The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy.

Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26.

Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.

Single-cell profiling of prurigo nodularis demonstrates immune-stromal crosstalk driving profibrotic responses and reversal with nemolizumab.

BACKGROUND: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by bilaterally distributed pruritic hyperkeratotic nodules on extremities and trunk. Neuroimmune dysregulation and chronic scratching are believed to both induce and maintain the characteristic lesions. OBJECTIVES: This study sought to provide a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathologic processes and the cell types involved. Features that distinguish PN skin from the skin of patients with atopic dermatitis were of particular interest. We further aimed to determine the impact of the IL31RA antagonist, nemolizumab, and its specificity at the single-cell level. METHODS: Single-cell RNA-sequencing of skin from 15 healthy donors and nonlesional and lesional skin from 6 patients each with PN and atopic dermatitis, combined with spatial-sequencing using the 10x Visium platform. Integration with bulk RNA-sequencing data from patients treated with nemolizumab. RESULTS: This study demonstrates that PN is an inflammatory skin disease characterized by both keratinocyte proliferation and activation of profibrotic responses. This study also demonstrates that the COL11A1+ fibroblast subset is a major contributor to fibrosis and is predominantly found in the papillary dermis of PN skin. Activation of fibrotic responses is the main distinguishing feature between PN and atopic dermatitis skin. This study further shows the broad effect of nemolizumab on PN cell types, with a prominent effect driving COL11A1+ fibroblast and keratinocyte responses toward normal. CONCLUSIONS: This study provides a high-resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effect of nemolizumab on pathological processes in PN skin.

The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response.

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

IL-1ß Induces the Rapid Secretion of the Antimicrobial Protein IL-26 from Th17 Cells.

Th17 cells play a critical role in the adaptive immune response against extracellular bacteria, and the possible mechanisms by which they can protect against infection are of particular interest. In this study, we describe, to our knowledge, a novel IL-1ß dependent pathway for secretion of the antimicrobial peptide IL-26 from human Th17 cells that is independent of and more rapid than classical TCR activation. We find that IL-26 is secreted 3 hours after treating PBMCs with Mycobacterium leprae as compared with 48 hours for IFN-γ and IL-17A. IL-1ß was required for microbial ligand induction of IL-26 and was sufficient to stimulate IL-26 release from Th17 cells. Only IL-1RI+ Th17 cells responded to IL-1ß, inducing an NF-κB-regulated transcriptome. Finally, supernatants from IL-1ß-treated memory T cells killed Escherichia coli in an IL-26-dependent manner. These results identify a mechanism by which human IL-1RI+ "antimicrobial Th17 cells" can be rapidly activated by IL-1ß as part of the innate immune response to produce IL-26 to kill extracellular bacteria.

IL-12+IL-18 Cosignaling in Human Macrophages and Lung Epithelial Cells Activates Cathelicidin and Autophagy, Inhibiting Intracellular Mycobacterial Growth.

The ability of Mycobacterium tuberculosis to block host antimicrobial responses in infected cells provides a key mechanism for disease pathogenesis. The immune system has evolved to overcome this blockade to restrict the infection, but it is not clear whether two key innate cytokines (IL-12/IL-18) involved in host defense can enhance antimycobacterial mechanisms. In this study, we demonstrated that the combination of IL-12 and IL-18 triggered an antimicrobial response against mycobacteria in infected macrophages (THP-1 and human primary monocyte-derived macrophages) and pulmonary epithelial A549 cells. The inhibition of intracellular bacterial growth required p38-MAPK and STAT4 pathways, the vitamin D receptor, the vitamin D receptor-derived antimicrobial peptide cathelicidin, and autophagy, but not caspase-mediated apoptosis. Finally, the ability of IL-12+IL-18 to activate an innate antimicrobial response in human primary macrophages was dependent on the autonomous production of IFN-γ and the CAMP/autophagy pathway. Together, these data suggest that IL-12+IL-18 cosignaling can trigger the antimicrobial protein cathelicidin and autophagy, resulting in inhibition of intracellular mycobacteria in macrophages and lung epithelial cells.

Mycobacterium tuberculosis Transfer RNA Induces IL-12p70 via Synergistic Activation of Pattern Recognition Receptors within a Cell Network.

Upon recognition of a microbial pathogen, the innate and adaptive immune systems are linked to generate a cell-mediated immune response against the foreign invader. The culture filtrate of Mycobacterium tuberculosis contains ligands, such as M. tuberculosis tRNA, that activate the innate immune response and secreted Ags recognized by T cells to drive adaptive immune responses. In this study, bioinformatics analysis of gene-expression profiles derived from human PBMCs treated with distinct microbial ligands identified a mycobacterial tRNA-induced innate immune network resulting in the robust production of IL-12p70, a cytokine required to instruct an adaptive Th1 response for host defense against intracellular bacteria. As validated by functional studies, this pathway contained a feed-forward loop, whereby the early production of IL-18, type I IFNs, and IL-12p70 primed NK cells to respond to IL-18 and produce IFN-γ, enhancing further production of IL-12p70. Mechanistically, tRNA activates TLR3 and TLR8, and this synergistic induction of IL-12p70 was recapitulated by the addition of a specific TLR8 agonist with a TLR3 ligand to PBMCs. These data indicate that M. tuberculosis tRNA activates a gene network involving the integration of multiple innate signals, including types I and II IFNs, as well as distinct cell types to induce IL-12p70.

Plasticity of antimicrobial and phagocytic programs in human macrophages.

Macrophage (MΦ) polarization is triggered during the innate immune response to defend against microbial pathogens, but can also contribute to disease pathogenesis. In a previous study, we found that interleukin-15 (IL-15) -derived classically activated macrophages (M1 MΦ) have enhanced antimicrobial activity, whereas IL-10-derived alternatively activated macrophages (M2 MΦ) were highly phagocytic but lacked antimicrobial activity. Given that the ability to modulate MΦ polarization from M2 MΦ to M1 MΦ may promote a more effective immune response to infection, we investigated the plasticity of these MΦ programs. Addition of IL-10 to M1 MΦ induced M2-like MΦ, but IL-15 had little effect on M2 MΦ. We determined the set of immune receptors that are present on M2 MΦ, elucidating two candidates for inducing plasticity of M2 MΦ, Toll-like receptor 1 (TLR1) and interferonγ (IFN-γ) receptor 1. Stimulation of M2 MΦ with TLR2/1 ligand (TLR2/1L) or IFN-γ alone was not sufficient to alter M2 MΦ phenotype or function. However, co-addition of TLR2/1L and IFN-γ re-educated M2 MΦ towards the M1 MΦ phenotype, with a decrease in the phagocytosis of lipids and mycobacteria, as well as recovery of the vitamin-D-dependent antimicrobial pathway compared with M2 MΦ maintained in polarizing conditions. Similarly, treatment of M2 MΦ with both TLR2/1L and anti-IL-10 neutralizing antibodies led to polarization to the M1-like MΦ phenotype and function. Together, our data demonstrate an approach to induce MΦ plasticity that provides the potential for re-educating MΦ function in human mycobacterial disease to promote host defense and limit pathogenesis.

Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for Staphylococcus aureus cutaneous host defense.

Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs) to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways-the Toll-like receptor (TLR) and Stimulator of Interferon Gene (STING) pathways-were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed S. aureus. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS). Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus. TLR signaling was required for host defense, with its absence reducing interleukin (IL)-1ß production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by S. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus.

Jagged1 Instructs Macrophage Differentiation in Leprosy.

As circulating monocytes enter the site of disease, the local microenvironment instructs their differentiation into tissue macrophages (MΦ). To identify mechanisms that regulate MΦ differentiation, we studied human leprosy as a model, since M1-type antimicrobial MΦ predominate in lesions in the self-limited form, whereas M2-type phagocytic MΦ are characteristic of the lesions in the progressive form. Using a heterotypic co-culture model, we found that unstimulated endothelial cells (EC) trigger monocytes to become M2 MΦ. However, biochemical screens identified that IFN-γ and two families of small molecules activated EC to induce monocytes to differentiate into M1 MΦ. The gene expression profiles induced in these activated EC, when overlapped with the transcriptomes of human leprosy lesions, identified Jagged1 (JAG1) as a potential regulator of MΦ differentiation. JAG1 protein was preferentially expressed in the lesions from the self-limited form of leprosy, and localized to the vascular endothelium. The ability of activated EC to induce M1 MΦ was JAG1-dependent and the addition of JAG1 to quiescent EC facilitated monocyte differentiation into M1 MΦ with antimicrobial activity against M. leprae. Our findings indicate a potential role for the IFN-γ-JAG1 axis in instructing MΦ differentiation as part of the host defense response at the site of disease in human leprosy.

S100A12 Is Part of the Antimicrobial Network against Mycobacterium leprae in Human Macrophages.

Triggering antimicrobial mechanisms in macrophages infected with intracellular pathogens, such as mycobacteria, is critical to host defense against the infection. To uncover the unique and shared antimicrobial networks induced by the innate and adaptive immune systems, gene expression profiles generated by RNA sequencing (RNAseq) from human monocyte-derived macrophages (MDMs) activated with TLR2/1 ligand (TLR2/1L) or IFN-γ were analyzed. Weighed gene correlation network analysis identified modules of genes strongly correlated with TLR2/1L or IFN-γ that were linked by the "defense response" gene ontology term. The common TLR2/1L and IFN-γ inducible human macrophage host defense network contained 16 antimicrobial response genes, including S100A12, which was one of the most highly induced genes by TLR2/1L. There is limited information on the role of S100A12 in infectious disease, leading us to test the hypothesis that S100A12 contributes to host defense against mycobacterial infection in humans. We show that S100A12 is sufficient to directly kill Mycobacterium tuberculosis and Mycobacterium leprae. We also demonstrate that S100A12 is required for TLR2/1L and IFN-γ induced antimicrobial activity against M. leprae in infected macrophages. At the site of disease in leprosy, we found that S100A12 was more strongly expressed in skin lesions from tuberculoid leprosy (T-lep), the self-limiting form of the disease, compared to lepromatous leprosy (L-lep), the progressive form of the disease. These data suggest that S100A12 is part of an innate and adaptive inducible antimicrobial network that contributes to host defense against mycobacteria in infected macrophages.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

BACKGROUND: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. RESULTS: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. CONCLUSIONS: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

TLR2 looks at lipoproteins.

Toll-like receptors (TLRs) have evolved to recognize lipoproteins from diverse species of bacteria. In this issue of Immunity, Kang et al. (2009) provide insight into the mechanisms by which TLR2-TLR6 heterodimers recognize diacylated liporoteins, whereas TLR2-TLR1 heterodimers recognize triacylated lipopeptides.

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

SaVanT: a web-based tool for the sample-level visualization of molecular signatures in gene expression profiles.

BACKGROUND: Molecular signatures are collections of genes characteristic of a particular cell type, tissue, disease, or perturbation. Signatures can also be used to interpret expression profiles generated from heterogeneous samples. Large collections of gene signatures have been previously developed and catalogued in the MSigDB database. In addition, several consortia and large-scale projects have systematically profiled broad collections of purified primary cells, molecular perturbations of cell types, and tissues from specific diseases, and the specificity and breadth of these datasets can be leveraged to create additional molecular signatures. However, to date there are few tools that allow the visualization of individual signatures across large numbers of expression profiles. Signature visualization of individual samples allows, for example, the identification of patient subcategories a priori on the basis of well-defined molecular signatures. RESULT: Here, we generate and compile 10,985 signatures (636 newly-generated and 10,349 previously available from MSigDB) and provide a web-based Signature Visualization Tool (SaVanT; http://newpathways.mcdb.ucla.edu/savant ), to visualize these signatures in user-generated expression data. We show that using SaVanT, immune activation signatures can distinguish patients with different types of acute infections (influenza A and bacterial pneumonia). Furthermore, SaVanT is able to identify the prominent signatures within each patient group, and identify the primary cell types underlying different leukemias (acute myeloid and acute lymphoblastic) and skin disorders. CONCLUSIONS: The development of SaVanT facilitates large-scale analysis of gene expression profiles on a patient-level basis to identify patient subphenotypes, or potential therapeutic target pathways.

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.

RATIONALE: The development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis. OBJECTIVES: To identify T-cell-dependent mechanisms that are protective in tuberculosis. METHODS: Multicolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood. MEASUREMENTS AND MAIN RESULTS: At two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%). CONCLUSIONS: Our data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis.

Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae.

The innate immune system recognizes microbial pathogens via pattern recognition receptors. One such receptor, NOD2, via recognition of muramyl dipeptide (MDP), triggers a distinct network of innate immune responses, including the production of interleukin-32 (IL-32), which leads to the differentiation of monocytes into dendritic cells (DC). NOD2 has been implicated in the pathogenesis of human leprosy, yet it is not clear whether Mycobacterium leprae, which has a distinct MDP structure, can activate this pathway. We investigated the effect of MDP structure on the innate immune response, finding that infection of monocytes with M. leprae induces IL-32 and DC differentiation in a NOD2-dependent manner. The presence of the proximal l-Ala instead of Gly in the common configuration of the peptide side chain of M. leprae did not affect recognition by NOD2 or cytokine production. Furthermore, amidation of the d-Glu residue did not alter NOD2 activation. These data provide experimental evidence that NOD2 recognizes naturally occurring structural variants of MDP.