Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

Two sequential activation modules control the differentiation of protective T helper-1 (Th1) cells.

Interferon-γ (IFN-γ)-producing CD4+ T helper-1 (Th1) cells are critical for protection from microbes that infect the phagosomes of myeloid cells. Current understanding of Th1 cell differentiation is based largely on reductionist cell culture experiments. We assessed Th1 cell generation in vivo by studying antigen-specific CD4+ T cells during infection with the phagosomal pathogen Salmonella enterica (Se), or influenza A virus (IAV), for which CD4+ T cells are less important. Both microbes induced T follicular helper (Tfh) and interleukin-12 (IL-12)-independent Th1 cells. During Se infection, however, the Th1 cells subsequently outgrew the Tfh cells via an IL-12-dependent process and formed subsets with increased IFN-γ production, ZEB2-transcription factor-dependent cytotoxicity, and capacity to control Se infection. Our results indicate that many infections induce a module that generates Tfh and poorly differentiated Th1 cells, which is followed in phagosomal infections by an IL-12-dependent Th1 cell amplification module that is critical for pathogen control.

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation.

B cells within germinal centers (GCs) enter cycles of antibody affinity maturation or exit the GC as memory cells or plasma cells. Here, we examined the contribution of interleukin (IL)-4 on B cell fate decisions in the GC. Single-cell RNA-sequencing identified a subset of light zone GC B cells expressing high IL-4 receptor-a (IL4Ra) and CD23 and lacking a Myc-associated signature. These cells could differentiate into pre-memory cells. B cell-specific deletion of IL4Ra or STAT6 favored the pre-memory cell trajectory, and provision of exogenous IL-4 in a wild-type context reduced pre-memory cell frequencies. IL-4 acted during antigen-specific interactions but also influenced bystander cells. Deletion of IL4Ra from follicular dendritic cells (FDCs) increased the availability of IL-4 in the GC, impaired the selection of affinity-matured B cells, and reduced memory cell generation. We propose that GC FDCs establish a niche that limits bystander IL-4 activity, focusing IL-4 action on B cells undergoing selection and enhancing memory cell differentiation.

Salmonella Persist in Activated Macrophages in T Cell-Sparse Granulomas but Are Contained by Surrounding CXCR3 Ligand-Positioned Th1 Cells.

Salmonella enterica (Se) bacteria cause persistent intracellular infections while stimulating a robust interferon-γ-producing CD4+ T (Th1) cell response. We addressed this paradox of concomitant infection and immunity by tracking fluorescent Se organisms in mice. Se bacteria persisted in nitric oxide synthase (iNOS)-producing resident and recruited macrophages while inducing genes related to protection from nitric oxide. Se-infected cells occupied iNOS+ splenic granulomas that excluded T cells but were surrounded by mononuclear phagocytes producing the chemokines CXCL9 and CXCL10, and Se epitope-specific Th1 cells expressing CXCR3, the receptor for these chemokines. Blockade of CXCR3 inhibited Th1 occupancy of CXCL9/10-dense regions, reduced activation of the Th1 cells, and led to increased Se growth. Thus, intracellular Se bacteria survive in their hosts by counteracting toxic products of the innate immune response and by residing in T cell-sparse granulomas, away from abundant Th1 cells positioned via CXCR3 in a bordering region that act to limit infection.

T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity.

T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naive CD4(+) T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant because systemic expression of a self peptide reduced the size of a naive cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naive T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations and might allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection.

Chrysalis: A New Method for High-Throughput Histo-Cytometry Analysis of Images and Movies.

Advances in imaging have led to the development of powerful multispectral, quantitative imaging techniques, like histo-cytometry. The utility of this approach is limited, however, by the need for time consuming manual image analysis. We therefore developed the software Chrysalis and a group of Imaris Xtensions to automate this process. The resulting automation allowed for high-throughput histo-cytometry analysis of three-dimensional confocal microscopy and two-photon time-lapse images of T cell-dendritic cell interactions in mouse spleens. It was also applied to epi-fluorescence images to quantify T cell localization within splenic tissue by using a "signal absorption" strategy that avoids computationally intensive distance measurements. In summary, this image processing and analysis software makes histo-cytometry more useful for immunology applications by automating image analysis.

TCR Affinity Biases Th Cell Differentiation by Regulating CD25, Eef1e1, and Gbp2.

Naive CD4+ T lymphocytes differentiate into various Th cell subsets following TCR binding to microbial peptide:MHC class II (p:MHCII) complexes on dendritic cells (DCs). The affinity of the TCR interaction with p:MHCII plays a role in Th differentiation by mechanisms that are not completely understood. We found that low-affinity TCRs biased mouse naive T cells to become T follicular helper (Tfh) cells, whereas higher-affinity TCRs promoted the formation of Th1 or Th17 cells. We explored the basis for this phenomenon by focusing on IL-2R signaling, which is known to promote Th1 and suppress Tfh cell differentiation. SIRP⍺+ DCs produce abundant p:MHCII complexes and consume IL-2, whereas XCR1+ DCs weakly produce p:MHCII but do not consume IL-2. We found no evidence, however, of preferential interactions between Th1 cell-prone, high-affinity T cells and XCR1+ DCs or Tfh cell-prone, low-affinity T cells and SIRP⍺+ DCs postinfection with bacteria expressing the peptide of interest. Rather, high-affinity T cells sustained IL-2R expression longer and expressed two novel Th cell differentiation regulators, Eef1e1 and Gbp2, to a higher level than low-affinity T cells. These results suggest that TCR affinity does not influence Th cell differentiation by biasing T cell interactions with IL-2-consuming DCs, but instead, directly regulates genes in naive T cells that control the differentiation process.

Many Th Cell Subsets Have Fas Ligand-Dependent Cytotoxic Potential.

CD4+ Th cells can have cytotoxic activity against cells displaying relevant peptide-MHC class II (p:MHCII) ligands. Cytotoxicity may be a property of Th1 cells and depends on perforin and the Eomes transcription factor. We assessed these assertions for polyclonal p:MHCII-specific CD4+ T cells activated in vivo in different contexts. Mice immunized with an immunogenic peptide in adjuvant or infected with lymphocytic choriomeningitis virus or Listeria monocytogenes bacteria induced cytotoxic Th cells that killed B cells displaying relevant p:MHCII complexes. Cytotoxicity was dependent on Fas expression by target cells but was independent of Eomes or perforin expression by T cells. Although the priming regimens induced different proportions of Th1, Th17, regulatory T cells, and T follicular helper cells, the T cells expressed Fas ligand in all cases. Reciprocally, Fas was upregulated on target cells in a p:MHCII-specific manner. These results indicate that many Th subsets have cytotoxic potential that is enhanced by cognate induction of Fas on target cells.

Regulatory T Cell Induction and Retention in the Lungs Drives Suppression of Detrimental Type 2 Th Cells During Pulmonary Cryptococcal Infection.

Lethal disease caused by the fungus Cryptococcus neoformans is a consequence of the combined failure to control pulmonary fungal replication and immunopathology caused by induced type 2 Th2 cell responses in animal models. In order to gain insights into immune regulatory networks, we examined the role of regulatory T (Treg) cells in suppression of Th2 cells using a mouse model of experimental cryptococcosis. Upon pulmonary infection with Cryptococcus, Treg cells accumulated in the lung parenchyma independently of priming in the draining lymph node. Using peptide-MHC class II molecules to identify Cryptococcus-specific Treg cells combined with genetic fate-mapping, we noted that a majority of the Treg cells found in the lungs were induced during the infection. Additionally, we found that Treg cells used the transcription factor, IFN regulatory factor 4, to dampen harmful Th2 cell responses, as well as mediate chemokine retention of Treg cells in the lungs. Taken together, induction and IFN regulatory factor 4-dependent localization of Treg cells in the lungs allow Treg cells to suppress the deleterious effects of Th2 cells during cryptococcal infection.

Polymicrobial Sepsis Diminishes Dendritic Cell Numbers and Function Directly Contributing to Impaired Primary CD8 T Cell Responses In Vivo.

Patients surviving acute stages of sepsis often display impaired adaptive-immune responses. Using the cecal ligation and puncture model, we demonstrated that sepsis leads to substantial and long-lasting changes in the naive CD8 T cell repertoire, affecting the capacity of the host to respond to new infections. However, the identity of CD8 T cell-extrinsic factor(s) and mechanism(s) that contribute to impaired CD8 T cell responses after sepsis is unknown. Priming of naive CD8 T cells is critically dependent on the ability of dendritic cells (DCs) to provide Ag, costimulation, and inflammatory signal 3 cytokines; therefore, the sepsis-induced changes in the DC compartment might represent a contributing factor leading to diminished CD8 T cell immunity in septic hosts. In a direct test of this hypothesis, we show that, in addition to numerical decline, sepsis leads to functional impairments in DCs, diminishing their capacity to produce cytokines upon TLR stimulation in vitro or postinfection in vivo. Importantly, we demonstrated a direct link between DC dysfunction and impairments in CD8 T cell immunity after sepsis by directly targeting Ag to DCs. Finally, postsepsis Flt3 ligand treatment increased the number of DCs and improved DC function, including the ability to sense inflammation and produce IL-12, leading to improved primary CD8 T cell responses to newly encountered Ags. Thus, sepsis-induced numerical and functional loss of DCs contributes to the observed defects in CD8 T cell immunity, and therapeutic approaches designed to improve the status of the DC compartment after sepsis might facilitate the recovery of CD8 T cell immunity.

Immunosuppression is a conserved driver of tuberculosis susceptibility.

Mycobacterium tuberculosis ( Mtb ) causes 1.6 million deaths a year 1 . However, no individual mouse model fully recapitulates the hallmarks of human tuberculosis disease. Here we report that a comparison across three different susceptible mouse models identifies Mtb -induced gene signatures that predict active TB disease in humans significantly better than a signature from the standard C57BL/6 mouse model. An increase in lung myeloid cells, including neutrophils, was conserved across the susceptible mouse models, mimicking the neutrophilic inflammation observed in humans 2,3 . Myeloid cells in the susceptible models and non-human primates exhibited high expression of immunosuppressive molecules including the IL-1 receptor antagonist, which inhibits IL-1 signaling. Prior reports have suggested that excessive IL-1 signaling impairs Mtb control 4-6 . By contrast, we found that enhancement of IL-1 signaling via deletion of IL-1 receptor antagonist promoted bacterial control in all three susceptible mouse models. IL-1 signaling enhanced cytokine production by lymphoid and stromal cells, suggesting a mechanism for IL-1 signaling in promoting Mtb control. Thus, we propose that myeloid cell expression of immunosuppressive molecules is a conserved mechanism exacerbating Mtb disease in mice, non-human primates, and humans.

Disruption of Aldehyde Dehydrogenase 2 protects against bacterial infection.

The ALDH2*2 (rs671) allele is one of the most common genetic mutations in humans, yet the positive evolutionary selective pressure to maintain this mutation is unknown, despite its association with adverse health outcomes. ALDH2 is responsible for the detoxification of metabolically produced aldehydes, including lipid-peroxidation end products derived from inflammation. Here, we demonstrate that host-derived aldehydes 4-hydroxynonenal (4HNE), malondialdehyde (MDA), and formaldehyde (FA), all of which are metabolized by ALDH2, are directly toxic to the bacterial pathogens Mycobacterium tuberculosis and Francisella tularensis at physiological levels. We find that Aldh2 expression in macrophages is decreased upon immune stimulation, and that bone marrow-derived macrophages from Aldh2 -/- mice contain elevated aldehydes relative to wild-type mice. Macrophages deficient for Aldh2 exhibited enhanced control of Francisella infection. Finally , mice lacking Aldh2 demonstrated increased resistance to pulmonary infection by M. tuberculosis , including in a hypersusceptible model of tuberculosis, and were also resistant to Francisella infection. We hypothesize that the absence of ALDH2 contributes to the host's ability to control infection by pathogens such as M. tuberculosis and F. tularensis , and that host-derived aldehydes act as antimicrobial factors during intracellular bacterial infections. One sentence summary: Aldehydes produced by host cells contribute to the control of bacterial infections.

MHC class II tetramers engineered for enhanced binding to CD4 improve detection of antigen-specific T cells.

The ability to identify T cells that recognize specific peptide antigens bound to major histocompatibility complex (MHC) molecules has enabled enumeration and molecular characterization of the lymphocytes responsible for cell-mediated immunity. Fluorophore-labeled peptide:MHC class I (p:MHCI) tetramers are well-established reagents for identifying antigen-specific CD8+ T cells by flow cytometry, but efforts to extend the approach to CD4+ T cells have been less successful, perhaps owing to lower binding strength between CD4 and MHC class II (MHCII) molecules. Here we show that p:MHCII tetramers engineered by directed evolution for enhanced CD4 binding outperform conventional tetramers for the detection of cognate T cells. Using the engineered tetramers, we identified about twice as many antigen-specific CD4+ T cells in mice immunized against multiple peptides than when using traditional tetramers. CD4 affinity-enhanced p:MHCII tetramers, therefore, allow direct sampling of antigen-specific CD4+ T cells that cannot be accessed with conventional p:MHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire.

Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons.

Type I interferons (IFNs) are essential for anti-viral immunity, but often impair protective immune responses during bacterial infections. An important question is how type I IFNs are strongly induced during viral infections, and yet are appropriately restrained during bacterial infections. The Super susceptibility to tuberculosis 1 (Sst1) locus in mice confers resistance to diverse bacterial infections. Here we provide evidence that Sp140 is a gene encoded within the Sst1 locus that represses type I IFN transcription during bacterial infections. We generated Sp140-/- mice and found that they are susceptible to infection by Legionella pneumophila and Mycobacterium tuberculosis. Susceptibility of Sp140-/- mice to bacterial infection was rescued by crosses to mice lacking the type I IFN receptor (Ifnar-/-). Our results implicate Sp140 as an important negative regulator of type I IFNs that is essential for resistance to bacterial infections.

Peptide:MHCII Tetramer-Based Cell Enrichment for the Study of Epitope-Specific CD4+ T Cells.

Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiological conditions. © 2019 by John Wiley & Sons, Inc.

BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors.

CD4+ T helper 17 (Th17) cells protect vertebrate hosts from extracellular pathogens at mucosal surfaces. Th17 cells form from naive precursors when signals from the T cell antigen receptor (TCR) and certain cytokine receptors induce the expression of the RORγt transcription factor, which activates a set of Th17-specific genes. Using T cell-specific loss-of-function experiments, we find that two components of the Polycomb repressive complex 1.1 (PRC1.1), BCL6 corepressor (BCOR) and KDM2B, which helps target the complex to unmethylated CpG DNA islands, are required for optimal Th17 cell formation in mice after Streptococcus pyogenes infection. Genome-wide expression and BCOR chromatin immunoprecipitation studies revealed that BCOR directly represses Lef1, Runx2, and Dusp4, whose products inhibit Th17 differentiation. Together, the results suggest that the PRC1.1 components BCOR and KDM2B work together to enhance Th17 cell formation by repressing Th17 fate suppressors.

Most microbe-specific naïve CD4⁺ T cells produce memory cells during infection.

Infection elicits CD4(+) memory T lymphocytes that participate in protective immunity. Although memory cells are the progeny of naïve T cells, it is unclear that all naïve cells from a polyclonal repertoire have memory cell potential. Using a single-cell adoptive transfer and spleen biopsy method, we found that in mice, essentially all microbe-specific naïve cells produced memory cells during infection. Different clonal memory cell populations had different B cell or macrophage helper compositions that matched effector cell populations generated much earlier in the response. Thus, each microbe-specific naïve CD4(+) T cell produces a distinctive ratio of effector cell types early in the immune response that is maintained as some cells in the clonal population become memory cells.

Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.