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Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c+CD5+ DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5+ DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5hi T helper and CD8+ T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5+ DCs are an essential component of optimal ICB therapy.

Follicular CD8+CXCR5+ T cells are a specialized CD8+ T cell subset with unique follicular-homing capabilities that have been reported to display effector functions in viral immunity, tumor immunity, and autoimmunity. CD8+CXCR5+ T cells exhibit B cell helper functions and express CD40L, ICOS, programmed cell death protein 1 (PD-1), and BCL-6, the transcriptional regulator of CD4+CXCR5+ T follicular helper cells and of germinal center B cells. HIV is known to be sequestered in lymphoid follicles, and CD8+CXCR5+ T cell frequency is a marker for disease severity, given that HIV-infected patients with lower numbers of circulating CD8+CXCR5+ T cells display lower CD4+ T cell counts. Likewise, several groups have reported a direct correlation between the quantity of CD8+CXCR5+ T cells and suppression of HIV viral load. In this study, we observed elevated absolute numbers of CD8+CXCR5+ and CD8+CXCR5+BCL-6+PD-1+ T cells in the blood of HIV-infected participants of the Multicenter AIDS Cohort Study. We further demonstrated in vitro that activated human CD8+CXCR5+ T cells isolated from peripheral blood and tonsil from healthy donors show increased CD40L expression and induce the production of PD ligand 1 (PD-L1)+IgG+ B cells. Moreover, absolute numbers of CD8+CXCR5+ T cells significantly and positively correlated with numbers of PD-L1+ B cells found in blood of HIV-infected individuals. Altogether, these results show that activated CD8+CXCR5+ T cells have the ability to activate B cells and increase the percentage of PD-L1+ and PD-L1+IgG+ B cells, which provides insights into the early events of B cell activation and differentiation and may play a role in disease progression and lymphomagenesis in HIV-infected individuals.

Humoral immune response is important in fighting pathogens by the production of specific antibodies by B cells. In germinal centers, T follicular helper (TFH) cells provide important help to B-cell antibody production but also contribute to HIV persistence. T follicular regulatory (TFR) cells, which inhibit the function of TFH cells, express similar surface markers. Since FOXP3 is the only marker that distinguishes TFR from TFH cells it is unknown whether the increase in TFH cells observed in HIV infection and HIV persistence may be partly due to an increase in TFR cells. Using multicolor flow cytometry to detect TFH and TFR cells in cryopreserved peripheral blood mononuclear cells from HIV-infected and non-infected participants in the UCLA Multicenter AIDS Cohort Study (MACS), we identified CD3+CXCR5+CD4+CD8-BCL6+ peripheral blood TFH (pTFH) cells and CD3+CXCR5+CD4+CD8-FOXP3+ peripheral blood TFR (pTFR) cells. Unlike TFR cells in germinal centers, pTFR cells do not express B cell lymphoma 6 (BCL6), a TFH cell master transcriptional regulator. Our major findings are that the frequency of pTFH cells, but not pTFR cells was higher in HIV-infected participants of the MACS and that pTFH cells expressed less CCR5 in HIV-infected MACS participants. Constitutive expression of CCR5 in TFR cells supports their potential to contribute to HIV persistence.

Objective: Investigating the changes in the oxidative stress levels and helper T lymphocyte (Th) subsets in patients with periodontitis and IgA nephropathy (IgAN) to determine their relationship. Background: IgAN has a high prevalence, poor prognosis, and no effective cure. Accumulating evidence has implicated a close relationship between periodontitis and chronic kidney diseases, in which both IgAN and chronic periodontitis show chronic inflammation and abnormal metabolism. However, few studies have been conducted on the relationship between the two diseases from this perspective. Methods: We divided 86 IgAN patients into patients with healthy periodontium (IgAN‐H, n = 34) and patients with periodontitis IgAN (IgAN‐P, n = 52); moreover, we divided 72 systemically healthy participants into patients with periodontitis (H‐P, n = 35) and participants with healthy periodontium (H‐H, n = 37). The proportions of Th subsets in peripheral blood were estimated using flow cytometry. Cytokine levels in plasma were assessed using cytokine assay kits. Enzyme‐linked immunosorbent assay was used to evaluate the plasma levels of oxidative stress. Results: Our results from analyzing the Th cell subsets indicated that Th2 cell counts in the IgAN‐P group were significantly lower than those in the IgAN‐H group, while Th17 cell counts were increased (p < 0.05). Moreover, the Th1/Th2 ratio and interleukin‐6 levels in the IgAN‐P group were significantly higher than those in the H‐H group (p < 0.01). Compared with that in the H‐H group, in the remaining three groups, plasma total oxidation state (TOS) levels were increased (p < 0.01), while plasma total antioxidant state (TAS) levels were decreased (p < 0.05). Furthermore, estimated glomerular filtration rate was negatively correlated with the probing depth and gingival bleeding index. IgAN was a risk factor for periodontitis, while TAS was a protective factor for periodontitis. The oxidative stress index (OSI) might be valuable for distinguishing periodontitis patients from healthy controls (area under the receiver operator characteristic curve = 0.951). Conclusion: IgAN is an independent risk factor of periodontitis, and the Th17 cell‐mediated inflammatory response might be associated with the occurrence of periodontitis in patients with IgAN. Patients with coexisting IgAN and periodontitis exhibit increased oxidative stress, in which TOS and OSI are potential biomarkers for diagnosing periodontitis. [ABSTRACT FROM AUTHOR]

Antibody-mediated adaptive immunity must provide effective long-term protection with minimal adverse effects, against rapidly mutating pathogens, in a human population with diverse ages, genetics, and immune histories. In order to grasp and leverage the complexities of the antibody response, we advocate for a mechanistic understanding of the multiscale germinal center (GC) reaction - the process by which precursor B-cells evolve high-affinity antigen-specific antibodies, forming an effector repertoire of plasma and memory cells for decades-long protection. The regulatory dynamics of B-cells within the GC are complex, and unfold across multiple interacting spatial and temporal scales. At the organism scale, over weeks to years, the antibody sequence repertoire formed by various B-cell clonal lineages modulates antibody quantity and quality over time. At the tissue and cellular scale, over hours to weeks, B-cells undergo selection via spatially distributed interactions with local stroma, antigen, and helper T-cells. At the molecular scale, over seconds to days, intracellular signaling, transcriptional, and epigenetic networks modulate B-cell fates and shape their clonal lineages. We summarize our current understanding within each of these scales, and identify missing links in connecting them. We suggest that quantitative multi-scale mathematical models of B-cell and GC reaction dynamics provide predictive frameworks that can apply basic immunological knowledge to practical challenges such as rational vaccine design.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

Systemic lupus erythematosus (SLE) is an incurable autoimmune disease disproportionately affecting women. A major obstacle in finding targeted therapies for SLE is its remarkable heterogeneity in clinical manifestations as well as in the involvement of distinct cell types. To identify cell-specific targets as well as cross-correlation relationships among expression programs of different cell types, we here analyze six major circulating immune cell types from SLE patient blood. Our results show that presence of an interferon response signature stratifies patients into two distinct groups (IFNneg vs. IFNpos). Comparing these two groups using differential gene expression and differential gene coexpression analysis, we prioritize a relatively small list of genes from classical monocytes including two known immune modulators: TNFSF13B/BAFF (target of belimumab, an approved therapeutic for SLE) and IL1RN (the basis of anakinra, a therapeutic for rheumatoid arthritis). We then develop a multi-cell type extension of the weighted gene coexpression network analysis (WGCNA) framework, termed mWGCNA. Applying mWGCNA to RNA-seq data from six sorted immune cell populations (15 SLE, 10 healthy donors), we identify a coexpression module with interferon-stimulated genes (ISGs) among all cell types and a cross-cell type correlation linking expression of specific T helper cell markers to B cell response as well as to TNFSF13B expression from myeloid cells, all of which in turn correlates with disease severity of IFNpos patients. Our results demonstrate the power of a hypothesis-free and data-driven approach to discover drug targets and to reveal novel cross-correlation across cell types in SLE with implications for other autoimmune diseases.

Ambient air pollution exposure is associated with cardiovascular dysregulation and immune system alterations, yet no study has investigated both simultaneously in children. Understanding the multifaceted impacts may provide early clues for clinical intervention prior to actual disease presentation. We therefore determined the associations between exposure to multiple air pollutants and both immunological outcomes (methylation and protein expression of immune cell types associated with immune regulation) and cardiovascular outcomes (blood pressure) in a cohort of school-aged children (6-8 years; n = 221) living in a city with known elevated pollution levels. Exposure to fine particular matter (PM2.5), carbon monoxide (CO), and ozone (O3) was linked to altered methylation of most CpG sites for genes Foxp3, IL-4, IL-10 and IFN-g, all involved in immune regulation (e.g. higher PM2.5 exposure 1 month prior to the study visit was independently associated with methylation of the IL-4 CpG24 site (est = 0.16; P = 0.0095). Also, immune T helper cell types (Th1, Th2 and Th17) were associated with short-term exposure to PM2.5, O3 and CO (e.g. Th1 cells associated with PM2.5 at 30 days: est = - 0.34, P

Background: Decidual macrophages participate in immune regulation at the maternal–fetal interface. Abnormal M1/M2 polarization of decidual macrophages might predispose immune maladaptation in recurrent pregnancy loss (RPL). However, the mechanism of decidual macrophage polarization is unclear. We explored the role of Estradiol (E2)-sensitive serum-glucocorticoid regulated kinase (SGK) 1 in promoting macrophage polarization and suppressing inflammation at the maternal–fetal interface. Methods: We assessed serum levels of E2 and progesterone during first trimester of pregnancy in women with or without threatened miscarriages (ended in live birth, n = 448; or early miscarriages, n = 68). For detection of SGK1 in decidual macrophages, we performed immunofluorescence labeling and western blot analysis applying decidual samples from RPL (n = 93) and early normal pregnancy (n = 66). Human monocytic THP-1 cells were differentiated into macrophages and treated with Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS), E2, inhibitors or siRNA for in vitro analysis. Flow cytometry analysis were conducted to detect macrophages polarization. We also applied ovariectomized (OVX) mice with hormones exploring the mechanisms underlying the regulation of SGK1 activation by E2 in the decidual macrophages in vivo. Results: SGK1 expression down regulation in the decidual macrophages of RPL was consistent with the lower concentration and slower increment of serum E2 from 4 to 12 weeks of gestation seen in these compromised pregnancies. LPS reduced SGK1 activities, but induced the pro-inflammatory M1 phenotype of THP-1 monocyte-derived macrophages and T helper (Th) 1 cytokines that favored pregnancy loss. E2 pretreatment promoted SGK1 activation in the decidual macrophages of OVX mice in vivo. E2 pretreatment amplified SGK1 activation in TLR4-stimulated THP-1 macrophages in vitro through the estrogen receptor beta (ERβ) and PI3K pathway. E2-sensitive activation of SGK1 increased M2 macrophages and Th2 immune responses, which were beneficial to successful pregnancy, by inducing ARG1 and IRF4 transcription, which are implicated in normal pregnancy. The experiments on OVX mice have shown that pharmacological inhibition of E2 promoted nuclear translocation of NF-κB in the decidual macrophages. Further more, pharmacological inhibition or knockdown of SGK1 in TLR4-stimulated THP-1 macrophages activated NF-κB by promoting its nuclear translocation, leading to increased secretion of pro-inflammatory cytokines involved in pregnancy loss. Conclusion: Our findings highlighted the immunomodulatory roles of E2-activated SGK1 in Th2 immune responses by priming anti-inflammatory M2 macrophages at the maternal–fetal interface, resulting in a balanced immune microenvironment during pregnancy. Our results suggest new perspectives on future preventative strategies for RPL. [ABSTRACT FROM AUTHOR]

Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

BackgroundCommon variable immunodeficiency (CVID) is characterized by low immunoglobulin G and IgA/IgM, decreased switched memory B cells, impaired response to vaccine, and an increased susceptibility to infections and autoimmunity. TFH cells play an important role in germinal center reaction where it supports isotype switching, somatic hypermutation, generation of memory B cells, and differentiation of B cells to plasma cells. The objective was to study the distribution of three subsets of TFH cells and their relationship with autoimmune diseases associated with CVID.MethodsTFH cells have been divided into TFH 1 (interleukin 21 [IL-21] and interferon γ), TFH 2 (IL-21 and IL-4), and TFH 17 (IL-21 and IL-17) cells. Mononuclear cells from 25 patients with CVID and age and gender-matched controls were stained with various monoclonal antibodies (anti-CD4 APC, anti-CXCR5 FITC, anti-CCR6 PerCP, and anti-CXCR3 PE) and isotype controls and analyzed for TFH 1 (CD4+ CXCR5+ CXCR3+ CCR6- ), TFH 2 (CD4+ CXCR5+ CXCR3- CCR6- ), and TFH 17 (CD4+ CXCR5+ CXCR3- CCR6+ ) cells by multicolor flow cytometry. Twenty thousand cells were acquired and analyzed by FlowJo software. Statistical analysis of comparison of patients and healthy controls was performed by paired t test using PRISM 7 software.ResultsTFH 2 and TFH 17 cells subpopulations of TFH cells were significantly decreased (P

The absence of alloantibodies is a feature of transplantation tolerance. Although the lack of T cell help has been evoked to explain this absence, herein we provide evidence for B cell-intrinsic tolerance mechanisms. Using a murine model of heart tolerance, we showed that alloreactive B cells were not deleted but rapidly lost their ability to differentiate into germinal center B cells and secrete donor-specific antibodies. We inferred that tolerant alloreactive B cells retained their ability to sense alloantigen because they continued to drive T cell maturation into CXCR5+PD-1+ T follicular helper cells. Unexpectedly, dysfunctional alloreactive B cells acquired the ability to inhibit antibody production by new naive B cells in an antigen-specific manner. Thus, tolerant alloreactive B cells contribute to transplantation tolerance by foregoing germinal center responses while retaining their ability to function as antigen-presenting cells and by actively suppressing de novo alloreactive B cell responses.

Control of diverse pathogens requires an adaptive antibody response, dependent on cellular division of labor to allocate antigen-dependent B- and CD4+ T-cell fates that collaborate to control the quantity and quality of antibody. This is orchestrated by the dynamic action of key transcriptional regulators mediating gene expression programs in response to pathogen-specific environmental inputs. We describe a conserved, likely ancient, gene regulatory network that intriguingly operates contemporaneously in B and CD4+ T cells to control their cell fate dynamics and thus, the character of the antibody response. The remarkable output of this network derives from graded expression, designated by antigen receptor signal strength, of a pivotal transcription factor that regulates alternate cell fate choices.

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) - an autoimmune disease of the CNS - increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.

Over the last century, the alarming surge in allergy and autoimmune disease has led to the hypothesis that decreasing exposure to microbes, which has accompanied industrialization and modern life in the Western world, has fundamentally altered the immune response. In its current iteration, the "hygiene hypothesis" suggests that reduced microbial exposures during early life restricts the production and differentiation of immune cells suited for immune regulation. Although it is now well-appreciated that the increase in hypersensitivity disorders represents a "perfect storm" of many contributing factors, we argue here that two important considerations have rarely been explored. First, the window of microbial exposure that impacts immune development is not limited to early childhood, but likely extends into the womb. Second, restricted microbial interactions by an expectant mother will bias the fetal immune system toward hypersensitivity. Here, we extend this discussion to hypothesize that the cell types sensing microbial exposures include fetal hematopoietic stem cells, which drive long-lasting changes to immunity.

Follicular helper T (TFH) cells are essential for generating protective humoral immunity. To date, microRNAs (miRNAs) have emerged as important players in regulating TFH cell biology. Here, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated TFH cell frequencies upon different immune challenges, whereas overexpression of this miRNA family led to reduced TFH cell responses. Mechanistically, miR-23~27~24 clusters coordinately control TFH cells through targeting a network of genes that are crucial for TFH cell biology. Among them, thymocyte selection-associated HMG-box protein (TOX) was identified as a central transcription regulator in TFH cell development. TOX is highly up-regulated in both mouse and human TFH cells in a BCL6-dependent manner. In turn, TOX promotes the expression of multiple molecules that play critical roles in TFH cell differentiation and function. Collectively, our results establish a key miRNA regulon that maintains optimal TFH cell responses for resultant humoral immunity.

BackgroundIn 6.5 years of newborn screening for severe combined immunodeficiency in California, 3,252,156 infants had DNA from dried blood spots (DBSs) assayed for T-cell receptor excision circles. Infants with T-cell receptor excision circle values of less than a designated cutoff on a single DBS, 2 DBS samples with insufficient PCR amplification, or known genetic risk of immunodeficiency had peripheral blood complete blood counts and lymphocyte subsets assayed in a single flow cytometry laboratory. Cases in which immune defects were ruled out were available for analysis.ObjectiveWe sought to determine reference intervals for lymphocyte subsets in racially/ethnically diverse preterm and term newborns who proved to be unaffected by any T-lymphopenic immune disorder.MethodsEffective gestational age (GA) was defined as GA at birth plus postnatal age at the time of sample collection. After determining exclusion criteria, we analyzed demographic and clinical information, complete and differential white blood cell counts, and lymphocyte subsets for 301 infants, with serial measurements for 33 infants. Lymphocyte subset measurements included total T cells, helper and cytotoxic T-cell subsets, naive and memory phenotype of each T-cell subset, B cells, and natural killer cells.ResultsReference intervals were generated for absolute numbers and lymphocyte subsets from infants with effective GAs of 22 to 52 weeks. Sex and ethnicity were not significant determinants of lymphocyte subset counts in this population. Lymphocyte counts increased postnatally.ConclusionThis study provides a baseline for interpreting comprehensive lymphocyte data in preterm and term infants, aiding clinicians to determine which newborns require further evaluations for immunodeficiency.

BackgroundCheckpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling.MethodsTo facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses.ResultsWe show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery.ConclusionsWe show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFβ-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFβ combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFβ/α-PD-1 combination therapy (NCT02947165).

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

The pleiotropic cytokine IL-6 plays an integral role not only in innate inflammatory responses but also in the activation and differentiation of lymphocyte subsets. In this study, by using a conditional knockout (cKO) model with selective IL-6 receptor deletion in T cells (IL-6R-cKO), we demonstrated that T cell-specific IL-6R signaling is essential for viral control during persistent lymphocytic choriomeningitis virus clone 13 infection. Strikingly, we observed that in contrast to previous studies with ubiquitous IL-6 deletion or blockade, specific IL-6R deletion in T cells did not affect T follicular helper (Tfh) cell accumulation unless IL-6R-deficient T cells were competing with wild-type cells in mixed bone marrow chimeras. In contrast, Tfh cells from IL-6R-cKO-infected mice exhibited reduced ICOS expression in both chimeric and nonchimeric settings, and this sole identifiable Tfh defect was associated with reduced germinal centers, compromised Ig switch and low avidity of lymphocytic choriomeningitis virus-specific Abs despite intact IL-6R expression in B cells. We posit that IL-6R cis-signaling is absolutely required for appropriate ICOS expression in Tfh cells and provides a competitive advantage for Tfh accumulation, enabling generation of optimal B cell and Ab responses, and ultimately viral control during in vivo chronic infection.

The sympathetic nervous system (SNS) serves to maintain homeostasis of vital organ systems throughout the body, and its dysfunction plays a major role in human disease. The SNS also links the central nervous system to the immune system during different types of stress via innervation of the lymph nodes, spleen, thymus, and bone marrow. Previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP, gene name adcyap1) exhibits anti-inflammatory properties in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Because PACAP is known to regulate SNS function, we hypothesized that part of the immunoprotective action of PACAP is due to its neuromodulatory effects on sympathetic neurons. To examine this, we used an inducible, targeted approach to conditionally disrupt not only the PACAP-preferring PAC1 receptor gene (adcyap1r1) in dopamine β-hydroxylase-expressing cells, which includes postganglionic sympathetic neurons, but also catecholaminergic neurons in the brain and adrenomedullary chromaffin cells. In contrast to our previous EAE studies using PACAP global knockout mice which developed severe and prolonged EAE, we found that mice with conditional loss of PAC1 receptors in catecholaminergic cells developed a delayed time course of EAE with reduced helper T cell type 1 (Th1) and Th17 and enhanced Th2 cell polarization. At later time points, similar to mice with global PACAP loss, mice with conditional loss of PAC1 exhibited more severe clinical disease than controls. The latter was associated with a reduction in the abundance of thymic regulatory T cells (Tregs). These studies indicate that PAC1 receptor signaling acts in catecholaminergic cells in a time-dependent manner. At early stages of disease development, it enhances the ability of the SNS to polarize the Th response towards a more inflammatory state. Then, after disease is established, it enhances the ability of the SNS to dampen the inflammatory response via Tregs. The lack of concordance in results between global PACAP KO mice and mice with the PAC1 deletion targeted to catecholaminergic cells during early EAE may be explained by the fact that PACAP acts to regulate inflammation via multiple receptor subtypes and multiple targets, including inflammatory cells.

Intestinal adaptive immune responses influence host health, yet only a few intestinal bacteria species that induce cognate adaptive immune responses during homeostasis have been identified. Here, we show that Akkermansia muciniphila, an intestinal bacterium associated with systemic effects on host metabolism and PD-1 checkpoint immunotherapy, induces immunoglobulin G1 (IgG1) antibodies and antigen-specific T cell responses in mice. Unlike previously characterized mucosal responses, T cell responses to A. muciniphila are limited to T follicular helper cells in a gnotobiotic setting, without appreciable induction of other T helper fates or migration to the lamina propria. However, A. muciniphila-specific responses are context dependent and adopt other fates in conventional mice. These findings suggest that, during homeostasis, contextual signals influence T cell responses to the microbiota and modulate host immune function.

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.

CD4+ Foxp3+ T regulatory (Treg) cells are key players in preventing lethal autoimmunity. Tregs undertake differentiation processes and acquire diverse functional properties. However, how Treg's differentiation and functional specification are regulated remains incompletely understood. Here, we report that gradient expression of TCF1 and LEF1 distinguishes Tregs into three distinct subpopulations, particularly highlighting a subset of activated Treg (aTreg) cells. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to systemic autoimmunity. TCF1 and LEF1 are dispensable for Treg's suppressive capacity but essential for maintaining a normal aTreg pool and promoting Treg's competitive survival. As a consequence, the development of T follicular regulatory (Tfr) cells, which are a subset of aTreg, is abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmunity.

B-cell responses are dynamic processes that depend on multiple types of interactions. Rare antigen-specific B cells must encounter antigen and specialized systems are needed-unique to each lymphoid tissue type-to ensure this happens efficiently. Lymphoid tissue barrier cells act to ensure that pathogens, while being permitted entry for B-cell recognition, are blocked from replication or dissemination. T follicular helper (Tfh) cells often need to be primed by dendritic cells before supporting B-cell responses. For most responses, antigen-specific helper T cells and B cells need to interact, first to initiate clonal expansion and the plasmablast response, and later to support the germinal center (GC) response. Newly formed plasma cells need to travel to supportive niches. GC B cells must become confined to the follicle center, organize into dark and light zones, and interact with Tfh cells. Memory B cells need to be positioned for rapid responses following reinfection. Each of these events requires the actions of multiple G-protein coupled receptors (GPCRs) and their ligands, including chemokines and lipid mediators. This review will focus on the guidance cue code underlying B-cell immunity, with an emphasis on findings from our laboratory and on newer advances in related areas. We will discuss our recent identification of geranylgeranyl-glutathione as a ligand for P2RY8. Our goal is to provide the reader with a focused knowledge about the GPCRs guiding B-cell responses and how they might be therapeutic targets, while also providing examples of how multiple types of GPCRs can cooperate or act iteratively to control cell behavior.

T helper 9 (TH9) cells are important for the development of inflammatory and allergic diseases. The TH9 transcriptional network converges signals from cytokines and antigen presentation but is incompletely understood. Here, we identified TL1A, a member of the TNF superfamily, as a strong inducer of mouse and human TH9 differentiation. Mechanistically, TL1A induced the expression of the transcription factors BATF and BATF3 and facilitated their binding to the Il9 promoter leading to enhanced secretion of IL-9. BATF- and BATF3-deficiencies impaired IL-9 secretion under TH9 and TH9-TL1A-polarizing conditions. In vivo, using a T-cell transfer model, we demonstrated that TL1A promoted IL-9-dependent, TH9 cell-induced intestinal and lung inflammation. Neutralizing IL-9 antibodies attenuated TL1A-driven mucosal inflammation. Batf3-/- TH9-TL1A cells induced reduced inflammation and cytokine expression in vivo compared to WT cells. Our results demonstrate that TL1A promotes TH9 cell differentiation and function and define a role for BATF3 in T-cell-driven mucosal inflammation.

Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a Gα13-coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues1,2. P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma1,3-6, and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-L-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues.

BackgroundThe prevalence of asthma and allergy is increasing in US children. In utero exposure to chemicals used in personal care products and plastics may contribute to increase in these diseases.MethodsWe quantified urinary concentrations of eight phthalate metabolites and bisphenol A in mothers twice during pregnancy in 1999-2000 in Salinas, California. We assessed probable asthma, aeroallergies, eczema, and spirometry in their children at age 7, and measured T helper 1 and T helper 2 cells in blood at ages 2, 5, and 7 (N = 392). We employed Bayesian model averaging to select confounders from additional biomarkers measured in this population and controlled for them in logistic and linear regressions.ResultsMonocarboxyisooctyl phthalate was associated with increased odds for probable asthma (odds ratio: 1.54, 95% CI: 1.12, 2.12), and with lower forced expiratory volume in one second (β: -0.09 L, 95% CI: -0.15, -0.03) and forced expiratory flow from 25% to 75% of forced vital capacity (β: -7.06 L/s, 95% CI: -11.04, -2.90). Several other associations were attenuated in final models that controlled for additional biomarkers.ConclusionMonocarboxyisooctyl phthalate was associated with lower respiratory health after controlling for related chemical exposure, which suggests that confounding by multiple chemical exposures should be considered in future research.

Gain-of-function variants in p110δ, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) expressed in lymphocytes, cause activated PI3-kinase δ syndrome (APDS), a primary immunodeficiency that is characterized by recurrent infections, viremia, lymphadenopathy, and autoimmunity. The mechanism of autoimmunity in APDS has not been well-understood. Here, we show the profound skewing of peripheral CD4+ T cells to a T follicular helper (TFH) phenotype in a patient with APDS bearing a novel p110δ variant, Y524S. We also saw a diminishment of transient Foxp3 expression in activated T cells. Mechanistic studies revealed that both the new variant and a previously described, pathogenic variant (E81K) enhanced an interaction between intracellular Osteopontin and p85α. This interaction had been shown in mice to promote TFH differentiation. Our results demonstrate a new influence of PI3K on human T cell differentiation that is unrelated to its lipid-kinase activity and suggest that TFH should be monitored in APDS patients.

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

This manuscript reviews selected topics in primary immunodeficiency diseases (PIDDs) published in 2017. These include (1) the role of follicular T cells in the differentiation of B cells and development of optimal antibody responses; (2) impaired nuclear factor κB subunit 1 signaling in the pathogenesis of common variable immunodeficiency, revealing an association between impaired B-cell maturation and development of inflammatory conditions; (3) autoimmune and inflammatory manifestations in patients with PIDDs in T- and B-cell deficiencies, as well as in neutrophil disorders; (4) newly described gene defects causing PIDDs, including exostosin-like 3 (EXTL3), TNF-α-induced protein 3 (TNFAIP3 [A20]), actin-related protein 2/3 complex-subunit 1B (ARPC1B), v-Rel avian reticuloendotheliosis viral oncogene homolog A (RELA), hypoxia upregulated 1 (HYOU1), BTB domain and CNC homolog 2 (BACH2), CD70, and CD55; (5) use of rapamycin and the phosphoinositide 3-kinase inhibitor leniolisib to reduce autoimmunity and regulate B-cell function in the activated phosphoinositide 3-kinase δ syndrome; (6) improved outcomes in hematopoietic stem cell transplantation for severe combined immunodeficiency (SCID) in the last decade, with an overall 2-year survival of 90% in part caused by early diagnosis through implementation of universal newborn screening; (7) demonstration of the efficacy of lentiviral vector-mediated gene therapy for patients with adenosine deaminase-deficient SCID; (8) the promise of gene editing for PIDDs using CRISPR/Cas9 and zinc finger nuclease technology for SCID and chronic granulomatous disease; and (9) the efficacy of thymus transplantation in Europe, although associated with an unexpected high incidence of autoimmunity. The remarkable progress in the understanding and management of PIDDs reflects the current interest in this area and continues to improve the care of immunodeficient patients.

Reciprocal interactions between B and follicular T helper (Tfh) cells orchestrate the germinal center (GC) reaction, a hallmark of humoral immunity. Abnormal GC responses could lead to the production of pathogenic autoantibodies and the development of autoimmunity. Here we show that miR-146a controls GC responses by targeting multiple CD40 signaling pathway components in B cells; by contrast, loss of miR-146a in T cells does not alter humoral responses. However, specific deletion of both miR-146a and its paralog, miR-146b, in T cells increases Tfh cell numbers and enhanced GC reactions. Thus, our data reveal differential cell-intrinsic regulations of GC B and Tfh cells by miR-146a and miR-146b. Together, members of the miR-146 family serve as crucial molecular brakes to coordinately control GC reactions to generate protective humoral responses without eliciting unwanted autoimmunity.

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality.

Qualitative differences in the innate and adaptive responses elicited by different HIV vaccine candidates have not been thoroughly investigated. We tested the ability of the Aventis Pasteur live recombinant canarypox vector (ALVAC)-SIV, DNA-SIV and Ad26-SIV vaccine prime modalities together with two ALVAC-SIV + gp120 protein boosts to reduce the risk of SIVmac251 acquisition in rhesus macaques. We found that the DNA and ALVAC prime regimens were effective, but the Ad26 prime was not. The activation of hypoxia and the inflammasome in CD14+CD16- monocytes, gut-homing CCR5-negative CD4+ T helper 2 (TH2) cells and antibodies to variable region 2 correlated with a decreased risk of SIVmac251 acquisition. By contrast, signal transducer and activator of transcription 3 activation in CD16+ monocytes was associated with an increased risk of virus acquisition. The Ad26 prime regimen induced the accumulation of CX3CR1+CD163+ macrophages in lymph nodes and of long-lasting CD4+ TH17 cells in the gut and lungs. Our data indicate that the selective engagement of monocyte subsets following a vaccine prime influences long-term immunity, uncovering an unexpected association of CD14+ innate monocytes with a reduced risk of SIVmac251 acquisition.

Follicular helper (TFH) and regulatory (TFR) cells are critical players in managing germinal center (GC) reactions that accomplish effective humoral immune responses. Transcriptome analyses were done comparing gene regulation of TFH and TFR cells isolated from Peyer's Patches (PP) and immunized peripheral lymph nodes (pLNs) revealing many regulatory patterns common to all follicular cells. However, in contrast to TFH cells, the upregulation or downregulation of many genes was attenuated substantially in pLN TFR cells when compared to those of PP. Additionally, PP but not pLN TFR cells were largely unresponsive to IL2 and expressed Il4 as well as Il21. Together with fundamental differences in gene expression that were found between cells of both compartments this emphasizes specific adaptations of follicular T cell functions to their micro-milieu. Moreover, although GL7 expression distinguishes matured follicular T cells, GL7+ as well as GL7- cells are present in the GC.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

We have previously demonstrated that anti-beta amyloid DNA vaccine (AV-1959D) based on our proprietary MultiTEP platform technology is extremely immunogenic in mice, rabbits, and monkeys. Importantly, MultiTEP platform enables development of vaccines targeting pathological molecules involved in various neurodegenerative disorders. Taking advantage of the universality of MultiTEP platform, we developed DNA vaccines targeting 3 B-cell epitopes (amino acids [aa]85-99, aa109-126, and aa126-140) of human alpha-synuclein (hα-Syn) separately or all 3 epitopes simultaneously. All 4 DNA vaccines (1) generate high titers of anti-hα-Syn antibodies and (2) induce robust MultiTEP-specific T-helper cell responses without activation of potentially detrimental autoreactive anti-hα-Syn T-helper cells. Generated antibodies recognize misfolded hα-Syn produced by neuroblastoma cells, hα-Syn in the brain tissues of transgenic mouse strains and in the brain tissues of dementia with Lewy body cases. Based on these results, the most promising vaccine targeting 3 B-cell epitopes of hα-Syn simultaneously (PV-1950D) has been chosen for ongoing preclinical assessment in mouse models of hα-Syn with the aim to translate it to the human clinical trials.

Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses toward antibody production via Tfh cells or inflammation by Teff cells. Tfh-Teff cell fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6-Blimp-1 counter-antagonism. We found that the TCR-signal-induced transcription factor Irf4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised Irf4 amounts and promoted Teff cell fates at the expense of Tfh ones. Importantly, orthogonal induction of Irf4 expression redirected Tfh cell fate trajectories toward those of Teff. Mechanistically, we linked greater Irf4 abundance with its recruitment toward low-affinity binding sites within Teff cell cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the "reader" of TCR signal strength, and in turn, concentration-dependent activity of Irf4 "writes" T helper fate choice.

The maturation process of high-affinity antibodies is a result of intricate interactions between B cells and follicular helper T (Tfh) cells occurring in lymphoid germinal centers. HIV infection induces significant chronic immune activation, phenotypic skewing, and inflammation driven by years of continuous viral replication. High levels of viremia as well as immune activation and dysfunction have been demonstrated to have a perturbing impact on the B cell memory compartment and contribute to B cell exhaustion. Counterintuitively, the factors associated with perturbation of the B cell compartment seem to be favorable for the generation of highly affinity-matured Env-specific antibodies in a minority of HIV-infected individuals. Thus, the impact of HIV antigenemia on B cells and Tfh cell interactions warrants further exploration. We therefore studied immunophenotypes of HIV-specific B cells in individuals with differing levels of viral control using HIV Env gp120 probes and characterized the functionality of matched T cells in peripheral blood. While CXCR5+ CD4+ T cells were significantly diminished in HIV progressors, we found that a small subset of gp120-specific interleukin-21 (IL-21)-secreting CXCR5+ CD4+ T cells were significantly associated with gp120-specific B cell frequencies. In contrast, neither bulk CXCR5+ CD4+ T cells nor other HIV antigen specificities were associated with gp120-specific B cell levels. HIV-specific B cells derived from elite controllers displayed greater amounts of gp120-specific B cells in the resting memory subset, whereas HIV-specific B cells in progressors accumulated in tissue-like and activated memory subsets. Furthermore, CXCR5+ CD4+ T cells from elite controllers showed a stronger ex vivo capacity to induce B cell maturation and immunoglobulin class switching than cells from HIV progressors.IMPORTANCE Dissecting the factors that are involved in B cell maturation and antibody development is important for HIV vaccine design. In this study, we found that HIV Env-specific CXCR5+ CD4+ T cells that secrete interleukin-21 are strongly associated with B cell memory phenotypes and function. Moreover, we found that the immune responses of HIV controllers showed intrinsically better helper activity than those of HIV progressors.

Background & aimsPeptic ulcer disease and gastric cancer are caused most often by Helicobacter pylori strains that harbor the cag pathogenicity island, which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host cells. cagY is an essential gene in the T4SS and has an unusual DNA repeat structure that predicts in-frame insertions and deletions. These cagY recombination events typically lead to a reduction in T4SS function in mouse and primate models. We examined the role of the immune response in cagY-dependent modulation of T4SS function.MethodsH pylori T4SS function was assessed by measuring CagA translocation and the capacity to induce interleukin (IL)8 in gastric epithelial cells. cagY recombination was determined by changes in polymerase chain reaction restriction fragment-length polymorphisms. T4SS function and cagY in H pylori from C57BL/6 mice were compared with strains recovered from Rag1-/- mice, T- and B-cell-deficient mice, mice with deletion of the interferon gamma receptor (IFNGR) or IL10, and Rag1-/- mice that received adoptive transfer of control or Ifng-/- CD4+ T cells. To assess relevance to human beings, T4SS function and cagY recombination were assessed in strains obtained sequentially from a patient after 7.4 years of infection.ResultsH pylori infection of T-cell-deficient and Ifngr1-/- mice, and transfer of CD4+ T cells to Rag1-/- mice, showed that cagY-mediated loss of T4SS function requires a T-helper 1-mediated immune response. Loss of T4SS function and cagY recombination were more pronounced in Il10-/- mice, and in control mice infected with H pylori that expressed a more inflammatory form of cagY. Complementation analysis of H pylori strains isolated from a patient over time showed changes in T4SS function that were dependent on recombination in cagY.ConclusionsAnalysis of H pylori strains from mice and from a chronically infected patient showed that CagY functions as an immune-sensitive regulator of T4SS function. We propose that this is a bacterial adaptation to maximize persistent infection and transmission to a new host under conditions of a robust inflammatory response.

Chronic HIV infection is associated with accumulation of germinal center (GC) T follicular helper (Tfh) cells in the lymphoid tissue. The GC Tfh cells can be heterogeneous based on the expression of chemokine receptors associated with T helper lineages, such as CXCR3 (Th1), CCR4 (Th2), and CCR6 (Th17). However, the heterogeneous nature of GC Tfh cells in the lymphoid tissue and its association with viral persistence and Ab production during chronic SIV/HIV infection are not known. To address this, we characterized the expression of CXCR3, CCR4, and CCR6 on GC Tfh cells in lymph nodes following SIVmac251 infection in rhesus macaques. In SIV-naive rhesus macaques, only a small fraction of GC Tfh cells expressed CXCR3, CCR4, and CCR6. However, during chronic SIV infection, the majority of GC Tfh cells expressed CXCR3, whereas the proportion of CCR4(+) cells did not change, and CCR6(+) cells decreased. CXCR3(+), but not CXCR3(-), GC Tfh cells produced IFN-γ (Th1 cytokine) and IL-21 (Tfh cytokine), whereas both subsets expressed CD40L following stimulation. Immunohistochemistry analysis demonstrated an accumulation of CD4(+)IFN-γ(+) T cells within the hyperplastic follicles during chronic SIV infection. CXCR3(+) GC Tfh cells also expressed higher levels of ICOS, CCR5, and α4β7 and contained more copies of SIV DNA compared with CXCR3(-) GC Tfh cells. However, CXCR3(+) and CXCR3(-) GC Tfh cells delivered help to B cells in vitro for production of IgG. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC Tfh cells, which are phenotypically and functionally distinct from conventional GC Tfh cells and contribute to hypergammaglobulinemia and viral reservoirs.

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4(+) T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and function.

To maintain a symbiotic relationship between the host and its resident intestinal microbiota, appropriate mucosal T cell responses to commensal antigens must be established. Mice acquire both IgG and IgA maternally; the former has primarily been implicated in passive immunity to pathogens while the latter mediates host-commensal mutualism. Here, we report the surprising observation that mice generate T cell-independent and largely Toll-like receptor (TLR)-dependent IgG2b and IgG3 antibody responses against their gut microbiota. We demonstrate that maternal acquisition of these antibodies dampens mucosal T follicular helper responses and subsequent germinal center B cell responses following birth. This work reveals a feedback loop whereby T cell-independent, TLR-dependent antibodies limit mucosal adaptive immune responses to newly acquired commensal antigens and uncovers a broader function for maternal IgG.

The discovery that eosinophilia and steroid responsiveness are dominant signals in patients with asthma led to the conclusion that inflammation characterized by up-regulation of the type 2 cytokines that mediate eosinophilia (IL -4, -5, and -13) (type 2 inflammation) is central to asthma pathogenesis in all patients and resulted in a singular emphasis on animal models of type 2 inflammation to unravel disease mechanisms. This in turn led to great progress in identifying drug targets and in developing inhibitors of type 2 inflammation. Despite this significant and clinically important progress, there has been a growing body of evidence that airway type 2 inflammation is not a ubiquitous pathologic feature of asthma and a growing acceptance that the type 2-centric asthma paradigm has held back understanding of mechanisms of disease in patients who do not have type 2 inflammation (helper T-cell type 2 [Th2]-low asthma). This "tyranny of the dominant paradigm" effect means that asthma clinicians have no effective controller treatments to offer their many patients with Th2-low asthma. It also means that asthma researchers are struggling to understand the mechanisms of disease that operate in Th2-low asthma and how these mechanisms might be modeled in vitro and in vivo to identify novel drug targets and provide a broader range of asthma treatments.

BackgroundActivation and differentiation of T-helper (Th) cells into Th1 and Th2 types is a complex process orchestrated by distinct gene activation programs engaging a number of genes. This process is crucial for a robust immune response and an imbalance might lead to disease states such as autoimmune diseases or allergy. Therefore, identification of genes involved in this process is paramount to further understand the pathogenesis of, and design interventions for, immune-mediated diseases.MethodsWe aimed at identifying protein-coding genes and long non-coding RNAs (lncRNAs) involved in early differentiation of T-helper cells by transcriptome analysis of cord blood-derived naïve precursor, primary and polarized cells.ResultsHere, we identified lineage-specific genes involved in early differentiation of Th1 and Th2 subsets by integrating transcriptional profiling data from multiple platforms. We have obtained a high confidence list of genes as well as a list of novel genes by employing more than one profiling platform. We show that the density of lineage-specific epigenetic marks is higher around lineage-specific genes than anywhere else in the genome. Based on next-generation sequencing data we identified lineage-specific lncRNAs involved in early Th1 and Th2 differentiation and predicted their expected functions through Gene Ontology analysis. We show that there is a positive trend in the expression of the closest lineage-specific lncRNA and gene pairs. We also found out that there is an enrichment of disease SNPs around a number of lncRNAs identified, suggesting that these lncRNAs might play a role in the etiology of autoimmune diseases.ConclusionThe results presented here show the involvement of several new actors in the early differentiation of T-helper cells and will be a valuable resource for better understanding of autoimmune processes.

Fungal infection stimulates the canonical C-type lectin receptor (CLR) signaling pathway via activation of the tyrosine kinase Syk. Here we identify a crucial role for the tyrosine phosphatase SHP-2 in mediating CLR-induced activation of Syk. Ablation of the gene encoding SHP-2 (Ptpn11; called Shp-2 here) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated the expression of genes encoding pro-inflammatory molecules following fungal stimulation. Mechanistically, SHP-2 operated as a scaffold, facilitating the recruitment of Syk to the CLR dectin-1 or the adaptor FcRγ, through its N-SH2 domain and a previously unrecognized carboxy-terminal immunoreceptor tyrosine-based activation motif (ITAM). We found that DC-derived SHP-2 was crucial for the induction of interleukin 1β (IL-1β), IL-6 and IL-23 and anti-fungal responses of the TH17 subset of helper T cells in controlling infection with Candida albicans. Together our data reveal a mechanism by which SHP-2 mediates the activation of Syk in response to fungal infection.