Showing papers in "Science immunology in 2016"

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients

Abstract: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are important regulators of immune responses in cancer and have been directly implicated in promotion of tumor progression. However, the heterogeneity of these cells and lack of distinct markers hampers the progress in understanding of the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation we determined that low density PMN-MDSC and high density neutrophils from the same cancer patients had a distinct gene profile. Most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Surprisingly, low-density lipoprotein (LDL) was one of the most increased regulators and its receptor oxidized LDL receptor 1 OLR1 was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor 1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5-15% of total neutrophils in cancer patients and 15-50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1- counterparts, LOX-1+ neutrophils had gene signature, potent immune suppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSC. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSC. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insight to the biology and potential therapeutic targeting of these cells.

ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways

Abstract: The interferon-inducible protein Z-DNA binding protein 1 (ZBP1, also known as DNA-dependent activator of IFN-regulatory factors (DAI) and DLM-1) was identified as a dsDNA sensor, which instigates innate immune responses. However, this classification has been disputed and whether ZBP1 functions as a pathogen sensor during an infection has remained unknown. Herein, we demonstrated ZBP1-mediated sensing of the influenza A virus (IAV) proteins NP and PB1, triggering cell death and inflammatory responses via the RIPK1-RIPK3-Caspase-8 axis. ZBP1 regulates NLRP3 inflammasome activation as well as induction of apoptosis, necroptosis and pyroptosis in IAV-infected cells. Importantly, ZBP1 deficiency protected mice from mortality during IAV infection owing to reduced inflammatory responses and epithelial damage. Overall, these findings indicate that ZBP1 is an innate immune sensor of IAV and highlight its importance in the pathogenesis of IAV infection.

The TCF1-Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness.

Abstract: During chronic viral infections and in cancer, T cells become dysfunctional, a state known as T cell exhaustion. Although it is well recognized that memory CD8 T cells account for the persistence of CD8 T cell immunity after acute infection, how exhausted T cells persist remains less clear. Using chronic infection with lymphocytic choriomeningitis virus clone 13 and tumor samples, we demonstrate that CD8 T cells differentiate into a less exhausted TCF1high and a more exhausted TCF1low population. Virus-specific TCF1high CD8 T cells, which resemble T follicular helper (TFH) cells, persist and recall better than do TCF1low cells and act as progenitor cells to replenish TCF1low cells. We show that TCF1 is both necessary and sufficient to support this progenitor-like CD8 subset, whereas cell-intrinsic type I interferon signaling suppresses their differentiation. Accordingly, cell-intrinsic TCF1 deficiency led to a loss of these progenitor CD8 T cells, sharp contraction of virus-specific T cells, and uncontrolled viremia. Mechanistically, TCF1 repressed several pro-exhaustion factors and induced Bcl6 in CD8 T cells, which promoted the progenitor fate. We propose that the TCF1-Bcl6 axis counteracts type I interferon to repress T cell exhaustion and maintain T cell stemness, which is critical for persistent antiviral CD8 T cell responses in chronic infection. These findings provide insight into the requirements for persistence of T cell immune responses in the face of exhaustion and suggest mechanisms by which effective T cell–mediated immunity may be enhanced during chronic infections and cancer.

Rapid profiling of RSV antibody repertoires from the memory B cells of naturally infected adult donors

Abstract: Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in young children and the elderly. There are currently no licensed RSV vaccines, and passive prophylaxis with the monoclonal antibody palivizumab is restricted to high-risk infants in part due to its modest efficacy. Although it is widely agreed that an effective RSV vaccine will require the induction of a potent neutralizing antibody response against the RSV fusion (F) glycoprotein, little is known about the specificities and functional activities of RSV F-specific antibodies induced by natural infection. Here, we have comprehensively profiled the human antibody response to RSV F by isolating and characterizing 364 RSV F-specific monoclonal antibodies from the memory B cells of three healthy adult donors. In all donors, the antibody response to RSV F is comprised of a broad diversity of clones that target several antigenic sites. Nearly half of the most potent antibodies target a previously undefined site of vulnerability near the apex of the prefusion conformation of RSV F (preF), providing strong support for the development of RSV vaccine candidates that preserve the membrane-distal hemisphere of the preF protein. Additionally, the antibodies targeting this new site display convergent sequence features, thus providing a future means to rapidly detect the presence of these antibodies in human vaccine samples. Many of the antibodies that bind preF-specific surfaces are over 100 times more potent than palivizumab, and several cross-neutralize human metapneumovirus (HMPV). Taken together, the results have implications for the design and evaluation of RSV vaccine candidates and offer new options for passive prophylaxis.

Class I HLA haplotypes form two schools that educate NK cells in different ways

Abstract: Natural killer (NK) cells are lymphocytes having vital functions in innate and adaptive immunity, as well as placental reproduction. Controlling education and functional activity of human NK cells are various receptors that recognize HLA class I on the surface of tissue cells. Epitopes of polymorphic HLA-A,-B and -C are recognized by equally diverse killer cell immunoglobulin-like receptors (KIR). In addition, a peptide cleaved from the leader sequence of HLA-A,-B or -C must bind to HLA-E for it to become a ligand for the conserved CD94:NKG2A receptor. Methionine/threonine dimorphism at position -21 of the leader sequence divides HLA-B allotypes into a majority having -21T that do not supply HLA-E binding peptides and a minority having -21M, that do. Genetic analysis of human populations worldwide shows how haplotypes with -21M HLA-B rarely encode the KIR ligands: Bw4+HLA-B and C2+HLA-C KIR. Thus there are two fundamental forms of HLA haplotype: one preferentially supplying CD94:NKG2A ligands, the other preferentially supplying KIR ligands. -21 HLA-B dimorphism divides the human population into three groups: M/M, M/T and T/T. Mass cytometry and assays of immune function, shows how M/M and M/T individuals have CD94:NKG2A+ NK cells which are better educated, phenotypically more diverse and functionally more potent than those in T/T individuals. Fundamental new insights are given to genetic control of NK cell immunity and the evolution that has limited the number of NK cell receptor ligands encoded by an HLA haplotype. These finding suggest new ways to dissect the numerous clinical associations with HLA class I.

The intersection of radiotherapy and immunotherapy: Mechanisms and clinical implications

Abstract: By inducing DNA damage, radiotherapy both reduces tumor burden and enhances anti-tumor immunity. Here, we will review the mechanisms by which radiation induces anti-tumor immune responses that can be augmented using immunotherapies to facilitate tumor regression. Radiotherapy increases inflammation in tumors by activating the NF-κB and the Type I interferon response pathways to induce expression of pro-inflammatory cytokines. This inflammation coupled with antigen release from irradiated cells facilitates dendritic cell maturation and cross-presentation of tumor antigens to prime tumor-specific T cell responses. Radiation also sensitizes tumors to these T cell responses by enhancing T cell infiltration into tumors and the recognition of both malignant cancer cells and non-malignant stroma that present cognate antigen. Yet, these anti-tumor immune responses may be blunted by several mechanisms including regulatory T cells and checkpoint molecules that promote T cell tolerance and exhaustion. Consequently, the combination of immunotherapy using vaccines and/or checkpoint inhibitors with radiation is demonstrating early clinical potential. Overall, this review will provide a global view for how radiation and the immune system converge to target cancers and the early attempts to exploit this synergy in clinical practice.

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation

Abstract: Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host’s lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

Partial exhaustion of CD8 T cells and clinical response to teplizumab in new-onset type 1 diabetes

Abstract: Biologic treatment of type 1 diabetes (T1D) typically results in transient stabilization of C-peptide levels (a surrogate for endogenous insulin secretion) in some patients, followed by progression at the same rate as in untreated control groups. We used integrated systems biology and flow cytometry approaches with clinical trial blood samples to elucidate pathways associated with C-peptide stabilization in T1D patients treated with the anti-CD3 monoclonal antibody teplizumab. We identified a population of CD8 T cells that accumulated in patients with the best response to treatment (responders) and showed that these cells phenotypically resembled exhausted T cells by expressing high levels of the transcription factor EOMES, effector molecules, and multiple inhibitory receptors (IRs), including TIGIT and KLRG1. These cells expanded after treatment, with levels peaking after 3 to 6 months. To functionally characterize these exhausted-like T cells, we isolated memory CD8 TIGIT + KLRG1 + T cells from responders and showed that they exhibited expanded T cell receptor clonotypes (indicative of previous in vivo expansion), recognized a broad-based spectrum of environmental antigens and autoantigens, and were hypoproliferative during polyclonal stimulation, increasing expression of IR genes and decreasing cell cycle genes. Triggering these cells with a recombinant ligand for TIGIT during polyclonal stimulation further down-regulated their activation, demonstrating that their exhausted phenotype was not terminal. These findings identify and functionally characterize a partially exhausted cell type associated with response to teplizumab therapy and suggest that pathways regulating T cell exhaustion may play a role in successful immune interventions for T1D.

In-depth tissue profiling using multiplexed immunohistochemical consecutive staining on single slide

Abstract: Despite remarkable recent achievements of immunotherapy strategies in cancer treatment, clinical responses remain limited to subsets of patients. Predictive markers of disease course and response to immunotherapy are urgently needed. Recent results have revealed the potential predictive value of immune cell phenotype and spatial distribution at the tumor site, prompting the need for multidimensional immunohistochemical analyses of tumor tissues. To address this need, we developed a sample-sparing, highly multiplexed immunohistochemistry technique based on iterative cycles of tagging, image scanning, and destaining of chromogenic substrate on a single slide. This assay, in combination with a newly developed automated digital landscaping solution, democratizes access to high-dimensional immunohistochemical analyses by capturing the complexity of the immunome using routine pathology standards. Applications of the method extend beyond cancer to screen and validate comprehensive panels of tissue-based prognostic and predictive markers, perform in-depth in situ monitoring of therapies, and identify targets of disease.

Human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment

Abstract: In mice, conventional and plasmacytoid dendritic cells (DCs) derive from separate hematopoietic precursors before they migrate to peripheral tissues. Moreover, two classes of conventional DCs (cDC1 and cDC2 DCs) and one class of plasmacytoid DCs (pDCs) have been shown to be transcriptionally and functionally distinct entities. In humans, these three DC subtypes can be identified using the cell surface markers CD1c (cDC2), CD141 (cDC1), and CD303 (pDCs), albeit it remains elusive whether DC functionality is mainly determined by ontogeny or the tissue microenvironment. By phenotypic and transcriptional profiling of these three DC subtypes in different human tissues derived from a large number of human individuals, we demonstrate that DC subpopulations in organs of the lymphohematopoietic system (spleen, thymus, and blood) are strongly defined by ontogeny rather than by signals from the microenvironment. In contrast, DC subsets derived from human lung or skin differed substantially, strongly arguing that DCs react toward modulatory signals from tissue microenvironments. Collectively, the data obtained in this study may serve as a major resource to guide further studies into human DC biology during homeostasis and inflammation.

Long-term maintenance of human naïve T cells through in situ homeostasis in lymphoid tissue sites

Abstract: Naive T cells develop in the thymus and coordinate immune responses to new antigens; however, mechanisms for their long-term persistence over the human lifespan remain undefined. Here, we investigated human naive T cell development and maintenance in primary and secondary lymphoid tissues obtained from individual organ donors aged 3 months-73 years. In the thymus, the frequency of double-positive thymocytes declined sharply in donors over age 40 coincident with reduced recent thymic emigrants (RTE) in lymphoid tissues, while naive T cells were functionally maintained predominantly in lymph nodes (LN). Analysis of TCR clonal distribution by CDR3 sequencing of naive CD4+ and CD8+ T cells in spleen and LNs reveal site-specific clonal expansions of naive T cells from individuals >40 years of age with minimal clonal overlap between lymphoid tissues. We also identified biased naive T cell clonal distribution within specific lymph nodes based on VJ usage. Together these results suggest prolonged maintenance of naive T cells through in situ homeostasis and retention in lymphoid tissue.

IL-1β is an innate immune sensor of microbial proteolysis

Abstract: Interleukin-1β (IL-1β) is a key proinflammatory cytokine that drives antimicrobial immune responses. IL-1β is aberrantly activated in autoimmune diseases, and IL-1β inhibitors are used as therapeutic agents to treat patients with certain autoimmune disorders. Review of postmarketing surveillance of patients receiving IL-1β inhibitors found a disproportionate reporting of invasive infections by group A Streptococcus (GAS). IL-1β inhibition increased mouse susceptibility to GAS infection, but IL-1β was produced independent of canonical inflammasomes. Newly synthesized IL-1β has an amino-terminal prodomain that blocks signaling activity, which is usually proteolytically removed by caspase-1, a protease activated within the inflammasome structure. In place of host caspases, the secreted GAS cysteine protease SpeB generated mature IL-1β. During invasive infection, GAS isolates may acquire pathoadaptive mutations eliminating SpeB expression to evade detection by IL-1β. Pharmacological IL-1β inhibition alleviates this selective pressure, allowing invasive infection by nonpathoadapted GAS. Thus, IL-1β is a sensor that directly detects pathogen-associated proteolysis through an independent pathway operating in parallel with host inflammasomes. Because IL-1β function is maintained across species, yet cleavage by caspases does not appear to be, detection of microbial proteases may represent an ancestral system of innate immune regulation.

Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome

Abstract: One paradigm in transplantation is that graft-infiltrating T cells are largely nonalloreactive “bystander” cells. However, the origin and specificity of allograft T cells over time have not been investigated in detail in animals or humans. We used polychromatic flow cytometry and high-throughput T cell receptor sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intragraft host-versus-graft (HvG) and graft-versus-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted in the long term in the graft. Early expansion of GvH clones in the graft correlated with the rapid replacement of donor antigen-presenting cells by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28 + NKG2D Hi CD8 + αβ T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady-state tissue-resident phenotype but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in nonrejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.

Immune perturbations in HIV-1–infected individuals who make broadly neutralizing antibodies

Abstract: Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. bnAbs occur in some HIV-1–infected individuals and frequently have characteristics of autoantibodies. We have studied cohorts of HIV-1–infected individuals who made bnAbs and compared them with those who did not do so, and determined immune traits associated with the ability to produce bnAbs. HIV-1–infected individuals with bnAbs had a higher frequency of blood autoantibodies, a lower frequency of regulatory CD4 + T cells, a higher frequency of circulating memory T follicular helper CD4 + cells, and a higher T regulatory cell level of programmed cell death–1 expression compared with HIV-1–infected individuals without bnAbs. Thus, induction of HIV-1 bnAbs may require vaccination regimens that transiently mimic immunologic perturbations in HIV-1–infected individuals.

Inflammatory monocytes hinder antiviral B cell responses.

Abstract: Antibodies are critical for protection against viral infections However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms We analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of draining lymph nodes, where they extensively interact with CD11b + Ly6C hi inflammatory monocytes These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon– and CCR2-dependent fashion, and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide Depletion of inflammatory monocytes, inhibition of their lymph node recruitment, or impairment of their nitric oxide–producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production Our results identify inflammatory monocytes as critical gatekeepers that restrain antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host

Inhibition of HDAC8 and HDAC9 by microbial short-chain fatty acids breaks immune tolerance of the epidermis to TLR ligands

Abstract: Epidermal keratinocytes participate in immune defense through their capacity to recognize danger, trigger inflammation, and resist infection However, normal skin immune function must tolerate contact with an abundant community of commensal microbes without inflammation We hypothesized that microbial environmental conditions dictate the production of molecules that influence epigenetic events and cause keratinocytes to break innate immune tolerance Propionibacterium acnes, a commensal skin bacterium, produced the short-chain fatty acids (SCFAs) propionate and valerate when provided a lipid source in hypoxic growth conditions, and these SCFAs inhibited histone deacetylase (HDAC) activity Inhibition of HDAC activity in keratinocytes promoted cytokine expression in response to Toll-like receptor (TLR) ligands for TLR2 or TLR3 This response was opposite to the action of HDAC inhibition on production of inflammatory cytokines by monocytes and involved HDAC8 and HDAC9 because small interfering RNA silencing of these HDACs recapitulated the activity of SCFAs Analysis of cytokine expression in mice confirmed the response of the epidermis where application of SCFA on the skin surface promoted cytokine expression, whereas subcutaneous administration was inhibitory These findings show that the products of commensal microbes made under specific conditions will inhibit HDAC activity and break tolerance of the epidermis to inflammatory stimuli

Interferon-driven deletion of antiviral B cells at the onset of chronic infection.

Abstract: Inadequate antibody responses and perturbed B cell compartments represent hallmarks of persistent microbial infections, but the mechanisms whereby persisting pathogens suppress humoral immunity remain poorly defined Using adoptive transfer experiments in the context of a chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, we have documented rapid depletion of virus-specific B cells that coincided with the early type I interferon response to infection We found that the loss of activated B cells was driven by type I interferon (IFN-I) signaling to several cell types including dendritic cells, T cells and myeloid cells Intriguingly, this process was independent of B cell-intrinsic IFN-I sensing and resulted from biased differentiation of naive B cells into short-lived antibody-secreting cells The ability to generate robust B cell responses was restored upon IFN-I receptor blockade or, partially, when experimentally depleting myeloid cells or the IFN-I-induced cytokines interleukin 10 and tumor necrosis factor alpha We have termed this IFN-I-driven depletion of B cells "B cell decimation" Strategies to counter "B cell decimation" should thus help us better leverage humoral immunity in the combat against persistent microbial diseases

Type I interferon suppresses virus-specific B cell responses by modulating CD8 + T cell differentiation.

Abstract: Studies have established a role for T cells in resolving persistent viral infections, yet emerging evidence indicates that both T and B cells are required to control some viruses During persistent infection, a marked lag or failure to generate neutralizing antibodies is commonly observed and likely contributes to an inability to control certain pathogens Using lymphocytic choriomeningitis virus (LCMV) as a model, we have examined how a persistent viral infection can suppress neutralizing humoral immunity By tracking the fate of virus-specific B cells in vivo, we report that LCMV-specific B cells were rapidly deleted within a few days of persistent infection, and this deletion was completely reversed by blockade of type I interferon (IFN-I) signaling Early interference with IFN-I signaling promoted survival and differentiation of LCMV-specific B cells, which accelerated the generation of neutralizing antibodies This marked improvement in antiviral humoral immunity did not rely on the cessation of IFN-I signaling in B cells but on alterations in the virus-specific CD8+ T cell response Using two-photon microscopy and in vivo calcium imaging, we observed that cytotoxic T lymphocytes (CTLs) productively engaged and killed LCMV-specific B cells in a perforin-dependent manner within the first few days of infection Blockade of IFN-I signaling protected LCMV-specific B cells by promoting CTL dysfunction Therapeutic manipulation of this pathway may facilitate efforts to promote humoral immunity during persistent viral infection in humans Our findings illustrate how events that occur early after infection can disturb the resultant adaptive response and contribute to viral persistence

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Abstract: Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

The orphan nuclear receptor RORα and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease.

Abstract: Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. In Crohn's disease, this manifests itself as recurrent gastrointestinal strictures for which there is no effective therapy beyond surgical intervention. Using a model of infection-induced chronic gut inflammation, we show that Rora-deficient mice are protected from fibrosis; infected intestinal tissues display diminished pathology, attenuated collagen deposition, and reduced fibroblast accumulation. Although Rora is best known for its role in group 2 innate lymphoid cell (ILC2) development, we find that Salmonella-induced fibrosis is independent of eosinophils, signal transducer and activator of transcription 6 signaling, and T helper 2 cytokine production, arguing that this process is largely ILC2-independent. Instead, we observe reduced levels of ILC3- and T cell-derived interleukin-17A (IL-17A) and IL-22 in infected gut tissues. Furthermore, using Rorasg/sg /Rag1-/- bone marrow chimeric mice, we show that restoring ILC function is sufficient to reestablish IL-17A and IL-22 production and a profibrotic phenotype. Our results show that RORα (retinoic acid receptor-related orphan receptor α)-dependent ILC3 functions are pivotal in mediating gut fibrosis, and they offer an avenue for therapeutic intervention in Crohn's-like diseases.

Group B Streptococcus circumvents neutrophils and neutrophil extracellular traps during amniotic cavity invasion and preterm labor

Abstract: Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion of the amniotic cavity (MIAC) is associated with the majority of early preterm births, the temporal events that occur during MIAC and preterm labor are not known. Group B Streptococci (GBS) are β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur during MIAC, we utilized a unique chronically catheterized nonhuman primate model that closely emulates human pregnancy. This model allows monitoring of uterine contractions, timing of MIAC and immune responses during pregnancy-associated infections. Here, we show that adverse outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during infection with hemolytic GBS when compared to nonhemolytic GBS. Although MIAC was associated with systematic progression in chorioamnionitis beginning with chorionic vasculitis and progressing to neutrophilic infiltration, the ability of the GBS hemolytic pigment toxin to induce neutrophil cell death and subvert killing by neutrophil extracellular traps (NETs) in placental membranes in vivo facilitated MIAC and fetal injury. Furthermore, compared to maternal neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired phagocytic functions to GBS. Collectively, our studies demonstrate how a unique bacterial hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to induce fetal injury and preterm labor.

Exploiting a host-commensal interaction to promote intestinal barrier function and enteric pathogen tolerance

Abstract: Commensal intestinal bacteria can prevent pathogenic infection; however, limited knowledge of the mechanisms by which individual bacterial species contribute to pathogen resistance has restricted their potential for therapeutic application. Here, we examined how colonization of mice with a human commensal Enterococcus faecium protects against enteric infections. We show that E. faecium improves host intestinal epithelial defense programs to limit Salmonella enterica serotype Typhimurium pathogenesis in vivo in multiple models of susceptibility. E. faecium protection is mediated by a unique peptidoglycan hydrolase, SagA, and requires epithelial expression of pattern recognition receptor components and antimicrobial peptides. Ectopic expression of SagA in non-protective and probiotic bacteria is sufficient to enhance intestinal barrier function and confer resistance against S. Typhimurium and Clostridium difficile pathogenesis. These studies demonstrate that specific factors from commensal bacteria can be used to improve host barrier function and limit the pathogenesis of distinct enteric infections.

The discontinuity theory of immunity

Abstract: Similar to many other biological systems, the immune system can be seen as a change-detection system. According to the discontinuity theory of immunity, the immune system responds to sudden changes in antigenic stimulation and is rendered tolerant by slow or continuous stimulation. This basic principle, which is supported by recent data on immune checkpoints in viral infections, cancers, and allergies, can be seen as a unifying framework for diverse immune responses.

Myosin light chains 9 and 12 are functional ligands for CD69 that regulate airway inflammation.

Abstract: Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4+ T helper 2 cells play critical roles in the pathogenesis of allergic airway inflammation, and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention through inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and Myl12 are new functional ligands for CD69. Blockade of CD69-Myl9/12 interaction ameliorates allergic airway inflammation in ovalbumin-induced and house dust mite-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of Myl9/12 was increased and that platelet-derived Myl9/12 localized to the luminal surface of blood vessels and formed intravascular net-like structures. Analysis of nasal polyps of eosinophilic chronic rhinosinusitis patients revealed that Myl9/12 expression was increased in inflammatory lesions and was distributed within net-like structures in the intravascular space. In addition, we detected Myl9/12 in perivascular spaces where many CD69+ cells were positioned within Myl9/12 structures. Thus, CD69-Myl9/12 interaction is a key event in the recruitment of activated CD69+ T cells to inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases.

PTPN22 inhibition resets defective human central B cell tolerance.

Abstract: The 1858T protein tyrosine phosphatase nonreceptor type 22 (PTPN22 T) allele is one of the main risk factors associated with many autoimmune diseases and correlates with a defective removal of developing autoreactive B cells in humans. To determine whether inhibiting PTPN22 favors the elimination of autoreactive B cells, we first demonstrated that the PTPN22 T allele interfered with the establishment of central B cell tolerance using NOD-scid-common γ chain knockout (NSG) mice engrafted with human hematopoietic stem cells expressing this allele. In contrast, the inhibition of either PTPN22 enzymatic activity or its expression by RNA interference restored defective central B cell tolerance in this model. Thus, PTPN22 blockade may represent a therapeutic strategy for the prevention or treatment of autoimmunity.

Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies.

Abstract: A subset of bovine antibodies have an exceptionally long third heavy-chain complementarity determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. Three new bovine Fab crystal structures have been determined to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, while the knob regions share a conserved β-sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the knob structural diversity. The unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems.

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites

Abstract: Developmental checkpoints eliminate B cells that synthesize defective immunoglobulin (Ig) heavy (HC) and light (LC) chains. The first checkpoint tests μHCs paired with VpreB/λ5 in a pre-B cell receptor (pre-BCR) to determine whether the μHC will be able to bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with the μHC antigen-binding region complementarity-determining region (CDR)–H3; however, whether this site contributes to Ig repertoire selection and function is unknown. We analyzed the amino acid content of CDR-H3s from HCs cloned from living and apoptotic pre-B cells and from IgG-antigen structures. Using a panel of D H gene–targeted mice, we showed that progressively reducing CDR-H3 tyrosine content increasingly impaired pre-BCR checkpoint passage. Counting from cysteine at framework 3 position 96, we found that VpreB particularly selected for tyrosine at CDR-H3 position 101 and that Y101 also bound antigen in IgG-antigen structures. Therefore, in addition to its stabilization role in the pre-BCR, VpreB also acts as an early invariant antigen by selecting for particular CDR-H3 amino acids. These interactions shape the specificity of the IgG humoral response and may thus impose limitations on development of certain neutralizing antibodies.

Tissue-specific emergence of regulatory and intraepithelial T cells from a clonal T cell precursor

Abstract: Peripheral Foxp3+ regulatory T cells (pTregs) maintain immune homeostasis by controlling potentially harmful effector T cell responses toward dietary and microbial antigens. Although the identity of the T cell receptor (TCR) can impose commitment and functional specialization of T cells, less is known about how TCR identity governs pTreg development from conventional CD4+ T cells. To investigate the extent to which TCR identity dictates pTreg fate, we used somatic cell nuclear transfer to generate a transnuclear (TN) mouse carrying a monoclonal TCR from a pTreg (pTreg TN mice). We found that the pTreg TCR did not inevitably predispose T cells to become pTreg but instead allowed for differentiation of noninflammatory CD4+CD8αα+ intraepithelial lymphocytes (CD4IELs) in the small intestine. Only when we limited the number of T cell precursors that carried the TN pTreg TCR did we observe substantial pTreg development in the mesenteric lymph nodes and small intestine lamina propria of mixed bone marrow chimeras. Small clonal sizes and therefore decreased intraclonal competition were required for pTreg development. Despite bearing the same TCR, small intestine CD4IEL developed independently of precursor frequency. Both pTreg and CD4IEL development strictly depended on the resident microbiota. A single clonal CD4+ T cell precursor can thus give rise to two functionally distinct and anatomically segregated T cell subsets in a microbiota-dependent manner. Therefore, plasticity of the CD4 T cell compartment depends not only on the microbiota but also on specialized environmental cues provided by different tissues.

A TSLP-complement axis mediates neutrophil killing of methicillin-resistant Staphylococcus aureus

Abstract: Community-acquired Staphylococcus aureus infections often present as serious skin infections in otherwise healthy individuals and have become a worldwide epidemic problem fueled by the emergence of strains with antibiotic resistance, such as methicillin-resistant S. aureus (MRSA). The cytokine thymic stromal lymphopoietin (TSLP) is highly expressed in the skin and in other barrier surfaces and plays a deleterious role by promoting T helper cell type 2 (TH2) responses during allergic diseases; however, its role in host defense against bacterial infections has not been well elucidated. We describe a previously unrecognized non-TH2 role for TSLP in enhancing neutrophil killing of MRSA during an in vivo skin infection. Specifically, we demonstrate that TSLP acts directly on both mouse and human neutrophils to augment control of MRSA. Additionally, we show that TSLP also enhances killing of Streptococcus pyogenes, another clinically important cause of human skin infections. Unexpectedly, TSLP mechanistically mediates its antibacterial effect by directly engaging the complement C5 system to modulate production of reactive oxygen species by neutrophils. Thus, TSLP increases MRSA killing in a neutrophil- and complement-dependent manner, revealing a key connection between TSLP and the innate complement system, with potentially important therapeutic implications for control of MRSA infection.

The human thymus perivascular space is a functional niche for viral-specific plasma cells

Abstract: The human thymus is susceptible to viral infections that can severely alter thymopoiesis and compromise the mechanisms of acquired tolerance to self-antigens. In humans, plasma cells (PCs) residing primarily in the bone marrow confer long-lasting protection to common viruses by secreting antigen-specific antibodies. Because the thymus also houses B cells, we examined the phenotypic complexity of these thymic resident cells and their possible protective role against viral infections. Using tissue specimens collected from patients ranging in age from 5 days to 71 years, we found that, starting during the first year of life, CD138 + PCs begin accumulating in the thymic perivascular space (PVS), where they constitutively produce immunoglobulin G (IgG) without the need for additional stimulation. These thymic PCs almost exclusively secrete IgG1 and IgG3, the two main complement-fixing effector IgG subclasses. Moreover, using antigen-specific enzyme-linked immunospot assays, we demonstrated that thymic PCs include a high frequency of cells reactive to common viral proteins. Our study reveals an unrecognized role of the PVS as a functional niche for viral-specific PCs. The PVS is located between the thymic epithelial areas and the circulation. PCs located in this compartment may therefore provide internal protection against pathogen infections and preserve the integrity and function of the organ.