dependent mechanism by which systemic immunization-induced IgG protects the gut during enteric C. difficile infection. These findings may be beneficial for the targeting of C. difficile-specific IgG to the gut.

α-Galactosylceramide-Reactive NKT Cells Increase IgG1 Class Switch against a Clostridioides difficile Polysaccharide Antigen and Enhance Immunity against a Live Pathogen Challenge.

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and, when conjugated to a protein carrier, induces a protective antibody response in animal models. Given that CD1d-restricted natural killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increased the IgG1/IgG2c ratio. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18-/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen toward Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B-cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

Clostridioides difficile Infection Induces an Inferior IgG Response to That Induced by Immunization and Is Associated with a Lack of T Follicular Helper Cell and Memory B Cell Expansion.

The intracellularly active bacterial toxin TcdB is a major Clostridioides difficile virulence factor that contributes to inflammation and tissue damage during disease. Immunization with an inactive TcdB fragment prevents C. difficile infection (CDI)-associated pathology. The protective immune response against inactive TcdB involves development of antigen-specific memory B cells and long-lived plasma cells that encode TcdB-neutralizing antibodies. Unlike the response to inactive TcdB, very little is known about the host humoral immune response to C. difficile and TcdB during primary and recurrent infection. Here, we used a murine model of C. difficile disease recurrence to demonstrate that an initial infection induced a serum IgM and mucosal IgA response against the toxin, but a low serum IgG response, which is associated with a lack of protection against disease during reinfection. Infection induced a partial expansion of the T follicular helper cell compartment, essential for B cell memory responses, and, consistent with that, failed to significantly expand the memory B cell compartment. Further, infection failed to stimulate the memory B cell compartment in preimmunized mice, although they were protected against associated disease. These results delineate the key humoral immune events that follow primary and recurrent C. difficile infection and provide a compelling inverse correlation between B cell memory and disease recurrence.

Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination.

Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2Δ1769-1787, exhibited no detectable cytotoxicity. TcdB2Δ1769-1787 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2Δ1769-1787 induced a TcdB2-neutralizing antibody response and protected against C. difficile disease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2Δ1769-1787 is a promising candidate for vaccination.

T Cell Fates Zipped Up: How the Bach2 Basic Leucine Zipper Transcriptional Repressor Directs T Cell Differentiation and Function.

Recent data illustrate a key role for the transcriptional regulator bric-a-brac, tramtrack, and broad complex and cap'n'collar homology (Bach)2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super-enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation, and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulators such as B lymphocyte-induced maturation protein 1. In this review, we examine our present understanding of the role of Bach2 in T cell function and highlight the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation, and memory T cell generation.

Loss of natural killer T cells promotes pancreatic cancer in LSL-KrasG12D/+ mice.

The role of the unique T-cell population, natural killer T (NKT) cells, which have similar functions to NK cells in pancreatic cancer (PC), is not yet evaluated. To address the regulatory roles of NKT cells on tumour progression through tumour-associated macrophages (TAM) and their production of microsomal prostaglandin E synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) in (Kras)-driven pancreatic tumour (KPT) progression, we crossed CD1d-/- mice deficient in both invariant and variant NKT cells with the KrasG12D mice. Loss of NKT cells significantly increased pancreatic intraepithelial neoplasia (PanIN) lesions and also increased 5-LOX and mPGES-1 expression in M2-type macrophages and cancer stem-like cells in pancreatic tumours. Pharmacological inhibition of mPGES-1 and 5-LOX in M2 macrophages with specific inhibitor YS-121 in KPT-CD1d-/- mice decreased PanIN lesions and suppressed tumour growth in association with elevated levels of active CD8a cells. Hence, NKT cells regulate PC by modulating TAMs (M2) through mPGES-1 and 5-LOX; and the absence of NKT cells leads to aggressive development of PC.

Memory B Cells Encode Neutralizing Antibody Specific for Toxin B from the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 but with Superior Neutralization of VPI 10463 Toxin B.

Secreted toxin B (TcdB) substantially contributes to the pathology observed during Clostridium difficile infection. To be successfully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encoding memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vaccination against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD) fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI 10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 induced strong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferred to naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Further experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible with Bmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells (PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven antibody titers, and the CTD2 antibody was neutralizing in vitro, but its protection against lethal challenge with TcdB2 was limited to delaying time to death. Therefore, CTD from different C. difficile strains may be a good immunogen for stimulating B cell memory that encodes in vitro neutralizing Ab but may be limited by variable protection against intoxication in vivo.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids.

Adoptive transfer of regulatory T cells promotes intestinal tumorigenesis and is associated with decreased NK cells and IL-22 binding protein.

High number of regulatory T cells (Tregs), both circulating and at the tumor site, often indicates a poor prognosis in CRC patient's possibly impairing natural killer (NK) cell function. To determine the role of Tregs in CRC development and their effects on NK cells, we created novel transgenic Rag-Apc mice that lack T cells and develop spontaneous intestinal tumors, and we adoptively transferred Tregs or transiently depleted NK cells during initial stages of tumorigenesis. In 6-weeks old Rag-Apc mice containing microscopic intestinal tumors adoptive transfer of Tregs or transient NK cell depletion dramatically associated with an increase in intestinal tumor multiplicity and tumor size, with significantly decreased survival rates. Importantly, Treg transfer increased small intestinal polyp formation up to 65% (P < 0.0005) and increased colon tumors multiplicities by 84% (P < 0.0001) with a significant decrease in NK cells as compared to control mice. Similarly, in NK depleted mice, colon tumor multiplicities increased up to 40% and small intestinal polyp formation up to 60% (P < 0.0001). Treg transfer or NK cell transient depletion markedly increased interleukin (IL)-22 systemically and the inflammatory signaling molecules P2X7R, and STAT3 in the tumors; and impaired production of the tumor suppressor interferon (IFN)-γ systemically. Notably, IL-22 binding protein (IL-22 BP) was associated with NKs and a significant decrease was seen at the tumor site in mice adoptively transferred with Tregs or depleted of NK cells. Our results suggest that adoptive transfer of Tregs aggressively promote intestinal tumorigenesis by decreasing NK cell number and activity by modulating IL-22 BP.

BAFF- and APRIL-dependent maintenance of antibody titers after immunization with T-dependent antigen and CD1d-binding ligand.

CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide-containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell-derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell-derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.

Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism.

Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease.

G-CSF Is a Novel Mediator of T-Cell Suppression and an Immunotherapeutic Target for Women with Colon Cancer.

PURPOSE: G-CSF enhances colon cancer development. This study defines the prevalence and effects of increased G-CSF signaling in human colon cancers and investigates G-CSF inhibition as an immunotherapeutic strategy against metastatic colon cancer. EXPERIMENTAL DESIGN: Patient samples were used to evaluate G-CSF and G-CSF receptor (G-CSFR) levels by IHC with sera used to measure G-CSF levels. Peripheral blood mononuclear cells were used to assess the rate of G-CSFR+ T cells and IFNγ responses to chronic ex vivo G-CSF. An immunocompetent mouse model of peritoneal metastasis (MC38 cells in C57Bl/6J) was used to determine the effects of G-CSF inhibition (αG-CSF) on survival and the tumor microenvironment (TME) with flow and mass cytometry. RESULTS: In human colon cancer samples, the levels of G-CSF and G-CSFR are higher compared to normal colon tissues from the same patient. High patient serum G-CSF is associated with increases in markers of poor prognosis, (e.g., VEGF, IL6). Circulating T cells from patients express G-CSFR at double the rate of T cells from controls. Prolonged G-CSF exposure decreases T cell IFNγ production. Treatment with αG-CSF shifts both the adaptive and innate compartments of the TME and increases survival (HR, 0.46; P = 0.0237) and tumor T-cell infiltration, activity, and IFNγ response with greater effects in female mice. There is a negative correlation between serum G-CSF levels and tumor-infiltrating T cells in patient samples from women. CONCLUSIONS: These findings support G-CSF as an immunotherapeutic target against colon cancer with greater potential benefit in women.

Reduction of CD1d expression in vivo minimally affects NKT-enhanced antibody production but boosts B-cell memory.

The CD1d-binding glycolipid α-galactosylceramide exerts potent adjuvant effects on T-dependent humoral immunity. The mechanism is driven by cognate interaction between CD1d-expressing B cells and TCR-expressing type I CD1d-restricted NKT cells. Thus, far positive effects of alpha-galactosylceramide have been observed on initial and sustained antibody titers as well as B-cell memory. Following vaccination, each of these features is desirable, but good B-cell memory is of paramount importance for long-lived immunity. We therefore tested the hypothesis that CD1d expression in vivo differentially affects initial antibody titers versus B-cell memory responses. CD1d(+/+) and CD1d(+/-) mice were generated and immunized with antigen plus CD1d ligand before analysis of cytokine expression, CD40L expression, initial and longer term antibody responses and B-cell memory. As compared with CD1d(+/+) controls, CD1d(+/-) mice had equivalent numbers of total NKT cells, lower cytokine production, fewer CD40L-expressing NKT cells, lower initial antibody responses, similar long-term antibody responses and higher B-cell memory. Our data indicate that weak CD1d antigen presentation may facilitate good B-cell memory without compromising antibody responses. This work may impact vaccine design since over-stimulation of NKT cells at the time of vaccination may not lead to optimal B-cell memory.

Peptide Aggregation Induced Immunogenic Rupture (PAIIR).

Immunogenic cell death (ICD) arises when cells are under stress, and their membranes are damaged. They release damage-associated molecular patterns (DAMPs) that stimulate and drive the type and magnitude of the immune response. In the presence of an antigen, DAMPs ride the longevity and efficacy of antigen-specific immunity. Yet, no tool can induce the controlled ICD with predictable results. A peptide-based tool, [II], is designed that aggregates in the cell and causes cell membrane damage, generates ICD and DAMPs release on various cell types, and hence can act as an adjuvant. An influenza vaccine is prepared by combining [II] with influenza hemagglutinin (HA) subunit antigens. The results show that [II] induced significantly higher HA-specific immunoglobulin G1 (IgG1) and IgG2a antibodies than HA-only immunized mice, while the peptide itself did not elicit antibodies. This paper demonstrates the first peptide-aggregation induced immunogenic rupture (PAIIR) approach as a vaccine adjuvant. PAIIR is a promising adjuvant with a high potential to promote universal protection upon influenza HA vaccination.

Antigen presentation and interferon signatures in B cells driven by localized ablative cancer immunotherapy correlate with extended survival.

Rationale: B cells have emerged as key regulators in protective cancer immunity. However, the activation pathways induced in B cells during effective immunotherapy are not well understood. Methods: We used a novel localized ablative immunotherapy (LAIT), combining photothermal therapy (PTT) with intra-tumor delivery of the immunostimulant N-dihydrogalactochitosan (GC), to treat mice bearing mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT). We used single-cell RNA sequencing to compare the transcriptional changes induced by PTT, GC and PTT+GC in B cells within the tumor microenvironment (TME). Results: LAIT significantly increased survival in the tumor-bearing mice, compared to the treatment by PTT and GC alone. We found that PTT, GC and PTT+GC increased the proportion of tumor-infiltrating B cells and induced gene expression signatures associated with B cell activation. Both GC and PTT+GC elevated gene expression associated with antigen presentation, whereas GC elevated transcripts that regulate B cell activation and GTPase function and PTT+GC induced interferon response genes. Trajectory analysis, where B cells were organized according to pseudotime progression, revealed that both GC and PTT+GC induced the differentiation of B cells from a resting state towards an effector phenotype. The analyses confirmed upregulated interferon signatures in the differentiated tumor-infiltrating B cells following treatment by PTT+GC but not by GC. We also observed that breast cancer patients had significantly longer survival time if they had elevated expression of genes in B cells that were induced by PTT+GC therapy in the mouse tumors. Conclusion: Our findings show that the combination of local ablation and local application of immunostimulant initiates the activation of interferon signatures and antigen-presentation in B cells which is associated with positive clinical outcomes for breast cancer. These findings broaden our understanding of LAIT's regulatory roles in remodeling TME and shed light on the potentials of B cell activation in clinical applications.

Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.