Th2 Cytokines IL-4 Research Articles

Trichuris muris whey acidic protein induces type 2 protective immunity against whipworm.

Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm’s pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.

Abstract B055: Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function

Abstract Prostate cancer tumor growth and disease progression is highly influenced by noncancerous host cells within the tumor microenvironment. Tumor-associated macrophages (TAMs) are a critical component of this microenvironment, and high TAM infiltrate correlates with poor prognosis in prostate cancer. Macrophages can inhibit or support tumor growth depending on their polarization to an M1 or M2 state. M1 macrophages, as part of the Th1 immune response that fights bacterial or viral infection, promote helper and killer T-cell recruitment and activation and induce cell death of infected cells. In the tumor, M1s and their recruited T cells target the cancer cells for cell death. Conversely, as part of the Th2 immune response that repairs tissue injury, M2 macrophages promote cell growth and proliferation, inhibit helper and killer T-cell function, and induce regulatory T-cell differentiation. Protumor M2s TAMS make up the majority of macrophage infiltrate in prostate cancer tumors and protect the cancer cells from attack by the immune system. Techniques for studying TAMs in vitro are limited, which is confounded by the lack of procedure consensus for in vitro macrophage differentiation and polarization. The Pienta Lab established an optimized protocol for generating homogenous populations of M1 and M2 macrophages from human monocytes isolated from whole blood (doi: 10.2144/000114435). While these cell populations have been well characterized by their marker expression, their functional ability to modulate T-cell activation is critical to determining their validity as an in vitro TAM model. Following our optimized protocol, monocytes isolated from human blood were differentiated and cultured as resting macrophages or polarized into M1 or M2 macrophages. Conditioned media were collected at 24, 48, and 72 hours. The immunomodulatory capacity of the macrophages was evaluated by measuring the secreted levels of Th1 cytokines IL-1B and TNF known to recruit and activate helper and killer T cells and Th2 cytokine IL-10 known to prevent helper and killer T-cell function and promote regulatory T-cell induction. The in vitro-polarized human M1s secreted high levels of Th1 cytokines IL-1B and TNF, while levels from both the resting and M2-polarized macrophages were below the limit of detection. Likewise, conditioned media from M2-polarized macrophages showed high levels of Th2 cytokine IL-10, but none was detected in resting or M1 cultures. Taken together, this indicates that M1 and M2 macrophages isolated and polarized using our optimized protocol accurately reflect anticipated macrophage immunomodulatory function. Further work will confirm these data by RT-qPCR of RNA taken from M1, M2, and resting macrophage cultures, and conditioned media from serum-free cultures will be assessed to determine the secretion of additional immunomodulatory cytokines present at abundant levels in serum (e.g., TGFB1). Citation Format: Amber E. de Groot, Kenneth J. Pienta. Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B055.

Recombinant leptin attenuates abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice

Vascular disease can manifest as stenotic plaques or ectatic aneurysms. Human abdominal aortic aneurysms (AAA) comprise an inflammatory disease characterized by the predominance of T helper type 2 (Th2) cytokine expression. Leptin has been clearly demonstrated to play an important role in regulating Th0 cell to Th1. So, we hypothesize that leptin has a protective effect on aneurysm formation. In this study, we demonstrated that intraperitoneal injection of leptin attenuated Ang II-induced AAA formation in ApoE−/− mice with no effect on serum lipids and systolic blood pressure. To investigate the mechanisms involved, we found that leptin pretreatment exhibited decreased protein expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 and increased transforming growth factor-β1 (TGF-β1). We also examined potential mechanism of leptin as a modulator of the immune response. Our results proved that pretreatment with leptin downregulated protein expression of Th2 cytokine IL-4 and mRNA levels of GATA-3, the key transcription factor for Th2 polarization, and upregulated Th1 cytokine INF-γ and T-bet, the key transcription factor for Th1 polarization. Taken together, leptin, with the effect of regulation of Th1/Th2 cytokines, may have therapeutic potential for the treatment of AAA. Leptin may constitute a novel therapeutic strategy to prevent AAA formation.

Development of a hollow mesoporous silica nanoparticles vaccine to protect against house dust mite induced allergic inflammation

Allergen specific immunotherapy (SIT) is the only specific therapeutic way for house dust mite (HDM) allergy. To improve the efficacy of SIT, hollow mesoporous silica nanoparticles (HMSNs) were used as vehicles for HDM allergen. The HMSNs were prepared and characterized. The major HDM allergen (Der f2) was loaded onto HMSNs, and the drug loading capacity and release profile were determined. Then the Der f2 loaded HMSNs were injected subcutaneously to mouse model of Der f2 induced allergic asthma and the preventive effects were evaluated. Our results showed that HMSNs were spherical (100 nm) with pore diameter of 2.897 nm and successfully loaded with Der f2 protein. The loading capacity is 90 μg Der f2/1 mg HMSNs. The Der f2 loaded on HMSNs released slowly in 72 h. Treatment with Der f2 loaded HMSNs could efficiently decrease Der f2 specific IgE levels, inflammatory cells infiltration in lung tissue, and Th2 cytokine IL4 levels in BALF. In the meanwhile, it could increase the Der f2 specific IgG levels, Th1 cytokine IFN-γ levels, and induce proliferation of splenocytes to Der f2 accompanied by increased IFN-γ levels. These results showed that Der f2 loaded HMSNs were efficient in preventing allergic inflammation, and HMSNs may be potential vehicles for SIT of HDM allergy.

Activation of natural killer T cells contributes to triptolide-induced liver injury in mice.

Triptolide (TP) is the main active ingredient of Tripterygium wilfordii Hook.f, which has attracted great interest due to its promising efficacy for autoimmune diseases and tumors. However, severe adverse reactions, especially hepatotoxicity, have restricted its approval in the market. In the present study we explored the role of hepatic natural killer T (NKT) cells in the pathogenesis of TP-induced liver injury in mice. TP (600 μg/kg/day, i.g.) was administered to female mice for 1, 3, or 5 days. We found that administration of TP dose-dependently induced hepatotoxicity, evidenced by the body weight reduction, elevated serum ALT and AST levels, as well as significant histopathological changes in the livers. However, the mice were resistant to the development of TP-induced liver injury when their NKT cells were depleted by injection of anti-NK1.1 mAb (200 μg, i.p.) on days -2 and -1 before TP administration. We further revealed that TP administration activated NKT cells, dominantly releasing Th1 cytokine IFN-γ, recruiting neutrophils and macrophages, and leading to liver damage. After anti-NK1.1 injection, however, the mice mainly secreted Th2 cytokine IL-4 in the livers and exhibited a significantly lower percentage of hepatic infiltrating neutrophils and macrophages upon TP challenge. The activation of NKT cells was associated with the upregulation of Toll-like receptor (TLR) signaling pathway. Collectively, these results demonstrate a novel role of NKT cells contributing to the mechanisms of TP-induced liver injury. More importantly, the regulation of NKT cells may promote effective measures that control drug-induced liver injury.

Anti-Allergic Inflammatory Activity of Interleukin-37 Is Mediated by Novel Signaling Cascades in Human Eosinophils.

IL-1 family regulatory cytokine IL-37b can suppress innate immunity and inflammatory activity in inflammatory diseases. In this study, IL-37b showed remarkable in vitro suppression of inflammatory tumor necrosis factor-α, IL-1β, IL-6, CCL2, and CXCL8 production in the coculture of human primary eosinophils and human bronchial epithelial BEAS-2B cells with the stimulation of bacterial toll-like receptor-2 ligand peptidoglycan, while antagonizing the activation of intracellular nuclear factor-κB, PI3K–Akt, extracellular signal-regulated kinase 1/2, and suppressing the gene transcription of allergic inflammation-related PYCARD, S100A9, and CAMP as demonstrated by flow cytometry, RNA-sequencing, and bioinformatics. Results therefore elucidated the novel anti-inflammation-related molecular mechanisms mediated by IL-37b. Using the house dust mite (HDM)-induced humanized asthmatic NOD/SCID mice for preclinical study, intravenous administration of IL-37b restored the normal plasma levels of eosinophil activators CCL11 and IL-5, suppressed the elevated concentrations of Th2 and asthma-related cytokines IL-4, IL-6, and IL-13 and inflammatory IL-17, CCL5, and CCL11 in lung homogenate of asthmatic mice. Histopathological results of lung tissue illustrated that IL-37b could mitigate the enhanced mucus, eosinophil infiltration, thickened airway wall, and goblet cells. Together with similar findings using the ovalbumin- and HDM-induced allergic asthmatic mice further validated the therapeutic potential of IL-37b in allergic asthma. The above results illustrate the novel IL-37-mediated regulation of intracellular inflammation mechanism linking bacterial infection and the activation of human eosinophils and confirm the in vivo anti-inflammatory activity of IL-37b on human allergic asthma.

Genetic polymorphisms of Th2 interleukins, history of asthma or eczema and childhood acute lymphoid leukaemia: Findings from the ESCALE study (SFCE)

BackgroundPrevious studies on the putative role of allergy in the aetiology of childhood leukaemia have reported contradictory results. The present study aimed to analyse the relation between a medical history of asthma or eczema and childhood acute lymphoid leukaemia (ALL) in light of potential candidate gene-environment interactions. MethodsAnalyses were based on a subset of 434 cases of ALL and 442 controls successfully genotyped and of European ancestry children enrolled in a French population-based case-control study conducted in 2003–2004. Information about medical history was obtained during a standardized interview with the mothers. Candidate polymorphisms in genes of the Th2 cytokines IL4, IL10, IL13 and IL4-receptor, were genotyped or imputed. ResultsNone of the variant alleles were directly associated with childhood acute lymphoid leukaemia. A medical history of asthma or eczema was reported more often in the control group (OR = 0.7 [0.5–1.0]). This association was mostly seen in the group of children not carrying the IL13-rs20541 variant allele (Interaction Odds Ratio IOR 1.9, p-interaction = 0.07) and in those carrying the IL10 triple variant haplotype (IOR 0.5, p-interaction = 0.04). No interaction was observed with the candidate polymorphisms in IL4 and IL4R. ConclusionThis study provides a new insight into the relationship between allergic symptoms and childhood acute lymphoid leukaemia, by suggesting this inverse association could be limited to children carrying certain genetic polymorphisms. If confirmed, these results could help better understand the biological mechanisms involved in the development of childhood acute lymphoid leukaemia.

Type 2 innate lymphoid cells contribute to a mixed-type allergic response following topical exposure to the quaternary ammonium compound didecyldimethylammonium chloride.

Abstract Didecyldimethylammonium chloride (DDAC) is an antimicrobial dialkyl-quaternary ammonium compound used in numerous industrial and commercial products. Clinical and animal data suggest that DDAC exposure elicits multiple types of hypersensitivity reactions. To examine the immunological mechanism behind this mixed type response and the potential involvement of ILCs, we assessed early immune responses in the skin following dermal DDAC exposure (0.25% and 0.5%) in a BALB/c murine model. DDAC exposure resulted in a rapid and dramatic increase in the Th2-skewing and ILC2 activating cytokine TSLP in the skin. Correspondingly, dermal ILC2s were activated 24 hours after DDAC exposure, resulting in increased levels of CD25, ICOS and KLRG1, and decreased CD127 on a per cell basis throughout a 7 day period of exposure. Following ILC2 activation, the Th2 cytokine IL-4 was found to be elevated compared to control mice in total ear protein lysate in the 0.5% DDAC treated mice. To determine a functional role for ILC2s in DDAC induced sensitization we used Rag2−/− mice, which lack mature T and B cells. ILC2s from Rag2−/− were similarly activated by DDAC and importantly, produced significant levels of IL-4 (0.5%DDAC) and IL-5 (0.25% and 0.5%) in the skin. These data indicate that ILC2s contribute to Th2 immune responses early after DDAC exposure. ILC2s have been previously implicated allergic responses, but to our knowledge have not been thoroughly investigated in chemical sensitization. These results indicate that skin resident ILC2s become activated and produce TH2 cytokines following exposure to DDAC, providing a possible mechanism for the development of a mixed-type allergic response commonly observed with chemical sensitizers.

Ocular autoimmunity develops despite concurrent absence of Th1 and Th17 signature cytokines

Abstract Th1 and Th17 cells are each a standalone effector phenotype, and each can elicit ocular autoimmunity independently of the other. In this study, we used mice deficient in IL-17A, or IFN-gamma (GKO), or both (DKO) to dissect the role of Th1 and Th17 cytokines in experimental autoimmune uveitis (EAU). EAU was induced by active immunization with the retinal autoantigen IRBP. Previous studies showed that EAU in GKO mice was exacerbated, whereas it was attenuated in IL-17A KO mice. Surprisingly, DKO mice were fully susceptible to EAU with scores similar to wild type (WT) controls, and displayed eosinophil-dominant infiltration along with enhancement of IL-17F, Th2, and proinflammatory cytokines. To test which cytokines contributed to pathology, EAU-challenged DKO mice were treated with blocking antibodies to IL-17F, TNF-a, GM-CSF or IL-4Ra (a shared subunit of receptor for IL-4 and IL-13). Blockade of IL-17F or TNF-a in DKO mice did not affect the severity of EAU. Treatment with an anti-GM-CSF antibody during both induction and expression stages significantly suppressed disease severity in DKO, but not in WT mice. By contrast, treatment of DKO mice with an anti-IL-4Ra antibody worsened EAU. Importantly, a single intravitreal injection of an anti-GM-CSF antibody after the disease onset reduced EAU severity in both WT and DKO mice. These results suggest that, in the absence of both IFN-gamma and IL-17A, (a) GM-CSF plays a major and nonredundant role as a pathogenic cytokine, (b) the Th2 cytokines IL-4 and/or IL-13 are protective, and (c) IL-17F and TNF-a may be dispensable. Our results highlight that ‘classical’ Th1 and Th17 cells are not the only pathogenic effectors, and that other inflammatory cytokine(s) can contribute to uveitis.

CRTAM shapes the microbiota and enhances the severity of gut infection.

Abstract Gut lymphocytes and the microbiota establish a reciprocal relationship that tunes the host immune response. For example, the gut microbiota is required for T helper (Th)-17 cell differentiation; Th17 cytokines, in turn, stimulate the intestinal epithelium to produce antimicrobial proteins that control the gut microbiota. Residency and maintenance of T cells in the gut is controlled by Class I-restricted T cell-associated molecule (CRTAM). We thus evaluated whether CRTAM influences the gut microbiota composition. We found that CRTAM expression caused a shift in several members of the gut microbiota under homeostatic conditions. As the gut microbiota and T cells contribute to host defense against intestinal pathogens, we tested whether CRTAM plays a role during infection with the enteric pathogen Salmonella. We found lower levels of Salmonella colonization in the gut of Crtam−/− mice, along with lower expression of the Th17 cytokines Il17 and Il22 in the cecum. Salmonella-infected Crtam−/− mice exhibited fewer gut T cells and IL-17-producing cells. To investigate whether the altered gut microbiota composition of Crtam−/− mice influenced the host response to infection, we transplanted the fecal microbiota from WT or Crtam−/− littermate mice into germ-free mice, and then infected the mice with Salmonella. Relative to mice transplanted with “WT” fecal microbiota, mice transplanted with “Crtam−/−” microbiota exhibited lower numbers of T cells and IL-17-producing cells in the gut upon infection. Our results thus suggest an interplay between CRTAM expression and the gut microbiota, which ultimately impacts the mucosal immune system and the host response to enteric pathogens.

Augmented Th17 Differentiation Leads to Cutaneous and Synovio-Entheseal Inflammation in a Novel Model of Psoriatic Arthritis.

To introduce a novel preclinical animal model of psoriatic arthritis (PsA) in R26Stat3Cstopfl/fl CD4Cre mice, and to investigate the role of Th17 cytokines in the disease pathogenesis. We characterized a novel murine model of Th17-driven cutaneous and synovio-entheseal disease directed by T cell-specific expression of a hyperactive Stat3 allele. By crossing R26Stat3Cstopfl/fl CD4Cre mice onto an interleukin-22 (IL-22)-knockout background or treating the mice with a neutralizing antibody against IL-17, we interrogated how these Th17 cytokines could contribute to the pathogenesis of PsA. R26Stat3Cstopfl/fl CD4Cre mice developed acanthosis, hyperkeratosis, and parakeratosis of the skin, as well as enthesitis/tendinitis and periarticular bone erosion in different joints, accompanied by osteopenia. T cell-specific expression of a hyperactive Stat3C allele was found to drive the augmented Th17 response in these animals. Careful characterization of the mouse bone marrow revealed an increase in osteoclast progenitor (OCP) and RANKL-producing cells, which contributed to the osteopenia phenotype observed in the mutant animals. Abrogation of the Th17 cytokines IL-17 or IL-22 improved both the skin and bone phenotype in R26Stat3Cstopfl/fl CD4Cre mice, revealing a central role of Th17 cells in the regulation of OCP and RANKL expression on stromal cells. Perturbation of the IL-23/Th17 axis instigates Th17-mediated inflammation in R26Stat3Cstopfl/fl CD4Cre mice, leading to cutaneous and synovio-entheseal inflammation and bone pathologic features highly reminiscent of human PsA. Both IL-17A and IL-22 produced by Th17 cells appear to play critical roles in promoting the cutaneous and musculoskeletal inflammation that characterizes PsA.

Enhanced Pro-Inflammatory Response of Macrophages to Interleukin-33 in an Allergic Environment

Background: Allergic asthma is common in childhood and is associated with a T-helper type 2 (Th2)-biased immunological response. Exacerbations of asthma are characterised by increased inflammation of the airways, which appears to be driven by interleukin (IL)-33 that can activate pulmonary macrophages. The Th2 cytokine environment of allergic asthma may contribute to exaggerated airway inflammation. Objectives: To test this, we assessed whether production of pro-inflammatory cytokines by IL-33-stimulated macrophages was enhanced in cells pre-treated with the key Th2 cytokines IL-4 and IL-13. We also investigated whether this was associated with altered expression of regulatory microRNAs (miRNAs). Methods: RAW264.7 cells cultured with IL-4 and IL-13 for 48 h were stimulated with IL-33 for 4 h. Pro-inflammatory mediators were assessed using quantitative real-time PCR (RT-PCR). Expression of miRNAs was assessed using microarrays and RT-PCR. In further experiments, we examined whether resolvin E1 (RvE1), which promotes the resolution of experimental asthmatic inflammation in vivo, could suppress the enhanced response by treating cells with RvE1 concurrently with IL-33 stimulation. Results: In cells pre-treated with IL-4 and IL-13, expression of mRNA for Ccl3, Ccl5, Ccl17, Ccl24, and Il1b in response to IL-33 stimulation was significantly increased. This was paralleled by up-regulated expression of miR-155-5p, a miRNA that is predicted to regulate several aspects of allergic inflammation. RvE1 suppressed the enhanced production of Ccl3, Ccl5, Ccl24, and Il1b. Conclusions: We conclude that IL-33-activated macrophages may contribute to the exaggerated airway inflammation in exacerbations of allergic asthma, and that RvE1 has potential as a therapeutic agent that targets macrophages.

Gene Expression Analysis of the Effect of Microbial Tryptophan Metabolites on T‐cell Differentiation

The goal of this work is to elucidate the molecular basis underlying the effect of tryptophan‐derived microbiota metabolites on T‐cell differentiation and function. Naive T‐cells differentiate into various subsets with specialized functions, including pro‐tolerogenic regulatory T (Treg) cells and pro‐inflammatory T helper 17 (Th17) cells. An imbalance in the relative proportions of the different T‐cell subtypes is associated with a number of autoimmune and inflammatory diseases. While the role of host cytokines and growth factors in T‐cell differentiation are well established, recent studies have suggested that the gut microbiota metabolites also modulate T‐cell differentiation. Recent work from our laboratory has demonstrated that the microbiota metabolite indole, which has been previously shown to attenuate indicators of inflammation in intestinal epithelial cells, augments TGF‐β mediated Treg differentiation from naïve T‐cells as well as suppresses IL‐6 mediated Th17 differentiation, promoting an anti‐inflammatory phenotype. On the other hand, 5‐hydroxyindole (5HI) shows reciprocal effects, suppressing Treg differentiation and augmenting Th17 differentiation. However, the mechanisms by which indole and 5HI modulate T‐cell differentiation and their effects on function are unknown. Using microarrays, we identified 551 and 219 differentially expressed genes in indole treated Treg cells and 5‐HI treated Th17 cells, respectively, as compared to conventionally differentiated cells. Indole treatment resulted in a slight decrease in overall transcription, with 290 of the differentially expressed genes being upregulated, while 5HI increased overall transcription with 182 differentially expressed genes being upregulated. The differentially expressed genes in Treg cells with indole treatment was largely comprised of enzymes (14%), transcription factors (10%), and cytokines (7%), while 5HI resulted in differential regulation of enzymes (10%), cytokines (9%), and transmembrane receptors (5%) in Th17 cells. The glycine amidinotransferase (Gatm) gene that is involved in creatine biosynthesis and contains an Aryl Hydrocarbon Responsive Element II (AHRE‐II) in its 5’ regulatory region was up‐regulated 12‐fold in indole treated Treg cells, suggesting a role for the aryl hydrocarbon receptor (AhR) in indole signaling. The gene encoding the IL‐13 cytokine was the most strongly induced by indole treatment (82‐fold increase). IL‐13 is produced by Th2 cells and has both pro and anti‐inflammatory properties. Interestingly, other Th2 cytokines genes, Il4 and Il5, were also up‐regulated at 10‐ and 26‐fold, respectively. This unexpected pattern of signaling suggests that indole may induce the differentiation of a unique cell type with phenotypes characteristic of both Treg cells and Th2 cells. In 5HI treated Th17 cells, a 15‐ and 3‐fold increase in the expression of genes encoding the pro‐inflammatory cytokines IL‐22 and IFNg was observed. The gene for IL‐17, which is the characteristic cytokine of Th17 cells was upregulated 1.6 fold. These expression patterns support the induction of pro‐inflammatory signaling by 5HI. Overall, these results suggest that different tryptophan‐derived microbiota metabolites can have different effects on T‐cell differentiation and function. Further characterization of cell function and investigation into the role of the AhR the signaling of these metabolites is warranted.Support or Funding InformationNational Institutes of Health R01AI110642This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia.

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100ml peripheral blood from pediatric pre-B ALL patients was processed including CD4+/CD8+-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4+ = 50%, CD8+ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

Regulation of type 2 innate lymphoid cell–dependent airway hyperreactivity by butyrate

Allergic asthma is characterized by airway hyperreactivity (AHR) and inflammation driven by aberrant TH2 responses. Type 2 innate lymphoid cells (ILC2s) are a critical source of the TH2 cytokines IL-5 and IL-13, which promote acute asthma exacerbation. Short-chain fatty acids (SCFAs) have been shown to attenuate T cell-mediated allergic airway inflammation. However, their role in regulation of ILC2-driven AHR and lung inflammation remains unknown. We investigated the immunomodulatory role of SCFAs in regulation of ILC2-induced AHR and airway inflammation and delineated the mechanism involved. We assessed the role of SCFAs in regulating survival, proliferation, and cytokine production in lung sorted ILC2s. The SCFA butyrate was administered through drinking water or intranasally in BALB/c mice to evaluate its role in the ILC2-driven inflammatory response in IL-33 and Alternaria alternata models of allergic inflammation. We further confirmed our findings in human ILC2s. We show that butyrate, but not acetate or propionate, inhibited IL-13 and IL-5 production by murine ILC2s. Systemic and local administration of butyrate significantly ameliorated ILC2-driven AHR and airway inflammation. We further demonstrate that butyrate inhibited ILC2 proliferation and GATA3 expression but did not induce cell apoptosis, likely through histone deacetylase (HDAC) inhibition, because trichostatin A, a pan-HDAC inhibitor, exerted similar effects on ILC2s. Importantly, cotreatment with trichostatin Aand butyrate did not result in an additive effect. Finally, we show that butyrate reduces cytokine production in human ILC2s. Our findings identify butyrate as a critical regulator of ILC2 proliferation and function through its HDAC inhibitory activity and can serve as a potential therapeutic target for asthma.

What We're Reading

Getting stabilized (from cegoh via Pixabay)A link has been found between oncogenic RAS signaling and increased immuno-suppressive expression of PD-L1. The mechanism relies on stabilization of the PD-L1 transcript, with RAS signaling culminating in phosphorylation and inactivation of TTP, a factor involved in degrading PD-L1 transcripts. As TTP inactivation causes accumulation of PD-L1 mRNA, interfering with the RAS pathway increases TTP binding to AU-rich elements of the transcripts, decreases PD-L1 protein production, and provides a means to enhance antitumor immunity.Coelho MA, …, Downward J. Immunity 2017 Dec 19;47:1083–99.e6.Resolvin D2 (from Roadnottaken via Wikimedia Commons)The goal of radio-, chemo-, and targeted therapy of tumors is to destroy tumor cells. However, the debris left from cell destruction can promote tumor growth through induction of local inflammation and cytokine release. Resolvins, as their name implies, quietly clean up and tune down inflammation. Addition of RvD1, RvD2, or RvE1 successfully cleared debris in a process dependent on phagocytosis by macrophages and reduction of inflammatory cytokine production.Sulciner ML, …, Panigrahy D. J Exp Med 2018 Jan 2;215:115–140.Shielded from removal (from LavaSnake via Wikimedia Commons)The proportion of patients that respond to PD-1 blockade remains low. PD-1 inhibits through recruitment of SHP2 and subsequent dephosphorylation of tyrosines involved in propagating TCR signals. SAP was found bound to intracellular PD-1 complexes and shielded phosphates from dephosphorylation by SHP2. Increased expression of SAP correlated with decreased inhibitory signaling through PD-1. Thus, therapeutically targeting SHP2 or SAP to increase T-cell responses could provide an approach to enhance responses to PD-1 blockade.Peled M, …, Mor, A. Proc Natl Acad Sci USA 2018 Jan 16;115:E468–77.Targeted IFN delivery plus TNF (from Cauwels et al.)Interferons can support antitumor activity, but have pleiotropic effects. Injection of exogenous IFN is associated with adverse side effects. Removing IFN's ability to bind to its widely expressed natural receptor, and providing it with an alternative segment that binds to Clec9, a DC-specific protein, allowed targeting of minute quantities of IFN to tumoral DCs without toxicity, and, with other therapies, completely eradicated tumors considered to be poorly immunogenic.Cauwels A, …, Tavernier J. Cancer Res 2018 Jan 15;78:463–74.Neutrophil enablers (from BruceBlaus via Wikimedia Commons)A hallmark of progression in many solid tumors is a preponderance of myeloid-derived cells and an increase in circulating myeloid-lineage cells. Nonmetastatic lung adenocarcinomas were found to disrupt bone marrow homeostasis in humans and mice. Osteocalcin+ osteoblasts increase in the bone marrow, and are responsible for the expansion of tumor-promoting SiglecFhigh neutrophils that accumulate in the tumor. The results highlight that cancer is a systemic disease, with cross-talk between lung tumors and bone enhancing tumor progression.Engblom C, …, Pittet MJ. Science 2017 Dec 1;358:eaal5081.Gastric tumor (from MF Press et al, Mol Cancer Therapeutics 2017)Although CTLA-4 acts as an inhibitor of antitumor responses, paradoxically, people with genetically-reduced expression of CTLA-4 have a greater risk for gastric cancer (GC). Insufficiency of CTLA-4 expression in mice instigates an inflammatory state that becomes premalignant and progresses to GC. The progression is not caused by dysregulation of microbiota, and can be transferred with spleen cells to Rag1-deficient mice. Th2 cytokines IL4 and IL13 were essential for tumorigenesis, inducing epigenetic disruptions in human GC, and Th1 or Th17 cells were not involved.Miska J, …, Chen Z. J Exp Med 2018 Jan 26. DOI: 10.1084/jem.20171971.

Rhinovirus respiratory tract infection in hospitalized adult patients is associated with TH2 response irrespective of asthma

ObjectivesWe assessed the immunological response of hospitalized adult patients with rhinovirus infection, including critically-ill patients. MethodsThe differential white blood cell (WBC) count and the levels of 29 plasma cytokines/chemokines were compared between 50 adult hospitalized patients with rhinovirus infection and 100 age-matched controls with influenza virus infection. ResultsThe demographics and comorbidities were similar between rhinovirus and influenza patients, but severe disease was more common for the rhinovirus cohort. Rhinovirus patients had significantly higher WBC counts than influenza patients, especially for eosinophil (P = 3.1 × 10−8). The level of the TH2 cytokine IL-5 was significantly higher among rhinovirus patients, while the levels of 9 other cytokines/chemokines were significantly lower among rhinovirus patients. The levels of CXCL-10 (IP-10), CCL-2 (MCP-1), IFN-α2, IFN-γ, IL-10, and IL-15 remained significantly lower among rhinovirus patients after correction for multiple comparisons. Notably, CXCL-10 had the highest area under the receiver operating characteristic curve (AUC) in differentiating rhinovirus from influenza patients (AUC, 0.918). In the patient subgroup without asthma, the difference in the WBC count and cytokine/chemokine levels between rhinovirus and influenza patients remained statistically significant. ConclusionsRhinovirus infection was characterized by a prominent TH2 response, even in patients without asthma. CXCL-10 (IP-10) is a potential biomarker in differentiating rhinovirus from influenza infection.

Identification of T helper (Th)1- and Th2-associated antigens of Cryptococcus neoformans in a murine model of pulmonary infection

Cryptococcosis, caused by Cryptococcus neoformans, has been demonstrated to be controlled by T helper (Th)1 cells while Th2 cells are associated with fungal growth and dissemination. Although cryptococcal immunoreactive protein antigens were previously identified, their association with Th1 or Th2 immune responses was not provided. In mice, Th1-dependent IFN-γ induces the production of IgG2a, whereas the Th2 cytokine IL-4 stimulates the expression of IgG1 rendering each isotype an indicator of the underlying Th cell response. Therefore, we performed an immunoproteomic study that distinguishes Th1- and Th2-associated antigens by their reactivity with Th1-dependent IgG2a or Th2-dependent IgG1 antibodies in sera from C. neoformans-infected wild-type mice. We additionally analysed sera from Th2-prone IL-12-deficient and Th1-prone IL-4Rα-deficient mice extending the results found in wild-type mice. In total, ten, four, and three protein antigens associated with IgG1, IgG2a, or both isotypes, respectively, were identified. Th2-associated antigens represent promising candidates for development of immunotherapy regimens, whereas Th1-associated antigens may serve as candidates for vaccine development. In conclusion, this study points to intrinsic immunomodulatory effects of fungal antigens on the process of Th cell differentiation based on the identification of cryptococcal protein antigens specifically associated with Th1 or Th2 responses throughout mice of different genotypes.

ITGB4 is essential for containing HDM-induced airway inflammation and airway hyperresponsiveness.

Airway epithelial cells play a significant role in the pathogenesis of asthma. Although the structural and functional defects of airway epithelial cells have been postulated to increase asthma susceptibility and exacerbate asthma severity, the mechanism and implication of these defects remain uncertain. Integrin β4 (ITGB4) is a structural adhesion molecule that is downregulated in the airway epithelium of asthma patients. In this study, we demonstrated that ITGB4 deficiency leads to severe allergy-induced airway inflammation and airway hyper-responsiveness (AHR) in mice. After house dust mite (HDM) challenge, epithelial cell-specific ITGB4-deleted mice showed increased lymphocyte, eosinophil, and neutrophil infiltration into lung compared with that of the wild-type mice. ITGB4 deficiency also resulted in increased expression of the Th2 cytokine IL-4, IL-13, and the Th17 cytokine IL-17A in the lung tissue and in the T cells after HDM challenge. The aggravated inflammation in ITGB4 defect mice was partly caused by enhanced disrupted epithelial barrier integrity after HDM stress, which induced the increased thymic stromal lymphopoietin secretion from airway epithelial cells. This study therefore demonstrates that ITGB4 plays a pivotal role in containing allergen-mediated lung inflammation and airway hyper-responsiveness in allergic asthma.

Thymic stromal lymphopoietin drives the development of IL-13+ Th2 cells

T helper 2 (Th2) cells are pivotal in the development of allergy. Allergen exposure primes IL-4+ Th2 cells in lymph node, but production of effector cytokines including IL-5 and IL-13 is thought to require additional signals from antigen and the environment. Here we report that a substantial proportion of naive CD4+ T cells in spleen and lymph node express receptors for the epithelium-derived inflammatory cytokine thymic stromal lymphopoietin (TSLP). Culture of naive CD4+ T cells in anti-(a)CD3, aCD28, and TSLP-supplemented Th2 conditions enabled the development of a unique population of IL-13-single positive (IL-13-SP) CD4+ T cells; TSLP and Th2 conditions were both required for their development. Sorting experiments revealed that IL-13-SP Th2 cells originated from IL-4-negative precursors and coexpressed transcripts for the Th2 cytokines IL-5 and IL-9. In vivo, high TSLP levels acted directly on CD4+ T cells to induce the development of IL-13-SP and IL-4+IL-13+ double-positive populations in lymph node. These cells were phenotypically similar to Th2 effector cells and were CXCR5lowPD1low and expressed low levels of Bcl6 and Il21 transcripts and high levels of Gata3, Il3, and Il5 Our findings suggest a role of TSLP in directly promoting Th2 cell effector function and support the notion of TSLP as a key driver of Th2 inflammation.