The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

Gut microbiota modulates bleomycin-induced acute lung injury response in mice.

BACKGROUND: Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. METHODS: Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. RESULTS: Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors' response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. CONCLUSION: The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

IL-33-mediated Eosinophilia Protects against Acute Lung Injury.

Pneumonia-induced lung injury and acute respiratory distress syndrome can develop because of an inappropriate inflammatory response to acute infections, leading to a compromised alveolar barrier. Recent work suggests that hospitalized patients with allergies/asthma are less likely to die of pulmonary infections and that there is a correlation between survival from acute respiratory distress syndrome and higher eosinophil counts; thus, we hypothesized that eosinophils associated with a type 2 immune response may protect against pneumonia-induced acute lung injury. To test this hypothesis, mice were treated with the type 2-initiating cytokine IL-33 intratracheally 3 days before induction of pneumonia with airway administration of a lethal dose of Staphylococcus aureus. Interestingly, IL-33 pretreatment promoted survival by inhibiting acute lung injury: amount of BAL fluid proinflammatory cytokines and pulmonary edema were both reduced, with an associated increase in oxygen saturation. Pulmonary neutrophilia was also reduced, whereas eosinophilia was strongly increased. This eosinophilia was key to protection; eosinophil reduction eliminated both IL-33-mediated protection against mortality and inhibition of neutrophilia and pulmonary edema. Together, these data reveal a novel role for eosinophils in protection against lung injury and suggest that modulation of pulmonary type 2 immunity may represent a novel therapeutic strategy.

Temperature Trajectory Subphenotypes Correlate With Immune Responses in Patients With Sepsis.

OBJECTIVES: We recently found that distinct body temperature trajectories of infected patients correlated with survival. Understanding the relationship between the temperature trajectories and the host immune response to infection could allow us to immunophenotype patients at the bedside using temperature. The objective was to identify whether temperature trajectories have consistent associations with specific cytokine responses in two distinct cohorts of infected patients. DESIGN: Prospective observational study. SETTING: Large academic medical center between 2013 and 2019. SUBJECTS: Two cohorts of infected patients: 1) patients in the ICU with septic shock and 2) hospitalized patients with Staphylococcus aureus bacteremia. INTERVENTIONS: Clinical data (including body temperature) and plasma cytokine concentrations were measured. Patients were classified into four temperature trajectory subphenotypes using their temperature measurements in the first 72 hours from the onset of infection. Log-transformed cytokine levels were standardized to the mean and compared with the subphenotypes in both cohorts. MEASUREMENTS AND MAIN RESULTS: The cohorts consisted of 120 patients with septic shock (cohort 1) and 88 patients with S. aureus bacteremia (cohort 2). Patients from both cohorts were classified into one of four previously validated temperature subphenotypes: "hyperthermic, slow resolvers" (n = 19 cohort 1; n = 13 cohort 2), "hyperthermic, fast resolvers" (n = 18 C1; n = 24 C2), "normothermic" (n = 54 C1; n = 31 C2), and "hypothermic" (n = 29 C1; n = 20 C2). Both "hyperthermic, slow resolvers" and "hyperthermic, fast resolvers" had high levels of G-CSF, CCL2, and interleukin-10 compared with the "hypothermic" group when controlling for cohort and timing of cytokine measurement (p < 0.05). In contrast to the "hyperthermic, slow resolvers," the "hyperthermic, fast resolvers" showed significant decreases in the levels of several cytokines over a 24-hour period, including interleukin-1RA, interleukin-6, interleukin-8, G-CSF, and M-CSF (p < 0.001). CONCLUSIONS: Temperature trajectory subphenotypes are associated with consistent cytokine profiles in two distinct cohorts of infected patients. These subphenotypes could play a role in the bedside identification of cytokine profiles in patients with sepsis.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

T-cell phenotypes are associated with serum IgE levels in Amish and Hutterite children.

OBJECTIVES: Amish children raised on traditional farms have lower atopy and asthma risk than Hutterite children raised on modern farms. In our previous study we established that the Amish environment affects the innate immune response to decrease asthma and atopy risk. Here we investigated T-cell phenotypes in the same Amish and Hutterite children as in our earlier study to elucidate how this altered innate immunity affects adaptive T cells. METHODS: Blood was collected from 30 Amish and 30 Hutterite age- and sex-matched children; cells were cryopreserved until analysis. Flow cytometry was used to analyze cell subsets. Atopy was determined based on allergen-specific and total IgE levels. RESULTS: Children exposed to Amish farms had increased activated regulatory CD4+ T-cell phenotypes, whereas conventional CD4 T cells expressed lower levels of costimulation molecules and other activation markers. The increase in numbers of circulating activated regulatory CD4+ T cells was associated with an increase in inhibitory receptors on monocytes in Amish, but not Hutterite, children. Strikingly, the Amish children had a higher proportion of CD28null CD8 T cells than the Hutterite children (P < .0001, nonparametric t test), a difference that remained even after accounting for the effects of age and sex (conditional log regression exponential ß = 1.08, P = .0053). The proportion of these cells correlated with high T-cell IFN-γ production (rs = 0.573, P = .005) and low serum IgE levels (rs = -0.417, P = .025). Furthermore, CD28null CD8 T-cell numbers were increased in Amish children, with high expression of the innate genes TNF and TNF-α-induced protein 3 (TNFAIP3) in peripheral blood leukocytes. CONCLUSION: Amish children's blood leukocytes are not only altered in their innate immune status but also have distinct T-cell phenotypes that are often associated with increased antigen exposure.

Effects of an FcγRIIA polymorphism on leukocyte gene expression and cytokine responses to anti-CD3 and anti-CD28 antibodies.

The low affinity Fcγ receptor, FcγRIIA, harbors a common missense mutation, rs1801274 (G>A, Arg131His) that modifies binding affinity to human IgG2 and mouse IgG1 antibodies and is associated with increased risk of autoimmune disease. Despite the important role of the Arg131His variant, little is understood about heterozygous genotype effects on global gene expression and cytokine production during an FcγR-dependent response. To address this gap in knowledge, we treated human whole-blood samples from 130 individuals with mouse IgG1 anti-CD3 and anti-CD28 antibodies and characterized the genome-wide gene expression profiles and cytokine production among individuals stratified by rs1801274 genotype. Our analysis revealed that the levels of four cytokines (IFNγ, IL-12, IL-2, TNFα) and global gene expression patterns differed between all three genotype classes. Surprisingly, the heterozygotes showed suboptimal T cell activation compared to cells from individuals homozygous for the higher-affinity FcγRIIA allele (GG; Arg/Arg). The results of this study demonstrate that IgG response varies among all rs1801274 genotype classes and results in profound differences in both cytokine responses and gene expression patterns in blood leukocytes. Because even heterozygotes showed dampened global responses, our data may provide insight into the heterogeneity of outcomes in cytokine release assays and immunotherapy efficacy.

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children.

BACKGROUND: The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. METHODS: We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. RESULTS: Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS: The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Distinct T-helper cell responses to Staphylococcus aureus bacteremia reflect immunologic comorbidities and correlate with mortality.

BACKGROUND: The dysregulated host immune response that defines sepsis varies as a function of both the immune status of the host and the distinct nature of the pathogen. The degree to which immunocompromising comorbidities or immunosuppressive medications affect the immune response to infection is poorly understood because these patients are often excluded from studies about septic immunity. The objectives of this study were to determine the immune response to a single pathogen (Staphylococcus aureus) among a diverse case mix of patients and to determine whether comorbidities affect immune and clinical outcomes. METHODS: Blood samples were drawn from 95 adult inpatients at multiple time points after the first positive S. aureus blood culture. Cox proportional hazards modeling was used to determine the associations between admission neutrophil counts, admission lymphocyte counts, cytokine levels, and 90-day mortality. A nested case-control flow cytometric analysis was conducted to determine T-helper type 1 (Th1), Th2, Th17, and regulatory T-cell (Treg) subsets among a subgroup of 28 patients. In a secondary analysis, we categorized patients as either having immunocompromising disorders (human immunodeficiency virus and hematologic malignancies), receiving immunosuppressive medications, or being not immunocompromised. RESULTS: Higher neutrophil-to-lymphocyte count ratios and higher Th17 cytokine responses relative to Th1 cytokine responses early after infection were independently associated with mortality and did not depend on the immune state of the patient (HR 1.93, 95% CI 1.17-3.17, p = 0.01; and HR 1.13, 95% CI 1.01-1.27, p = 0.03, respectively). On the basis of flow cytometric analysis of CD4 T-helper subsets, an increasing Th17/Treg response over the course of the infection was most strongly associated with increased mortality (HR 4.41, 95% CI 1.69-11.5, p < 0.01). This type of immune response was most common among patients who were not immunocompromised. In contrast, among immunocompromised patients who died, a decreasing Th1/Treg response was most common. CONCLUSIONS: The association of both increased Th17 responses and increased neutrophil counts relative to lymphocyte counts with mortality suggests that an overwhelming inflammatory response is detrimental. However, the differential responses of patients according to immune state suggest that immune status is an important clinical indicator that should be accounted for in the management of septic patients, as well as in the development of novel immunomodulatory therapies.

The role of dendritic cells and monocytes in the maintenance and loss of respiratory tolerance.

Promoting tolerance to inhaled antigens is an active area of study with the potential to benefit the millions of Americans currently suffering from respiratory allergies and asthma. Interestingly, not all individuals with atopy are symptomatic, arguing that sensitization alone does not lead to an allergic clinical phenotype. Respiratory dendritic cells (rDCs), classically associated with inducing inflammatory responses, can actively promote tolerance. Tolerance can be broken when inflammatory stimuli, including viral infections and other environmental exposures, inhibit rDC-mediated tolerance by allowing innocuous antigen to be presented to initiate type-2 immunity. Importantly, rDCs are composed of multiple subsets, each with a unique response to an inhaled antigen that can lead to either tolerance or inflammation. In this review, we will discuss how rDC subsets actively maintain tolerance or, alternatively, break tolerance in response to environmental cues.

Signaling through FcRγ-associated receptors on dendritic cells drives IL-33-dependent TH2-type responses.

BACKGROUND: Although allergic sensitization can be generated against various allergens, it is unknown how such a diversity of antigens is able to promote TH2-mediated inflammation leading to atopy. Our previous studies demonstrated that allergen-specific IgG immune complexes (ICs) and house dust mite (HDM) extract both induced dendritic cells (DCs) to drive TH2-mediated inflammation, but the mechanism by which these diverse stimuli produce similar responses is unknown. OBJECTIVE: We sought to identify the DC signaling pathways used by TH2 stimuli to promote TH2-mediated inflammation. METHODS: C57BL/6, FcγRIII(-/-), FcRγ(-/-), and ST2(-/-) mice were sensitized and challenged with HDM, and inflammation was assessed based on results of flow cytometry and histology and cytokine production. Bone marrow-derived DCs from these strains were used in signaling and adoptive transfer experiments. RESULTS: Our findings indicate that 2 distinct TH2 stimuli, ICs and HDM, use the FcRγ-associated receptors FcγRIII and Dectin-2, respectively, to promote TH2-mediated lung inflammation. In this study we demonstrate that both ICs and HDM induce expression of IL-33, a critical mediator in asthma pathogenesis and the differentiation of TH2 cells, in DCs. Upregulation of IL-33 in DCs is dependent on FcRγ, Toll-like receptor 4, and phosphoinositide 3-kinase. Exogenous IL-33 is sufficient to restore the development of TH2 responses in FcRγ-deficient mice. Finally, adoptive transfer of allergen-pulsed FcRγ(+/-) bone-marrow derived DCs restores the development of TH2-type inflammation in FcRγ-deficient mice, demonstrating the necessity of this signaling pathway in DCs for allergen-induced inflammation. CONCLUSION: These data identify a mechanism whereby TH2 stimuli signal through FcRγ-associated receptors on DCs to upregulate IL-33 production and induce TH2-mediated allergic airway inflammation.

IRF4 expression by lung dendritic cells drives acute but not Trm cell-dependent memory Th2 responses.

Expression of the transcription factor interferon regulatory factor 4 (IRF4) is required for the development of lung conventional DCs type 2 (cDC2s) that elicit Th2 responses, yet how IRF4 functions in lung cDC2s throughout the acute and memory allergic response is not clear. Here, we used a mouse model that loses IRF4 expression after lung cDC2 development to demonstrate that mice with IRF4-deficient DCs display impaired memory responses to allergen. This defect in the memory response was a direct result of ineffective Th2 induction and impaired recruitment of activated effector T cells to the lung after sensitization. IRF4-deficient DCs demonstrated defects in their migration to the draining lymph node and in T cell priming. Finally, T cells primed by IRF4-competent DCs mediated potent memory responses independently of IRF4-expressing DCs, demonstrating that IRF4-expressing DCs are not necessary during the memory response. Thus, IRF4 controlled a program in mature DCs governing Th2 priming and effector responses, but IRF4-expressing DCs were dispensable during tissue-resident memory T cell-dependent memory responses.

Pro-lymphangiogenic VEGFR-3 signaling modulates memory T cell responses in allergic airway inflammation.

In allergic airway inflammation, VEGFR-3-mediated lymphangiogenesis occurs in humans and mouse models, yet its immunological roles, particularly in adaptive immunity, are poorly understood. Here, we explored how pro-lymphangiogenic signaling affects the allergic response to house dust mite (HDM). In the acute inflammatory phase, the lungs of mice treated with blocking antibodies against VEGFR-3 (mF4-31C1) displayed less inflammation overall, with dramatically reduced innate and T-cell numbers and reduced inflammatory chemokine levels. However, when inflammation was allowed to resolve and memory recall was induced 2 months later, mice treated with mF4-31C1 as well as VEGF-C/-D knockout models showed exacerbated type 2 memory response to HDM, with increased Th2 cells, eosinophils, type 2 chemokines, and pathological inflammation scores. This was associated with lower CCL21 and decreased TRegs in the lymph nodes. Together, our data imply that VEGFR-3 activation in allergic airways helps to both initiate the acute inflammatory response and regulate the adaptive (memory) response, possibly in part by shifting the TReg/Th2 balance. This introduces new immunomodulatory roles for pro-lymphangiogenic VEGFR-3 signaling in allergic airway inflammation and suggests that airway lymphatics may be a novel target for treating allergic responses.

Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Transcriptional programming and T cell receptor repertoires distinguish human lung and lymph node memory T cells.

Antigen-specific memory T cells persist for years after exposure to a pathogen and provide effective recall responses. Many memory T cell subsets have been identified and differ in abundance throughout tissues. This study focused on CD4 and CD8 memory T cells from paired human lung and lung draining lymph node (LDLN) samples and identified substantial differences in the transcriptional landscape of these subsets, including higher expression of an array of innate immune receptors in lung T cells which were further validated by flow cytometry. Using T cell receptor analysis, we determined the clonal overlap between memory T cell subsets within the lung and within the LDLN, and this was greater than the clonal overlap observed between memory T cell subsets compared across tissues. Our results suggest that lung and LDLN memory T cells originate from different precursor pools, recognize distinct antigens and likely have separate roles in immune responses.

T cell Co-Stimulatory molecules ICOS and CD28 stratify idiopathic pulmonary fibrosis survival.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease that kills as many Americans as breast cancer each year. This study investigated whether lung function decline and survival associates with adaptive immunity in patients with IPF, specifically the expression of checkpoint molecules ICOS, CD28 and PD-1 on circulating CD4 T cells. Clinical data, blood samples and pulmonary function tests were collected prospectively and longitudinally from 59 patients with IPF over a study period of 5 years. Patients were followed until death, lung transplantation, or study end, and cell surface expression of CD45RO, CD28, ICOS, and PD-1 was measured on CD4 T cells via flow cytometry. Repeated measures of ICOS and CD28 on CD4 T cells revealed significant associations between declining ICOS and CD28 expression, and declining lung function parameters FVC and DLCO, independent of age, sex, race, smoking history, or immunosuppressant use. Strikingly, patients in the highest quintile of ICOS at study entry had markedly improved survival, while those with low CD28 fared poorly. No change in PD-1 expression was found. Analysis of ICOS and CD28 from the first blood draw identified three populations of IPF patients; those at high risk for early death, those with intermediate risk, and those at low risk. These results highlight the role of T cell mediated immunity in IPF survival, finding the assessment of two T cell stimulatory checkpoint molecules, CD28 and ICOS, was sufficient to discriminate three distinct survival trajectories over 5 years of patient follow up.

Protection against Staphylococcus aureus bacteremia-induced mortality depends on ILC2s and eosinophils.

The dysregulated, unbalanced immune response of sepsis results in a mortality exceeding 20%, yet recent findings by our group indicate that patients with allergic, type 2-mediated immune diseases are protected from developing sepsis. We evaluated CD4+ Th cell polarization among patients with Staphylococcus aureus bacteremia and confirmed that survivors had a higher percentage of circulating Th2 cells but lower frequencies of Th17 cells and neutrophils early in the course of infection. To establish the mechanism of this protection, we used a mouse model of lethal S. aureus bacteremia and found that intratracheal pretreatment with the type 2-initiating cytokine IL-33 activated pulmonary type 2 innate lymphoid cells (ILC2s) and promoted eosinophilia. In addition, stimulation of type 2 immunity before lethal infection suppressed the pulmonary neutrophilic response to S. aureus. Mice lacking functional ILC2s did not respond to IL-33 and were not protected from lethal bacteremia, but treatment of these mice with the type 2 cytokines IL-5 and IL-13 rescued them from death. Depletion of eosinophils abrogated IL-33-mediated protection, indicating that eosinophilia is also necessary for the survival benefit. Thus, we have identified a potentially novel mechanism by which type 2 immunity can balance dysregulated septic inflammatory responses, thereby clarifying the protective benefit of type 2 immune diseases on sepsis mortality.

Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells.

The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax) induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10-8) and rs6451692 on chromosome 5 (p = 2.55 x 10-8), which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDR<0.10 for transcriptional response to 1,25D treatment, which include the transcriptional regulator ets variant 3-like (ETV3L) and EH-domain containing 4 (EHD4). In addition, we identified response eQTLs in vitamin D receptor binding sites near genes differentially expressed in response to 1,25D, such as FERM Domain Containing 6 (FRMD6), which plays a critical role in regulating both cell proliferation and apoptosis. Combining information from the GWAS of Imax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis.

Skewed Lung CCR4 to CCR6 CD4+ T Cell Ratio in Idiopathic Pulmonary Fibrosis Is Associated with Pulmonary Function.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease. While it has been suggested that T cells may contribute to IPF pathogenesis, these studies have focused primarily on T cells outside of the pulmonary interstitium. Thus, the role of T cells in the diseased lung tissue remains unclear. OBJECTIVE: To identify whether specific CD4+ T cell subsets are differentially represented in lung tissue from patients with IPF. METHODS: CD4+ T cell subsets were measured in lung tissue obtained from patients with IPF at the time of lung transplantation, and from age- and gender-matched organ donors with no known lung disease. Subsets were identified by their surface expression of CCR4, CCR6, and CXCR3 chemokine receptors. CD4+ T cell subsets were correlated with measurements of lung function obtained prior to transplantation. RESULTS: Compared to controls, IPF patients had a higher proportion of lung CD4+ T cells, a higher proportion of CCR4+ CD4+ T cells, and a lower proportion of CCR6+ CD4+ T cells. The increase in CCR4+ CD4+ T cells in IPF lung tissue was not due to increased Tregs. Intriguingly, the increase in the ratio of CCR4+ cells to CCR6+ cells correlated significantly with better lung function. CONCLUSION: Our findings suggest a new paradigm that not all T cell infiltrates in IPF lungs are detrimental, but instead, specialized subsets may actually be protective. Thus, augmentation of the chemokines that recruit protective T cells, while blocking chemokines that recruit detrimental T cells, may constitute a novel approach to IPF therapy.

IL-33-dependent induction of allergic lung inflammation by FcγRIII signaling.

Atopic asthma is a chronic inflammatory disease of the lungs generally marked by excessive Th2 inflammation. The role of allergen-specific IgG in asthma is still controversial; however, a receptor of IgG-immune complexes (IgG-ICs), FcγRIII, has been shown to promote Th2 responses through an unknown mechanism. Herein, we demonstrate that allergen-specific IgG-ICs, formed upon reexposure to allergen, promoted Th2 responses in two different models of IC-mediated inflammation that were independent of a preformed T cell memory response. Development of Th2-type airway inflammation was shown to be both FcγRIII and TLR4 dependent, and T cells were necessary and sufficient for this process to occur, even in the absence of type 2 innate lymphoid cells. We sought to identify downstream targets of FcγRIII signaling that could contribute to this process and demonstrated that bone marrow-derived DCs, alveolar macrophages, and respiratory DCs significantly upregulated IL-33 when activated through FcγRIII and TLR4. Importantly, IC-induced Th2 inflammation was dependent on the ST2/IL-33 pathway. Our results suggest that allergen-specific IgG can enhance secondary responses by ligating FcγRIII on antigen-presenting cells to augment development of Th2-mediated responses in the lungs via an IL-33-dependent mechanism.