Th1 Immunity Research Articles

Interplay of Chemokines Receptors, Toll-like Receptors, and Host Immunological Pathways.

A comprehensive framework has been established for understanding immunological pathways, which can be categorized into eradicated and tolerable immune responses. Toll-like receptors (TLRs) are associated with specific immune responses. TH1 immunity is related to TLR7, TLR8, and TLR9, while TH2 immunity is associated with TLR1, TLR2, and TLR6. TH22 immunity is linked to TLR2, TLR4, and TLR5, and THαβ (Tr1) immunity is related to TLR3, TLR7, and TLR9. The chemokine receptor CXCR5 is a marker of follicular helper T cells, and other chemokine receptors can also be classified within a framework based on host immunological pathways. On the basis of a literature review on chemokines and immunological pathways, the following associations were identified: CCR5 with TH1 responses, CCR1 with TH1-like responses, CCR4 (basophils) and CCR3 (eosinophils) with TH2 and TH9 responses, CCR10 with TH22 responses, CCR6 with TH17 responses, CXCR3 with THαβ responses, CCR8 with regulatory T cells (Treg), and CCR2 with TH3 responses. These findings contribute to the identification of biomarkers for immune cells and provide insights into host immunological pathways. Understanding the chemokine and Toll-like receptor system is crucial for comprehending the function of the innate immune system, as well as adaptive immune responses.

Blocking Th2 Signaling Pathway Alleviates the Clinical Symptoms and Inflammation in Allergic Conjunctivitis.

To explore the role of Th2 signaling pathway in allergic conjunctivitis (AC). Serum Th2 cytokines IL-4 or IL-13 of patients with AC were detected using the Meso scale discovery assay to verify the correlation of Th2 immunity and AC pathogenesis. Wistar Han rats were intraperitoneally and subcutaneously injected with ovalbumin (OVA) to establish an experimental AC model and the Th2 signaling pathway was blocked by an investigational neutralizing antibody (CM310). Serum IgE and OVA-specific IgE were detected by ELISA. Conjunctivitis inflammation, infiltration of eosinophils, and mast cell degranulation were detected by histological examination. Immortalized human conjunctival epithelial cells, a conjunctival epithelial cell line, and peripheral blood mononuclear cells of patients with AC were used as the target cells to study the impact of IL-4 or IL-13 on AC progression. Finally, a STAT6 reporter gene system was constructed using immortalized human conjunctival epithelial cells to confirm whether the downstream signaling pathway activated by IL-4 or IL-13. Serum IL-4 or IL-13 were increased in patients with AC versus healthy individuals. In an OVA-induced rat experimental AC model, blocking the Th2 signaling pathway with CM310, an investigational neutralizing antibody, alleviated the conjunctival symptoms, and decreased serum IgE, suppressed infiltration of eosinophils and mast cell degranulation. Further, an in vitro model showed CM310 suppressed the secretion of inflammatory cytokine from both immune cells and epithelial cells in both patients peripheral blood mononuclear cells and cell line. Blocking Th2 signaling pathway alleviates the clinical symptoms and inflammation in AC.

PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma.

This timely study assesses the immunosuppressive effects of surgery on cytotoxic Th1-like immunity and investigates if immune checkpoint blockade (ICB) can boost Th1-like immunity in the perioperative window in upper gastrointestinal cancer (UGI) patients. PBMCs were isolated from 11 UGI patients undergoing tumour resection on post-operative days (POD) 0, 1, 7 and 42 and expanded ex vivo using anti-CD3/28 and IL-2 for 5 days in the absence/presence of nivolumab or ipilimumab. T cells were subsequently immunophenotyped via flow cytometry to determine the frequency of T helper (Th)1-like, Th1/17-like, Th17-like and regulatory T cell (Tregs) subsets and their immune checkpoint expression profile. Lymphocyte secretions were also assessed via multiplex ELISA (IFN-γ, granzyme B, IL-17 and IL-10). The 48h cytotoxic ability of vehicle-, nivolumab- and ipilimumab-expanded PBMCs isolated on POD 0, 1, 7 and 42 against radiosensitive and radioresistant oesophageal adenocarcinoma tumour cells (OE33 P and OE33 R) was also examined using a cell counting kit-8 (CCK-8) assay to determine if surgery affected the killing ability of lymphocytes and whether the use of ICB could enhance cytotoxicity. Th1-like immunity was suppressed in expanded PBMCs in the immediate post-operative setting. The frequency of expanded circulating Th1-like cells was significantly decreased post-operatively accompanied by a decrease in IFN-γ production and a concomitant increase in the frequency of expanded regulatory T cells with an increase in circulating levels of IL-10. Interestingly, PD-L1 and CTLA-4 immune checkpoint proteins were also upregulated on expanded Th1-like cells post-operatively. Additionally, the cytotoxic ability of expanded lymphocytes against oesophageal adenocarcinoma tumour cells was abrogated post-surgery. Of note, the addition of nivolumab or ipilimumab attenuated the surgery-mediated suppression of lymphocyte cytotoxicity, demonstrated by a significant increase in tumour cell killing and an increase in the frequency of Th1-like cells and Th1 cytokine production. These findings support the hypothesis of a surgery-mediated suppression in Th1-like cytotoxic immunity and highlights a rationale for the use of ICB within the perioperative setting to abrogate tumour-promoting effects of surgery and ameliorate the risk of recurrence.

A phase II study of concurrent WOKVAC vaccination with neoadjuvant chemotherapy and HER2-targeted monoclonal antibody therapy.

TPS636 Background: HER2+ breast cancer (BCa) is an aggressive breast cancer subtype. The treatment of Stage I-III HER2+ BCa with neoadjuvant chemotherapy and HER2 monoclonal antibodies (trastuzumab and pertuzumab) induces a pathologic complete response in 43-67% of patients. Residual HER2+ disease (i.e. residual cancer burden) after neoadjuvant therapy is associated with a significantly higher risk of recurrence. Evidence supports that HER2 immunity is progressively lost in HER2 oncogenesis and that restoration of HER2 specific CD4+ Th1 immunity is associated with a lower risk of recurrence. Trastuzumab can partially restore HER2 specific immunity, but only in a minority of patients. We have demonstrated that HER2 specific vaccination can augment HER2 specific immunity. While taxane chemotherapy can deplete immunosuppressive cells (MDSCs) 13 – 17 days after treatment creating an optimal time for vaccination. The WOKVAC vaccine is a plasmid vaccine encoding Th1 selective epitopes from HER2 and two other high-risk cancer antigens IGFBP-2 and IGF-1R. The Phase I trial of this vaccine reported that it is safe and all doses induced antigen specific Type I immune responses. We hypothesize that addition of WOKVAC to neoadjuvant chemo/HER2 targeted therapy will induce a significant increase in the number of patients with Th1 CD4+ and CD8+ T cells after neoadjuvant vaccine/chemo/HER2 therapy. Methods: Trial Design: Phase II single arm trial of WOKVAC vaccination with concurrent neoadjuvant taxane based chemotherapy, trastuzumab, and pertuzumab in patients with Stage I-III HER2+ (HR+/-) breast cancer. Enrolled patients will receive WOKVAC vaccine 150mcg on day 13 of cycles 1-3. WOKVAC vaccination may be given with either TCHP or THP. A maximum of 16 patients will be enrolled in this study. Objectives: The primary objective is to evaluate whether concurrent WOKVAC vaccine with chemotherapy and HER2 antibody therapy can significantly increase T-bet+ CD4+ and CD8+ TIL. Secondary objectives are to determine: (1) whether concurrent WOKVAC with neoadjuvant chemo/HER2 therapy is safe, (2) if the magnitude of vaccine induced antigen specific Th1 immunity after neoadjuvant therapy is associated with pathologic response. Statistical Methods: (1) In order to detect a 30% increase in the number of patients with Tbet+ CD4+ and CD8+ TIL in the enrolled patients with 80% power and a one-side alpha of 0.05, our enrollment goal for this study will be 16 patients. (2) Safety will be assessed per CTCAE v5.0, Benchmarks for safety needed to advance this trial concept to a Phase III study will be a grade 3 toxicity rate of ≤13% and a grade 4 toxicity rate of ≤ 7%. (3) The induction of Th1 immunity will be compared against the presence or absence of a complete pathologic response to determine if there is a significant correlation using Pearson r correlation analysis. Targeted Accrual: The study will accrue up to 16 patients. Clinical trial information: NCT04329065 .

Phase I study of adoptive T cell therapy following HER2-pulsed dendritic cell vaccine and pepinemab/trastuzumab in patients with metastatic HER2-positive breast cancer (MBC).

TPS1113 Background: Despite major improvement of overall survival of HER2+ MBC with effective HER2 targeted therapies, many patients experience significant toxicities and develop progressive disease during treatment. Therefore, new and more effective therapeutic options are needed. This novel approach will evaluate whether the combination of three immunotherapies in addition to trastuzumab: dendritic cell (DC) vaccination, anti-SEMA4D blocking antibody (pepinemab) and CD4+ T cell adoptive transfer can lead to improved outcomes for patients with MBC refractory to HER2-targeted agents. BC have been considered as immunologically cold which is attributed to immune evasion and suppression of host effector immune cells homing into tumor bed. Progressive loss of Th1 immunity against HER2 oncodriver correlates with poor prognosis. HER2 peptide pulsed type I dendritic cells (HER2-DC1) restored anti-HER2 CD4+ Th1 immune response and improved pathologic complete response (pCR) in HER2+ BC. Antibodies to SEMA4D have been shown to modulate the TME by increasing effector cell infiltration and reducing immunosuppression. In preclinical studies, treatment with anti-SEMA4D and HER2-DC1 in mice bearing established HER2+ tumors improved DC homing, expansion of CD4+ T cells, and complete tumor regression, compared to treatment with anti-SEMA4D or HER2-DC1 alone. Further, subsequent expansion and adoptive transfer of CD4+ T cells induced synergistic anti-tumor activity by activating CD8+ T mediated cytotoxicity. Pepinemab was well-tolerated and showed signs of anti-tumor activity in in immunotherapy-resistant and PD-L1 negative/low non-small cell lung cancer patients when combined with checkpoint inhibitor (avelumab). Methods: This open label Phase 1 study is enrolling up to 28 patients with HER2+ MBC. Patients will be treated with 6 weekly injections of dendritic cell (DC1) vaccines in combination with trastuzumab and pepinemab. We hypothesize these therapies may elicit CD4+ HER2-specific T cell responses. HER2-specific T cells will be expanded ex vivo and subsequently infused to patients following lymphodepletion with cyclophosphamide. Trastuzumab and pepinemab will be given as maintenance in addition to booster DC1 vaccines. Patients (ECOG 0,1) must have had disease progression while on trastuzumab for the treatment of HER2+ MBC and received no more than 3 lines of chemotherapy in the setting of metastatic disease. Dose escalation will consist of 3-6 patients each with increasing amounts of transferred CD4+ T cells, followed by dose expansion of 10 patients at the MTD. The primary objective is safety and tolerability; secondary objectives will include evaluation of T cell immunity and immune subsets, efficacy, PK/PD/ADA of pepinemab, and biomarker assessments. Clinical trial information: NCT05378464 .

Exposure to violence and asthma in Puerto Rican youth with high Th2 immunity.

Little is known about the determinants of asthma among youth with high T helper 2 (Th2) immunity. We hypothesized that exposure to violence (ETV) and violence-related distress are associated with asthma in children and adolescents with high Th2 immunity. We analyzed data from Puerto Ricans with high Th2 immunity aged 9-20 years in the Puerto Rico Genetics of Asthma and Lifestyle (PR-GOAL) and the Epigenetic Variation of Childhood Asthma in Puerto Ricans (EVA-PR) studies, and in a prospective study (PROPRA). High Th2-immunity was defined as ≥1 positive allergen-specific IgE and/or a total IgE ≥ 100 IU/mL and/or an eosinophil count ≥ 150 cells/μL. Asthma was defined as physician-diagnosed asthma and current wheeze. ETV and violence-related distress were assessed with the validated ETV Scale and Checklist of Children's Distress Symptoms (CCDS) questionnaires, respectively. In multivariable analyses, each 1-point increment in ETV score was significantly associated with 1.13-1.17 times increased odds of asthma in PR-GOAL and in EVA-PR (both at p ≤ 0.01), and each 1-point increment in CCDS score was significantly associated with 1.53-1.54 increased odds of asthma in PR-GOAL and in EVA-PR (both at p ≤ 0.03). Further, a persistently high ETV score was significantly associated with asthma in PROPRA (odds ratio [OR] = 2.83, 95% confidence interval [CI] = 1.10-7.29). Similar results were obtained in a sensitivity analysis using an eosinophil count ≥ 300 cells/μL instead of ≥150 cells/μL to define high Th2 immunity. ETV during childhood is associated with increased risk of persistent or new-onset asthma in youth with high Th2 immunity.

Identification of cough-variant asthma phenotypes based on clinical and pathophysiologic data

Cough variant asthma (CVA) may respond differently to anti-asthmatic treatment. There is limited data on the heterogeneity of CVA. We aimed to classify patients with CVA using cluster analysis based on clinico-physiological parameters and to unveil the underlying molecular pathways of these phenotypes with transcriptomic data of sputum cells. K-mean clustering was applied to 342 newly physician-diagnosed patients with CVA from a prospective, multicenter, observational cohort using 10 pre-specified baseline clinic-pathophysiological variables. The clusters were compared based on clinical features, treatment response and sputum transcriptomic data. Three stable CVA clusters were identified. Cluster 1 (n= 176) was characterized by female predominance, late-onset, normal lung function, and a low proportion of complete resolution of cough (60.8%) after anti-asthmatic treatment. Cluster 2 (n=105) presented with young, nocturnal cough, atopy, type 2-high inflammation, and a high proportion of complete resolution of cough (73.3%) with highly upregulated co-expression gene network that related to type 2 immunity. Cluster 3 (n= 61) had a high body mass index, long duration, family history of asthma, low lung function, and low proportion of complete resolution of cough (54.1%). Th17 immunity and type 2 immunity co-expression gene networks were both upregulated in clusters 1 and 3. Three clusters of CVA were identified with different clinical, pathophysiological, and transcriptomic features and responses to anti-asthmatics treatment, which may improve our understanding of pathogenesis and develop individualized treatment of cough in asthma.

Bacterial catabolism of tryptophan to indole is required to induce collagen induced arthritis via the IL23/Th17 axis

Abstract While intestinal dysbiosis is linked to the pathophysiology of inflammatory arthritides like rheumatoid arthritis (RA) and spondyloarthritis (SpA), the mechanisms by which this occurs are unknown. Therefore, we used the collagen-induced arthritis (CIA) model to connect the microbiome with bacteria-produced metabolites in the development of disease. After initially correlating CIA severity with bacteria-dependent alterations in dietary tryptophan metabolism, and specifically indole, we demonstrate that both bacteria and dietary tryptophan are required for disease, and that indole supplementation fulfills these requirements. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β and Th17 cells. These findings were associated with increased anti-CII IgG2b, decreased anti-CII antibody sialyation, and increased complement fixation, suggesting that indole-mediated Th17 immunity may enhance autoantibody pathogenicity. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in the IL-17 signaling pathway in B and T cells. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of Th17 immunity. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA. Supported by grants from NIH: NIAMS R01AR075933 (KAK), NIAMS T32AR007534 (WKJ, BT, AJB), NIAID T32AI007405 (BJS), NIAID F30AI174817 (BJS); and the Rheumatology Research Foundation: Future Physician Scientist Award (BJS) and Scientist Development Award (AJB).

Neutrophil extracellular traps and extracellular histones mediate IL-17 inflammation and bone destruction in periodontitis

Abstract Neutrophil infiltration is a hallmark of the inflammatory disease periodontitis, a prevalent oral condition in which Th17 driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that in periodontitis, neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model of periodontitis, we demonstrate that neutrophils infiltrate disease lesions at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanism by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document, in periodontitis patients, significantly elevated levels of NET complexes and of extracellular histones bearing classic NET-associated post-translational modifications. Strikingly, concentrations of NET components in disease lesions and in circulation, significantly correlate with the severity of bone destruction in patients, even in the absence of known confounding systemic disease. Our findings suggest a feed-forward loop in which NETs trigger Th17 immunity to further amplify neutrophil immunopathology in a prevalent human inflammatory disease.

Liposome and QS-21 Combined Adjuvant Induces theHumoral and Cellular Responses of Acellular Pertussis Vaccine in a Mice Model.

The resurgence of pertussis in vaccinated communities may be related to the reduced long-term immunity induced by acellular pertussis vaccines. Therefore, developing improved pertussis vaccine candidates that could induce strong Th1 or Th17 cellular immunity is an urgent need. The use of new adjuvants may well meet this requirement. In this research, we developed a novel adjuvant candidate by combining liposome and QS-21 adjuvant. Adjuvant activity, protective efficacy, the level of neutralizing antibody against PT, and the resident memory T (TRM) cells in lung tissue after vaccination were studied. We then performed B. pertussis respiratory challenge in mice after they received vaccination with traditional aluminum hydroxide and the novel adjuvant combination. Results showed that the liposome + QS-21 adjuvant group had a rapid antibody and higher antibody (PT, FHA, Fim) level, induced anti-PT neutralizing antibody and recruited more IL-17A-secreting CD4+ TRM cells along with IL-17A-secreting CD8+ TRM cells in mice, which provided robust protection against B. pertussis infection. These results provide a key basis for liposome + QS-21 adjuvant as a promising adjuvant candidate for developing an acellular pertussis vaccine that elicits protective immunity against pertussis.

Evaluation of the Local Immune Response to Hydatid Cysts in Sheep Liver.

In order to characterize the inflammatory phenotype of livers of sheep naturally infected by cystic echinococcosis, 100 sheep livers have been macroscopically assessed for the presence of hydatid cysts and sampled for histopathological and molecular analysis. According to gross and microscopic examination, livers were subsequently classified into three groups: normal liver (Group A), liver with the presence of fertile hydatid cysts (Group B), and liver with the presence of sterile hydatid cysts (Group C). Immunohistochemical analyses were accomplished using primary antibodies anti-Iba1, anti-CD3, anti-CD20, anti-TGF-β, and anti-MMP9. Finally, real-time PCR was performed in order to estimate the concentration levels of tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin (IL)-12, IL-10, and TGF-β. Immunohistochemical analysis showed a diffuse immunolabelling of mononuclear cells for Iba-1 and TGF-β and a higher amount of CD20+ B cells compared to CD3+ T cells in both Groups B and C. The expression levels of Th-1-like immune cytokines TNF-α, INF-γ, and IL-12 did not show significant statistical differences. However, we found a significant increase in expression levels of Th-2 immune cytokines TGF-β and IL-10 in Groups B and C compared to Group A. Taken together, our findings suggest that macrophages have a predominant role in the local immune response to cystic echinococcosis. Moreover, we can speculate that Th2 immunity may be dominant, corroborating the idea that B cells are decisively essential in the control of the immune response during parasitic infection and that the immunomodulatory role of IL-10 and TGF-β may ensure the persistence of the parasite within the host.

Adaptive and innate immune responses in multiple sclerosis with anti-CD20 therapy: Gene expression and protein profiles.

Anti-CD20 is a highly effective therapy for multiple sclerosis (MS), a disease with multiple abnormalities in function of B and T cells and innate immune cells. Anti-CD20 therapy depletes B cells, which alters antibody production and has diverse effects on B cell immunity. These changes potentially affect immunity beyond B cells in MS. Determine if anti-CD20 therapy effects non-B cell, as well as B cell, gene expression, and serum protein levels. Samples were collected from 10 healthy controls and from clinically stable relapsing-remitting MS - 10 untreated, 9 interferon-β-treated, and 15 ocrelizumab-treated patients were studied before, and 2 weeks and 6 months after, the first anti-CD20 infusion. Peripheral blood mononuclear cells (PBMC) were analyzed with sensitive, 135,000-transcript RNA expression microarrays, using stringent criteria. Gene expression was compared to 43 MS-relevant serum immune and neurotrophic proteins, using multiplex protein assays. Anti-CD20 therapy reduced expression of 413 total genes and 185 B-cell-regulated genes at 2 weeks vs. pre-therapy. Expression of 19 (15%) of these B cell genes returned toward baseline by 6 months, including genes for the B cell activation protein, CD79A, and for immunoglobulin A, D, and G heavy chains. Expression pathways for Th17 and CD4 regulatory T-cell (Treg) development, differentiation, and proliferation also quieted. In contrast, expression increased in Th1 and myeloid cell antiviral, pro-inflammatory, and toll-like receptor (TLR) gene pathways. These findings have clinical implications. B cell gene expression diminishes 2 weeks after anti-CD20 antibody infusion, but begins to rebound by 6 months. This suggests that the optimum time for vaccination is soon before reinfusion of anti-CD20 therapy. In addition, at 6 months, there is enhanced Th1 cell gene expression and induction of innate immune response genes and TLR expression, which can enhance anti-viral and anti-tumor immunity. This may compensate for diminished B cell gene expression after therapy. These data suggest that anti-CD20 therapy has dynamic effect on B cells and causes a compensatory rise in Th1 and myeloid immunity.

Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cells

IL-17-producing antigen-specific human Tcells elicit potent antitumor activity in mice. Yet, refinement of this approach is needed to position it for clinical use. While activation signal strength regulates IL-17 production by CD4+ Tcells, the degree to which Tcell antigen receptor (TCR) and costimulation signal strength influences Th17 immunity remains unknown. We discovered that decreasing TCR/costimulation signal strength by incremental reduction of αCD3/costimulation beads progressively altered Th17 phenotype. Moreover, Th17 cells stimulated with αCD3/inducible costimulator (ICOS) beads produced more IL-17A, IFNγ, IL-2, and IL-22 than those stimulated with αCD3/CD28 beads. Compared with Th17 cells stimulated with the standard, strong signal strength (three beads per Tcell), Th17 cells propagated with 30-fold fewer αCD3/ICOS beads were less reliant on glucose and favored the central carbon pathway for bioenergetics, marked by abundant intracellular phosphoenolpyruvate (PEP). Importantly, Th17 cells stimulated with weak αCD3/ICOS beads and redirected with a chimeric antigen receptor that recognizes mesothelin were more effective at clearing human mesothelioma. Less effective CAR Th17 cells generated with high αCD3/ICOS beads were rescued by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1), a PEP regulator. Thus, Th17 therapy can be improved by using fewer activation beads during manufacturing, a finding that is cost effective and directly translatable to patients.

Topical Administration of Gardenia jasminoides Extract Regulates Th2 Immunity in OVA-Induced Mice.

A key feature of an allergic immune response is a T helper type 2 (Th2)-mediated response with production of allergen-specific IgE antibodies. Gardenia jasminoides extract with the crocin removed (GJExCR) has been shown to inhibit IgE-mediated allergic disease. To evaluate the efficacy and mechanism-of-action of this inhibition, GJExCR was used in an ovalbumin (OVA)-induced allergy model in BALB/C mice. Sensitization of BALB/C mice with OVA and aluminum hydroxide was performed on days 1 and 14 by intraperitoneal injection, followed by OVA challenge to the dorsal skin for 2 weeks before removal. Seven days post-challenge, mice were treated with GJExCR topically every day for 11 days. Enzyme-linked immunosorbent assay, flow cytometry analysis, real-time PCR, and western blot were performed to determine IgE and Th2 cytokine levels. Following OVA challenge, Th2 cytokine expression and both total and OVA-specific serum IgE levels increased, of which OVA-specific IgE and Th2 cytokine levels decreased after GJExCR treatment. Flow cytometry analysis revealed that GJExCR treatment decreased CD4+ and CD8+ T cell populations in the spleen and lymph nodes. In addition, treatment with GJExCR downregulated signal transducer and activator of transcription 1 (STAT1) activation and Th2 cytokine levels as compared to control. GJExCR containing geniposide downregulated STAT1 activation in HaCaT cells. These findings demonstrate that GJExCR exerts its anti-allergy effect via inhibition of STAT1 activation, thus regulating the immune response via modulation of Th2 cytokine release and IgE levels. Therefore, we propose GJExCR as a potential treatment for allergic hypersensitivity reactions.

Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs.

Most T lymphocytes, including regulatory T cells, express the CD27 costimulatory receptor in steady state conditions. There is evidence that CD27 engagement on conventional T lymphocytes favors the development of Th1 and cytotoxic responses in mice and humans, but the impact on the regulatory lineage is unknown. In this report, we examined the effect of constitutive CD27 engagement on both regulatory and conventional CD4+ T cells in vivo, in the absence of intentional antigenic stimulation. Our data show that both T cell subsets polarize into type 1 Tconvs or Tregs, characterized by cell activation, cytokine production, response to IFN-γ and CXCR3-dependent migration to inflammatory sites. Transfer experiments suggest that CD27 engagement triggers Treg activation in a cell autonomous fashion. We conclude that CD27 may regulate the development of Th1 immunity in peripheral tissues as well as the subsequent switch of the effector response into long-term memory.

B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice.

The current study reveals that in chronic TB, the B cell-deficient μMT strain, relative to wild-type (WT) C57BL/6 mice, displays in the lungs lower levels of inflammation that are associated with decreased CD4+ T cell proliferation, diminished Th1 response, and enhanced levels of interleukin (IL)-10. The latter result raises the possibility that B cells may restrict lung expression of IL-10 in chronic TB. These observations are recapitulated in WT mice depleted for B cells using anti-CD20 antibodies. IL-10 receptor (IL-10R) blockade reverses the phenotypes of decreased inflammation and attenuated CD4+ T cell responses in B cell-depleted mice. Together, these results suggest that in chronic murine TB, B cells, by virtue of their capacity to restrict expression of the anti-inflammatory and immunosuppressive IL-10 in the lungs, promote the development of a robust protective Th1 response, thereby optimizing anti-TB immunity. This vigorous Th1 immunity and restricted IL-10 expression may, however, allow the development of inflammation to a level that can be detrimental to the host. Indeed, decreased lung inflammation observed in chronically infected B cell-deficient mice, which exhibit augmented lung IL-10 levels, is associated with a survival advantage relative to WT animals. Collectively, the results reveal that in chronic murine TB, B cells play a role in modulating the protective Th1 immunity and the anti-inflammatory IL-10 response, which results in augmentation of lung inflammation that can be host-detrimental. Intriguingly, in tuberculous human lungs, conspicuous B cell aggregates are present in close proximity to tissue-damaging lesions manifesting necrosis and cavitation, suggesting the possibility that in human TB, B cells may contribute to the development of exacerbated pathology that is known to promote transmission. Since transmission is a major hindrance to TB control, investigating into whether B cells can shape the development of severe pulmonic pathological responses in tuberculous individuals is warranted.

A58 ENTERIC TUFT CELLS MODULATE LOCAL BUT NOT SYSTEMIC HOST IMMUNE RESPONSES TO CO-ORDINATE TIMELY EXPULSION OF HYMENOLEPIS DIMINUTA

Abstract Background The intestinal tuft cell is a versatile epithelial cell implicated in host detection and defence against enteric nematode and trematode parasites by producing interleukin(IL)-25 and cysteinyl leukotrienes. We have shown that immunocompetent mice develop small intestinal tuft cell hyperplasia during infection with the cestode, Hymenolepis diminuta. Whether tuft cells coordinate murine expulsion of H. diminuta and if this response is similar to other well-established parasitic models is unknown. Purpose To test 1) if tuft cells coordinate host detection and immune responses to H. diminuta; and, 2) if the lack of functional B, T cells and innate lymphoid cells (ILC2s) affect tuft cell hyperplasia and expulsion of the parasite. Method Male and female pou2f3-/-/-/+ littermates, rag-1-/- and C57BL6 mice were infected with 5 cysticercoids of H. diminuta. ILC2s were depleted in rag-1-/- with anti-CD90.2 (250μg/mouse, intraperitoneal). At necropsy, small intestines were flushed with cold PBS to count worms. IgG1, IgG2α and mast cell protease-1 (MCPT-1) was measured in serum and supernatants from splenic cells stimulated with ConA (48h) were assessed by ELISA for IL-4, -5, -10 and -13. Doublecortin-like kinase -1 (DCLK-1)+ tuft cells, goblet cells and eosinophils were identified in sections of paraffin embedded or cryopreserved mid-jejunum sections. mRNA of small intestinal epithelium enriched fractions isolated from mice at control and 11 days post-infection (dpi) was evaluated by qPCR. Result(s) Unlike pou2f3+/- and C57Bl6 mice that expel H. diminuta by 8-11 dpi., pou2f3-/- mice harbour H. diminuta at 11 dpi. Pou2f3-/- mice show similar splenic IL-4, -5, -10, -13, and serum IgG1, IgG2a and MCPT-1 levels at 8 dpi. and higher splenic IL-4, -10 at 11 dpi. compared to pou2f3+/- mice. In contrast, pou2f3+/- mice show higher jejunal goblet cell hyperplasia at 8 dpi., higher levels of small intestinal epithelium expression of dclk-1, alox5 and il-25 and jejunal eosinophilia at 11 dpi. compared to pou2f3-/- mice. At 11 dpi, ILCdepletedrag-1-/- develop tuft cell hyperplasia to the same extent as infected PBS/isotype controls and show no trace of intestinal worms in the lumen. Conclusion(s) Enteric tuft cells coordinate rapid expulsion of H. diminuta from mice, but are not essential for the host to detect and develop Th2 systemic responses in response to the worm. However, in the absence of tuft cells, deficiencies are observed in gut-specific effector immune events commonly associated with timely expulsion of enteric helminths; illustrating a disparity between local and systemic immunity following infection with this tapeworm parasite in a non permissive host. The lack of a functional adaptive immune system and surprisingly, depletion of ILC2s, does not completely abrogate worm expulsion or tuft cell hyperplasia in rag-1-/- at 11 dpi. with H. diminuta. This suggests that an innate immune cell population other than CD90.2+ ILC2s amplifies Th2 immunity to H. diminuta-infection. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; NSERC, , Eyes High doctoral scholarship, Alberta Graduate excellence scholarship Disclosure of Interest None Declared

Maternal helminth infection protects offspring from high-fat-diet-induced obesity through altered microbiota and SCFAs.

Helminth-induced Th2 immunity and gut microbiota have been recently shown to be highly effective in modulating metabolic syndromes in animal models. This study aimed to determine whether maternal immunity and microbial factors affect the induction and development of obesity in offspring. Here, Heligomosomoides polygyrus (Hp)-infected or control female C57BL/6J mice mated with normal males and their offspring were fed a high-fat diet (HFD) for 9 weeks after weaning. Our results showed that Hp-induced maternal outcomes during gestation and lactation significantly impacted offspring metabolic phenotypes. This was evidenced by results showing that offspring from helminth-infected mothers on an HFD (Hp-offspring + HFD) gained significantly less body weight than those from uninfected mothers (Cont-offspring + HFD). Hp-offspring + HFD exhibited no Th2 phenotype but displayed a pattern of gut microbiota composition similar to that of Hp-infected mothers. Cross-fostering experiments confirmed that the helminth-induced maternal attenuation of offspring obesity was mediated through both prenatal and postnatal effects. Our results further showed that helminth-infected dams and their offspring had a markedly altered gut microbiome composition, with increased production of short-chain fatty acids (SCFAs). Intriguingly, Hp-infected mothers and Hp-offspring + HFD showed increased SCFA receptor (GPR) expression in adipose and colonic tissues compared to noninfected mothers and Cont-offspring + HFD, respectively. Moreover, SCFA supplementation to the pups of uninfected control mothers during lactation protected against HFD-induced weight gain, which corresponded with changes in gut bacterial colonization. Collectively, our findings provide new insights into the complex interaction of maternal immune status and gut microbiome, Hp infection, and the immunity and gut microbiome in obese-prone offspring in infant life.

Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments.

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.

The inhibition of retinoic acid signaling in innate immune cells paradoxically suppresses respiratory syncytial virus infection through enhanced antiviral immunity

Respiratory syncytial virus (RSV) infection is a major cause of morbidity and mortality in children. It is also associated with enhanced Th2 immunity and asthma. Vitamin A deficiency weakens mucosal immunity because of the central role of retinoic acid (RA) in regulating different immune cell functions. We therefore sought to evaluate the effect of RA signaling deficiency on immune responses to RSV infection.