Helper Cell Activity Research Articles

Interleukin 9 mediates T follicular helper cell activation to promote antibody responses.

Antigen-specific humoral responses are orchestrated through complex interactions among immune cells in lymphoid tissues, including the collaboration between B cells and T follicular helper (Tfh) cells. Accumulating evidence indicates a crucial role for interleukin-9 (IL-9) in the formation of germinal centers (GCs), enhancing the generation of class-switched high-affinity antibodies. However, the exact function of IL-9 in Tfh cell regulation remains unclear. In this study, we examined the humoral immune responses of CD4Cre/+Il9rafl/fl mice, which lack an IL-9-specific receptor in Tfh cells. Upon intraperitoneal immunization with sheep red blood cells (SRBCs), CD4Cre/+Il9rafl/fl mice displayed diminished levels of SRBC-specific IgG antibodies in their sera, along with reduced levels of GC B cells and plasma cells. Notably, Il9ra-deficient Tfh cells in the spleen exhibited decreased expression of their signature molecules such as B-cell lymphoma 6, C-X-C chemokine receptor 5, IL-4, and IL-21 compared to control mice. In models of allergic asthma induced by house dust mite (HDM) inhalation, CD4Cre/+Il9rafl/fl mice failed to elevate serum levels of HDM-specific IgE and IgG. This was accompanied by reductions in Tfh cells, GC B cells, and plasma cells in mediastinal lymph nodes. Furthermore, group 2 innate lymphoid cells (ILC2s) were identified as producers of IL-9 under immunizing conditions, possibly induced by leukotrienes released by activated IgD+ B cells around the T-B border. These observations may indicate the critical role of IL-9 receptor signaling in the activation of Tfh cells, with ILC2s potentially capable of supplying IL-9 in organized lymphoid tissues.

IL-12 drives the differentiation of human T follicular regulatory cells.

T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.

Protection from environmental enteric dysfunction and growth improvement in malnourished newborns by amplification of secretory IgA

Environmental enteric dysfunction (EED) is a condition associated with malnutrition that can progress to malabsorption and villous atrophy. Severe EED results in linear growth stunting, slowed neurocognitive development, and unresponsiveness to oral vaccines. Prenatal exposure to malnutrition and breast feeding by malnourished mothers replicates EED. Pups are characterized by deprivation of secretory IgA (SIgA) and altered development of the gut immune system and microbiota. Extracellular ATP (eATP) released by microbiota limits T follicular helper (Tfh) cell activity and SIgA generation in Peyer's patches (PPs). Administration of a live biotherapeutic releasing the ATP-degrading enzyme apyrase to malnourished pups restores SIgA levels and ameliorates stunted growth. SIgA is instrumental in improving the growth and intestinal immune competence of mice while they are continuously fed a malnourished diet. The analysis of microbiota composition suggests that amplification of endogenous SIgA may exert a dominant function in correcting malnourishment dysbiosis and its consequences on host organisms, irrespective of the actual microbial ecology.

Innate immune responses are increased in children with acute asthma exacerbation.

Little is known about the immune responses during acute asthma exacerbation. In this study, we examined immune responses in children following an acute asthma exacerbation. We evaluated pro-inflammatory cytokine levels and gene expression profiles in blood samples from pediatric patients admitted for acute asthma exacerbation. Viral PCR was performed to differentiate between viral or non-viral-associated exacerbations. Following informed consent, clinical data were obtained from 20 children with asthma (median [interquartile range, IQR]: age 11.5 [8.0, 14.2]) years and 14 healthy age-matched controls (10.5 [7.0, 13.0]). Twelve had positive nasopharyngeal Polymerase chain reaction (PCR) for viral infection (11 rhinoviruses and 1 respiratory syncytial virus (RSV)). Nine were in the pediatric intensive care unit (PICU) and among them five required continuous positive airway pressure (CPAP). Mean (±SD) days on systemic steroids before drawing blood sample were 2.5 ± 1.6. Twelve had history of environmental allergies with 917 (274, 1396) IU/mL total IgE (median (IQR)). Compared with controls, IL-1RA and IL-10 levels were significantly increased and TNF-α significantly decreased in asthma subjects (p < .05 for all). RNA-seq analysis revealed 852 differentially expressed genes in subjects with asthma. Pathway analysis found upregulated genes and pathways involved in innate immune responses in subjects with asthma. Significantly reduced genes included pathways associated with T helper cell differentiation and activation. In acute asthma exacerbation, innate immune pathways remained increased while adaptive immune responses related to T helper cells are blunted and are independent of trigger or asthma severity. Our novel findings highlight the need to identify new therapies to target persistent innate immune responses to improve outcomes in acute asthma.

T cell activation and deficits in T regulatory cells are associated with major depressive disorder and severity of depression

Major depressive disorder (MDD) is associated with T cell activation, but no studies have examined the combined effects of T cell activation and deficits in T regulatory (Treg) cells on the severity of acute phase MDD. Using flow cytometry, we determined the percentage and median fluorescence intensity of CD69, CD71, CD40L, and HLADR-bearing CD3+, CD4+, and CD8+ cells, and cannabinoid type 1 receptor (CB1), CD152 and GARP (glycoprotein A repetitions predominant)-bearing CD25+ FoxP3 T regulatory (Treg) cells in 30 MDD patients and 20 healthy controls in unstimulated and stimulated (anti-CD3/CD28) conditions. Based on cytokine levels, we assessed M1 macrophage, T helper (Th)-1 cell, immune-inflammatory response system (IRS), T cell growth, and neurotoxicity immune profiles. We found that the immune profiles (including IRS and neurotoxicity) were significantly predicted by decreased numbers of CD152 or GARP-bearing CD25+ FoxP3 cells or CD152 and GARP expression in combination with increases in activated T cells (especially CD8+ CD40L+ percentage and expression). MDD patients showed significantly increased numbers of CD3+ CD71+, CD3+ CD40L+, CD4+ CD71+, CD4+ CD40L+, CD4+ HLADR+, and CD8+ HLADR+ T cells, increased CD3+ CD71+, CD4+ CD71+ and CD4+ HLADR+ expression, and lowered CD25+ FoxP3 expression and CD25+ FoxP+ CB1+ numbers as compared with controls. The Hamilton Depression Rating Scale score was strongly predicted (between 30 and 40% of its variance) by a lower number of CB1 or GARP-bearing Treg cells and one or more activated T cell subtypes (especially CD8+ CD40L+). In conclusion, increased T helper and cytotoxic cell activation along with decreased Treg homeostatic defenses are important parts of MDD that lead to enhanced immune responses and, as a result, neuroimmunotoxicity.

Abstract 6553: Targeting macrophages and regulatory T cells improves response to chemotherapy in high-grade serous ovarian cancer

Abstract Despite the initial response to chemotherapy, most high-grade serous ovarian cancer (HGSOC) patients will suffer disease relapse within two years of diagnosis with no currently successful immunotherapy options.We performed single-cell RNA sequencing (scRNAseq) on omental metastasis from HGSOC patients. Analysis of 64,097 immune cells showed that neoadjuvant chemotherapy (NACT) induced immunosuppressive mechanisms that counteracted its potentially positive effects. We showed that while NACT enhanced phagocytosis and antigen presentation, this was counterbalanced by upregulation of Stabilin1 (STAB1) in eight out of eleven macrophage subpopulations we identified. The NACT associated increase in macrophage STAB1 expression was validated in a separate cohort of eighty HGSOC patients at the protein level. STAB1 is a macrophage scavenger receptor that transports phagocytosed targets to undergo significant lysosomal degradation reducing the efficiency of antigen presentation. In vitro anti-stabilin1 antibody (anti-stab1 ab) -treated macrophages had significantly reduced antigen degradation and higher CD11C expression than isotype-treated ones. Furthermore, T cells co-cultured with anti-stab1 ab -treated macrophages had increased T cell proliferation and killing.We identified thirteen CD4 subpopulations by scRNAseq, five of which were Tregs. We showed that NACT significantly upregulated and activated Tregs. Tregs displayed a shared developmental trajectory with naïve and effector T cells suggesting that those are induced rather than natural Tregs. This was supported by TCR clonal sharing between Tregs and other effector cells. In vitro T cells treated with FOXP3 anti-sense oligonucleotide (ASO), AZD8701, had an anti-tumor cytokine profile and enhanced tumor cell killing. Analysis of 69,781 scRNAseq cells from control and chemotherapy-treated HGSOC syngeneic mouse model showed similar responses. In two HGSOC syngeneic mouse models (HGS2 and 30200), combinations of chemotherapy with anti-stab1 ab and/or Foxp3-ASO significantly increased median survival of mice with established peritoneal disease compared to chemotherapy alone. Bulk RNAseq of tumors treated with anti-stab1 ab were enriched with CXCL9 positive macrophages, while Foxp3-ASO treatment showed significant increase in T helper cell infiltration and activation. Potentially cured long-term survivors (300 days+) were resistant to tumor rechallenge.In a third HGSOC mouse model (60577), in which tumors relapsed after a good initial response to chemotherapy, combinations of chemotherapy with anti-stab1 ab and/or Foxp3-ASO significantly increased the post relapse survival compared to chemotherapy alone.We believe that modulating Tregs and STAB1 could improve response to chemotherapy and can have translational potential as each AZD8701 and a humanized anti-stab1 ab are in phase I clinical trials. Citation Format: Samar Elorbany, Chiara Berlato, Larissa Carnevalli, Simon Barry, Eleni Maniati, Jun Wang, Ranjit Manchanda, Julia Kzhyshkowska, Frances Balkwill. Targeting macrophages and regulatory T cells improves response to chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6553.

Propionate functions as a feeding state-dependent regulatory metabolite to counter proinflammatory signaling linked to nutrient load and obesity.

Generally, fasting and refeeding confer anti- and proinflammatory effects, respectively. In humans, these caloric-load interventions function, in part, via regulation of CD4+ T cell biology. However, mechanisms orchestrating this regulation remain incomplete. We employed integrative bioinformatics of RNA sequencing and high-performance liquid chromatography-mass spectrometry data to measure serum metabolites and gene expression of peripheral blood mononuclear cells isolated from fasting and refeeding in volunteers to identify nutrient-load metabolite-driven immunoregulation. Propionate, a short chain fatty acid (SCFA), and the SCFA-sensing G protein-coupled receptor 43 (ffar2) were coordinately and inversely regulated by fasting and refeeding. Propionate and free fatty acid receptor agonists decreased interferon-γ and interleukin-17 and significantly blunted histone deacetylase activity in CD4+ T cells. Furthermore, propionate blunted nuclear factor κB activity and diminished interleukin-6 release. In parallel, propionate reduced phosphorylation of canonical T helper 1 (TH1) and TH17 regulators, STAT1 and STAT3, respectively. Conversely, knockdown of free fatty acid receptors significantly attenuated the anti-inflammatory role of propionate. Interestingly, propionate recapitulated the blunting of CD4+ TH cell activation in primary cells from obese individuals, extending the role of this metabolite to a disease associated with low-grade inflammation. Together, these data identify a nutrient-load responsive SCFA-G protein-coupled receptor linked pathway to regulate CD4+ TH cell immune responsiveness.

The role of miRNAs in T helper cell development, activation, fate decisions and tumor immunity.

T helper (Th) cells are central members of adaptive immunity and comprise the last line of defense against pathogen infection and malignant cell invasion by secreting specific cytokines. These cytokines then attract or induce the activation and differentiation of other immune cells, including antibody-producing B cells and cytotoxic CD8+ T cells. Therefore, the bidirectional communication between Th cells and tumor cells and their positioning within the tumor microenvironment (TME), especially the tumor immune microenvironment (TIME), sculpt the tumor immune landscape, which affects disease initiation and progression. The type, number, and condition of Th cells in the TME and TIME strongly affect tumor immunity, which is precisely regulated by key effectors, such as granzymes, perforins, cytokines, and chemokines. Moreover, microRNAs (miRNAs) have emerged as important regulators of Th cells. In this review, we discuss the role of miRNAs in regulating Th cell mediated adaptive immunity, focusing on the development, activation, fate decisions, and tumor immunity.

To stay or not to stay intact as an allergen: the endolysosomal degradation assay used as tool to analyze protein immunogenicity and T cell epitopes.

Antigen uptake and processing of exogenous proteins is critical for adaptive immunity, particularly for T helper cell activation. Proteins undergo distinct proteolytic processing in endolysosomal compartments of antigen-presenting cells. The resulting peptides are presented on MHC class II molecules and specifically recognized by T cells. The in vitro endolysosomal degradation assay mimics antigen processing by incubating a protein of interest with a protease cocktail derived from the endolysosomal compartments of antigen presenting cells. The kinetics of protein degradation is monitored by gel electrophoresis and allows calculation of a protein's half-life and thus endolysosomal stability. Processed peptides are analyzed by mass spectrometry and abundant peptide clusters are shown to harbor T cell epitopes. The endolysosomal degradation assay has been widely used to study allergens, which are IgE-binding proteins involved in type I hypersensitivity. In this review article, we provide the first comprehensive overview of the endolysosomal degradation of 29 isoallergens and variants originating from the PR-10, Ole e 1-like, pectate lyase, defensin polyproline-linked, non-specific lipid transfer, mite group 1, 2, and 5, and tropomyosin protein families. The assay method is described in detail and suggestions for improved standardization and reproducibility are provided. The current hypothesis implies that proteins with high endolysosomal stability can induce an efficient immune response, whereas highly unstable proteins are degraded early during antigen processing and therefore not efficient for MHC II peptide presentation. To validate this concept, systematic analyses of high and low allergenic representatives of protein families should be investigated. In addition to purified molecules, allergen extracts should be degraded to analyze potential matrix effects and gastrointestinal proteolysis of food allergens. In conclusion, individual protein susceptibility and peptides obtained from the endolysosomal degradation assay are powerful tools for understanding protein immunogenicity and T cell reactivity. Systematic studies and linkage with in vivo sensitization data will allow the establishment of (machine-learning) tools to aid prediction of immunogenicity and allergenicity. The orthogonal method could in the future be used for risk assessment of novel foods and in the generation of protein-based immunotherapeutics.

Abstract 24 — Myricetin Impedes Tfh Cell Differentiation and Ameliorates the Tumorigenic Phenotype of Adjuvant-Induced Arthritis FLS in Rheumatoid Arthritis

Background Rheumatoid arthritis (RA) is an autoimmune disorder characterized by destructive polyarthritis and extra-articular manifestations. Myricetin, a flavonoid, possesses several pharmacological benefits, including anti-arthritic effects. However, the specific role and molecular basis of myricetin in IL-21 mediated T follicular helper (Tfh) cell differentiation and activation of fibroblast-like synoviocytes (FLS) in RA remain unknown. In the present study, we decoded the therapeutic intervention of myricetin as an anti-arthritic small-molecule drug for impeding Tfh cell differentiation and for abrogating the RA-FLS-transformed aggressive phenotype. Methods In vivo experiments were conducted using an adjuvant-induced arthritis animal model to evaluate the effects of myricetin on Tfh cell differentiation, IL-21 production, choline kinase (ChoK) activation, and the JAK/STAT signaling pathway. Results In this study, myricetin was found to significantly inhibit the interaction between IL-21 and IL-21R, and at the molecular level, it suppressed JAK/STAT signaling and the downstream transcription factor Bcl-6, which decreased Tfh cell differentiation. Additionally, myricetin suppressed the IL-21-induced hyperproliferation of AIA-FLSs by downregulating the ChoK signaling cascade (Ras, Ral-GDS, and PI3K). Myricetin treatment reduced ChoK enzymatic activity and the proliferative, migratory, and invasive properties of AIA-FLS. Conclusion Taken together, our results demonstrated that myricetin is a promising therapeutic compound that abates IL-21-induced Tfh cell differentiation and the invasive behavior of AIA-FLS.

Modulation of immune responses to liposomal vaccines by intrastructural help

The encapsulation of HIV-unrelated T helper peptides into liposomal vaccines presenting trimers of the HIV-1 envelope glycoprotein (Env) on the surface (T helper liposomes) may recruit heterologous T cells to provide help for Env-specific B cells. This mechanism called intrastructural help can modulate the HIV-specific humoral immune response. In this study, we used cationic T helper liposomes to induce intrastructural help effects in a small animal model. The liposomes were functionalized with Env trimers by a tag-free approach designed to enable a simplified GMP production. The pre-fusion conformation of the conjugated Env trimers was verified by immunogold electron microscopy (EM) imaging and flow cytometry. The liposomes induced strong activation of Env-specific B cells in vitro. In comparison to previously established anionic liposomes, cationic T helper liposomes were superior in CD4+ T cell activation after uptake by dendritic cells. Moreover, the T helper liposomes were able to target Env-specific B cells in secondary lymphoid organs after intramuscular injection. We also observed efficient T helper cell activation and proliferation in co-cultures with Env-specific B cells in the presence of cationic T helper liposomes. Mouse immunization experiments with cationic T helper liposomes further revealed a modulation of the Env-specific IgG subtype distribution and enhancement of the longevity of antibody responses by ovalbumin- and Hepatitis B (HBV)-specific T cell help. Thus, clinical evaluation of the concept of intrastructural help seems warranted.

Unraveling Mechanisms of Autoimmune Skin Blistering: Applying Single-Cell Transcriptomics to Pemphigus B Cells

Unraveling Mechanisms of Autoimmune Skin Blistering: Applying Single-Cell Transcriptomics to Pemphigus B Cells

Cladribine Effects on T and B Cells and T Cell Reactivity in Multiple Sclerosis.

Cladribine tablet therapy is an efficacious treatment for multiple sclerosis (MS), however, its mechanism of action on T and B cell subsets remains unclear. The purpose of this study was to investigate the treatment effects of cladribine on the peripheral pool of T and B cells subsets and reactivity toward central nervous system (CNS) antigens. In this cross-sectional exploratory study, frequencies and absolute counts of peripheral T and B cell subsets and B cell cytokine production from untreated patients with relapsing-remitting MS (RRMS) and patients treated with cladribine for 1 year were measured using flow cytometry. Autoreactivity was assessed using a FluoroSpot assay. We found that 1 year after initiation of cladribine treatment, a lower number of CD4+ T cells was persisting whereas CD19+ B cell counts were normalized compared to untreated patients with RRMS. Follicular helper T cells and their effecter subsets producing cytokines exerting distinct B cell helper activity were lower and, additionally, the peripheral B cell pool was skewed toward a naïve and anti-inflammatory phenotype. Finally, reactivity to the recently identified CNS-enriched autoantigen RAS guanyl-releasing protein 2 (RASGRP2), but not to myelin basic protein and myelin oligodendrocyte glycoprotein, was lower in cladribine-treated patients. Together, these investigations on T and B cell subsets suggest that cladribine treatment impairs the B-T cell crosstalk and reduces their ability to mediate pathogenic effector functions. This may result in specific reduction of autoreactivity to RASGRP2 which is expressed in B cells and brain tissue. ANN NEUROL 2023.

IgA deficiency destabilizes homeostasis toward intestinal microbes and increases systemic immune dysregulation.

The ability of most patients with selective immunoglobulin A (IgA) deficiency (SIgAD) to remain apparently healthy has been a persistent clinical conundrum. Compensatory mechanisms, including IgM, have been proposed, yet it remains unclear how secretory IgA and IgM work together in the mucosal system and, on a larger scale, whether the systemic and mucosal anti-commensal responses are redundant or have unique features. To address this gap in knowledge, we developed an integrated host-commensal approach combining microbial flow cytometry and metagenomic sequencing (mFLOW-Seq) to comprehensively define which microbes induce mucosal and systemic antibodies. We coupled this approach with high-dimensional immune profiling to study a cohort of pediatric patients with SIgAD and household control siblings. We found that mucosal and systemic antibody networks cooperate to maintain homeostasis by targeting a common subset of commensal microbes. In IgA-deficiency, we find increased translocation of specific bacterial taxa associated with elevated levels of systemic IgG targeting fecal microbiota. Associated features of immune system dysregulation in IgA-deficient mice and humans included elevated levels of inflammatory cytokines, enhanced follicular CD4 T helper cell frequency and activation, and an altered CD8 T cell activation state. Although SIgAD is clinically defined by the absence of serum IgA, the symptomatology and immune dysregulation were concentrated in the SIgAD participants who were also fecal IgA deficient. These findings reveal that mucosal IgA deficiency leads to aberrant systemic exposures and immune responses to commensal microbes, which increase the likelihood of humoral and cellular immune dysregulation and symptomatic disease in patients with IgA deficiency.

Antigen-specific downregulation of miR-150 in CD4 T cells promotes cell survival.

MicroRNA-150 (miR-150) has been shown to play a general role in the immune system, but very little is known about its role on CD4+ T cell responses. During T cell responses against superantigen Staphylococcal Enterotoxin A, miR-150 expression was down-regulated in antigen-specific CD4+ T cells but up-regulated in CD8+ T cells. CD4+ and CD8+ T cell clonal expansion was greater in miR-150-KO mice than in WT mice, but miR-150 selectively repressed IL-2 production in CD4+ T cells. Transcriptome analysis of CD4+ T cells demonstrated that apoptosis and mTOR pathways were highly enriched in the absence of miR-150. Mechanistic studies confirmed that miR-150 promoted apoptosis specifically in antigen-specific CD4+ T cells, but not in bystander CD4+ nor in CD8+ T cells. Furthermore, inhibition of mTOR-linked mitochondrial superoxidedismutase-2 increased apoptosis in miR-150-/- antigen-specific CD4+ T. Thus, miR-150 impacts CD4+ T cell helper activity by attenuating IL-2 production along with clonal expansion, and suppresses superoxidedismutase to promote apoptosis.

Cell penetrating peptide: A potent delivery system in vaccine development.

One of the main obstacles to most medication administrations (such as the vaccine constructs) is the cellular membrane's inadequate permeability, which reduces their efficiency. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) are well-known as potent biological nanocarriers to overcome this natural barrier, and to deliver membrane-impermeable substances into cells. The physicochemical properties of CPPs, the attached cargo, concentration, and cell type substantially influence the internalization mechanism. Although the exact mechanism of cellular uptake and the following processing of CPPs are still uncertain; but however, they can facilitate intracellular transfer through both endocytic and non-endocytic pathways. Improved endosomal escape efficiency, selective cell targeting, and improved uptake, processing, and presentation of antigen by antigen-presenting cells (APCs) have been reported by CPPs. Different in vitro and in vivo investigations using CPP conjugates show their potential as therapeutic agents in various medical areas such as infectious and non-infectious disorders. Effective treatments for a variety of diseases may be provided by vaccines that can cooperatively stimulate T cell-mediated immunity (T helper cell activity or cytotoxic T cell function), and immunologic memory. Delivery of antigen epitopes to APCs, and generation of a potent immune response is essential for an efficacious vaccine that can be facilitated by CPPs. The current review describes the delivery of numerous vaccine components by various CPPs and their immunostimulatory properties.

Aiolos supports TFH cell differentiation by antagonizing the IL-2/STAT5 signaling pathway

Abstract CD4+ T follicular helper (TFH) cells are critical for the generation of robust humoral immune responses, as they provide help to B cells to support the generation of both pathogen-neutralizing antibodies and long-lived plasma cell populations. To date, the mechanisms underlying TFH differentiation are incompletely understood. Here, we identify the transcription factor Aiolos as a key regulator of TFH responses. We find that Aiolos expression is increased in TFH cell populations generated during influenza infection and that Aiolos deficiency results in compromised TFH cell differentiation and B cell helper activity. Mechanistically, loss of Aiolos results in diminished expression of TFH genes, including those encoding the key transcription factors Bcl-6, TCF-1, and Tox. Conversely, expression of genes associated with IL-2/STAT5 signaling, a known antagonist of the TFH gene program, were significantly elevated in Aiolos-deficient settings. Consistent with these data, antigen-specific Aiolos-deficient effector CD4+ T cell populations generated in response to influenza infection exhibited significantly elevated IL-2Rα surface expression. Together, our findings suggest that repression of IL-2/STAT5 signaling represents a novel mechanism by which Aiolos regulates the TFH gene program. These findings are important, as they provide critical insight that may ultimately be leveraged for the development of novel TFH-focused immunotherapies and strategies to improve vaccination approaches. Supported by a grant from the NIAID (NIH; R01 AI134972), funds through The Ohio State University College of Medicine, and funds through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program

Scrub typhus weakens humoral immune responses through suppression of germinal center and B cell activation

Abstract Scrub typhus is a poorly studied, but life-threatening, disease due to uncontrolled growth of Orientia tsutsugamushi bacteria within endothelial cells and phagocytes, in addition to dysregulated Th1 immune responses and inflammation. Cellular and humoral immunity in infected patients and experimental animals are short-lived, and wane within 1–2 years; however, the underlying mechanism of this transient immunity is unclear. No reports have examined splenic germinal center (GC) or B cell activation during this infection. The study objective is to fill this knowledge gap by using endothelium-targeted C57BL/6 mouse models that mimic pathological features of human scrub typhus. Through NanoString, qRT-PCR, and flow cytometry, we found significant repression of key markers crucial for B cell development (CCR7, BAFFR, PAX5) and GC formation (SLAMF1, ICOS-L, CXCR5, BCL-6) in both mild and severe disease outcomes. The widespread impairments in B and T follicular helper (Tfh) cell activation correlated with excessive Th1 cell activation during disease progression. Immunofluorescence staining (B220, CD3, GL7) revealed evidence of GC formation with sublethal infection, but the near absence of organized GCs at late stages of lethal infection, which was consistent with results from our splenic multi-color flow cytometry and serum antigen-specific ELISA assays for IgG isotypes. Ongoing RNA-seq studies for splenic B cells isolated from sub-lethally or lethally infected mice will provide in-depth, B cell-specific insight during infection. In conclusion, this is the first study to show profound alterations in B cell activation and GC formation in murine models, which helps understand “poor” humoral immunity seen with human scrub typhus. Supported by grants from NIH (R01 EY028773 to JS, R01 AI132674 to LS, R21 AI156536 to LS)

Activation and expansion of T-follicular helper cells in chronic lymphocytic leukemia nurselike cell co-cultures.

Interactions between chronic lymphocytic leukemia (CLL) cells and T-cell subsets in the lymph node microenvironment are thought to play a central role in disease biology. To study these interactions in a model of the CLL lymph node microenvironment, we characterized T-cell subsets in CLL nurselike cell (NLC) co-cultures. We focused on T-follicular helper (Tfh) cells, which are characterized by CXCR5 expression and localization to B-cell follicles. In co-cultures from 28 different CLL patients, we detected an expansion of Tfh cells based on PD-1, BCL6, and ICOS expression, with increased IL-21 and downmodulated CD40L surface expression. Regulatory T cells (Treg), which promote immune tolerance, also expanded in NLC co-cultures. T-cell receptor (TR) gene repertoire analyses confirmed the clonal expansion of CD4+ T cells, with an enrichment of TR clonotypes commonly expanded also in primary CLL samples. Multicolor confocal microscopy revealed that Tfh, but not Treg co-localize with proliferating CLL cells in CLL lymph node sections. Collectively, these data provide new insight into the cellular and molecular cross-talk between CLL and T-cell subsets, resulting in clonal expansion of T-helper cells and interaction of Tfh cells with proliferating CLL cells which may open new avenues for therapeutic targeting.

Innate Immune Activation Via TLR9 May Enhance Antibody Formation Against Factors VIII and IX in Protein and Gene Therapy

In recent years, the potential impact of innate immune responses on inhibitor formation in hemophilia has increasingly received attention, as innate immune sensing may provide a signal that leads to antibody formation, thus seriously complicating treatment of hemophilia. Similar concerns exist with the newer gene therapy approaches, where the gene transfer vector stimulates innate immunity. Curiously, we found that antibody formation against factor IX (FIX) in gene therapy for hemophilia B is independent of TLR9 sensing of the adeno-associated viral (AAV) genome. This is despite the critical role of TLR9 (toll-like receptor 9, an innate endosomal sensor of DNA) in CD8+ T cell responses against this vector (Blood 129:3184-3195, 2017). These results are consistent with the notion that TLRs more typically promote Th1 than Th2 immunity. However, when we co-administered an CpG-deoxyoligonucleotide (ODN-1826) agonist of TLR9 with AAV1-FIX vector (1x1011 vg/mouse) to skeletal muscle of C57BL/6 mice, IgG1 formation against FIX was reproducibly observed at average titers of 3-5 ug/ml serum within 2 weeks. Control mice, which received vector only, formed <1 ug/ml to undetectable anti-FIX titers. Therefore, TLR9 activation via a different ligand (instead of the AAV genome) can induce antibody formation. Co-administration of lipopolysaccharides that are known ligands for TLR2 and TLR4 failed to induce an anti-FIX response. Although both TLR4 and TLR9 agonists upregulated co-stimulatory molecules on dendritic cells (DCs) upon intramuscular injection, only the TLR9 agonist increased the frequency of monocyte-derived DCs (moDCs), which are known to enhance T follicular helper (Tfh) cell activation, thereby enhancing germinal center and antibody formation. Indeed, we found that co-administration of ODN-1826 significantly increased frequencies if Tfh (PD-1hiBcl-6+CXCR5hiCD4+) cells. This occurred in the presence or absence of FoxP3+ Treg, although antibody formation was only increased in the presence of Treg, indication a critical role for Treg in modulating the response to the ODN. Interestingly, others had reported that ODN-1826 reduces antibody formation against FVIII in hemophilia A mice (JCI 125:3766-81, 2015), and proposed a mechanism by which Treg are induced upon TLR9 stimulation of plasmacytoid DCs, thereby suppressing anti-FVIII responses. We similarly injected FVIII (1IU, B domain deleted) intravenously once/week for 6 weeks into hemophilia A mice (F8e16-/-, C57BL6/129) with our without ODN-1826 (n=4/group). We indeed found a 4-fold lower IgG titer against FVIII in the presence of the ODN (ave. of 0.9 vs 3.6 ug/ml, P<0.05). However, inhibitor titers were substantially increased by ODN co-administration (ave. of 1675 BU vs 340 BU, P<0.001). Therefore, TLR9 activation reduced the overall antibody production but increased the formation of neutralizing antibodies. In summary, our data show that a pathway exists for TLR9-drived innate immune signaling to increase the risk of inhibitor formation in gene and protein therapy for hemophilia. DisclosuresHerzog:Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties; Novo Nordisk: Research Funding.