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SummaryInterstitial lung diseases (ILD) are heterogeneous conditions that may lead to progressive fibrosis and death of affected individuals. Despite diversity in clinical manifestations, enlargement of lung-associated lymph nodes (LLN) in fibrotic ILD patients predicts worse survival. Herein, we revealed a common adaptive immune landscape in LLNs of all ILD patients, characterized by highly activated germinal centers and antigen-activated T cells including regulatory T cells (Tregs). In support of these findings, we identified serum reactivity to 17 candidate auto-antigens in ILD patients through a proteome-wide screening using phage immunoprecipitation sequencing. Autoantibody responses to actin binding LIM protein 1 (ABLIM1), a protein highly expressed in aberrant basaloid cells of fibrotic lungs, were correlated with LLN frequencies of T follicular helper cells and Tregs in ILD patients. Together, we demonstrate that end-stage ILD patients have converging immune mechanisms, in part driven by antigen-specific immune responses, which may contribute to disease progression.

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., This work was supported by NIH grants R01 HL118758, R01 HL128075, R01 HL119577, R01 HL085197, U19 AI095230, UG3 OD023282 and UM1 AI114271.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

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 a strong regulatory 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 genotype at the asthma-associated 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 novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

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 a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. Supporting this notion, we show that genotype at an asthma-associated 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 novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

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., Schoettler et al. show that specific memory T cell subsets from the lung have distinct transcriptional programming and T cell receptor repertoires when compared with memory T cell subsets from the lung draining lymph nodes (LDLNs). This study suggests that memory T cell subsets from the lung and LDLN are derived from distinct progenitor pools.

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. Keywords: Idiopathic pulmonary fibrosis, T cells, ICOS, CD28, Immune checkpoint

CD43 interaction with ERM proteins regulates CD43 phosphorylation and T-cell migration. CD43 phosphorylation can also drive CD43 localization in T-cells independently of ERM association., Cell polarization is a key feature of cell motility, driving cell migration to tissues. CD43 is an abundantly expressed molecule on the T-cell surface that shows distinct localization to the migrating T-cell uropod and the distal pole complex (DPC) opposite the immunological synapse via association with the ezrin-radixin-moesin (ERM) family of actin regulatory proteins. CD43 regulates multiple T-cell functions, including T-cell activation, proliferation, apoptosis, and migration. We recently demonstrated that CD43 regulates T-cell trafficking through a phosphorylation site at Ser-76 (S76) within its cytoplasmic tail. Using a phosphorylation-specific antibody, we now find that CD43 phosphorylation at S76 is enhanced by migration signals. We further show that CD43 phosphorylation and normal T-cell trafficking depend on CD43 association with ERM proteins. Interestingly, mutation of S76 to mimic phosphorylation enhances T-cell migration and CD43 movement to the DPC while blocking ERM association, showing that CD43 movement can occur in the absence of ERM binding. We also find that protein kinase Cθ can phosphorylate CD43. These results show that while CD43 binding to ERM proteins is crucial for S76 phosphorylation, CD43 movement and regulation of T-cell migration can occur through an ERM-independent, phosphorylation–dependent mechanism.

Allergic asthma affects more than 300 million people worldwide and is characterized by airway hypersensitivity and eosinophilia. A prevalent feature of allergic asthma is increased serum IL-33 levels, and asthma GWAS have demonstrated that SNPs in the IL-33 locus are significantly associated with disease. While IL-33 and its downstream type 2 responses have been extensively studied in murine models of asthma, much less is known about the regulation of human IL33. Analysis of the mouse and human IL33 loci reveals little genomic conservation between the two species in non-coding regions. We generated a novel BAC transgenic mouse strain containing the human IL-33 locus with a fluorescent reporter to interrogate the regulation and expression of human IL-33. Surprisingly, the mice expressed human IL-33 primarily in endothelial cells, whereas murine IL-33 was expressed primarily in epithelial cells. These results mirror the expression profiles of IL-33 in primary human lung cells from the LungGENS database, thus demonstrating that our novel mouse model faithfully replicates human IL-33 expression. To understand how human IL-33 in the lung is regulated and expressed during inflammation, we examined BAC transgenic mice challenged with either house dust mite extract (HDM) or poly(I:C). In contrast to murine IL-33, expression of human IL-33 was reduced during allergic inflammation. We tested whether IL-33 is able to autoregulate itself via a negative feedback loop. Indeed, IL-33 administration downregulated the expression of human IL-33 in lung endothelial cells. Together, these data emphasize a distinct and novel role in humans for lung endothelial cells in allergic airway disease by producing and responding to IL-33.

Acute respiratory distress syndrome (ARDS) affects almost 200,000 people in the US each year with a mortality rate of 20–40%. ARDS occurs as a result of acute lung injury (ALI)-induced damage to pulmonary epithelium and/or endothelium. In a bleomycin model of ALI, we previously have shown that ICOS−/− mice, which have defective type-2 immune responses, are more susceptible to death. However, exogenous treatment of these mice with IL-5 reduces lung edema and mortality. We hypothesized that IL-5 signaling is necessary for survival from ALI and examined how mice deficient in IL-5Rα respond during ALI. IL-5Rα−/− mice had significantly increased lung edema at day 5 post-bleomycin challenge compared to WT mice (P=0.014), and had 100% mortality by day 15 (P=0.0002). IL-5Rα−/− mice had severely reduced numbers of lung eosinophils before and after challenge compared to WT mice, suggesting that eosinophils may provide protection from ALI. However, we found no difference in survival between eosinophil-deficient PHIL mice and WT mice (P=0.36). In WT mice, IL-5Rα was constitutively expressed by lung eosinophils and neutrophils. However, bleomycin challenge upregulated the receptor on myeloid cells and epithelial cells, indicating that IL-5 may regulate multiple cell populations during inflammation. Our study demonstrates that IL-5 is a critical mediator of protection against lung injury by maintaining barrier function in the lung. Surprisingly, eosinophils may not be required for the protective effect, and our data suggests that many lung cell types are capable of responding to IL-5 in the context of ALI. Thus, IL-5 may be a novel therapeutic strategy to protect against lung injury following pulmonary infection, sepsis, or trauma.

Background Although allergic sensitization can be generated against various allergens, it is unknown how such a diversity of antigens is able to promote T H 2-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 T H 2-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 T H 2 stimuli to promote T H 2-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 T H 2 stimuli, ICs and HDM, use the FcRγ-associated receptors FcγRIII and Dectin-2, respectively, to promote T H 2-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 T H 2 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 T H 2 responses in FcRγ-deficient mice. Finally, adoptive transfer of allergen-pulsed FcRγ +/− bone-marrow derived DCs restores the development of T H 2-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 T H 2 stimuli signal through FcRγ-associated receptors on DCs to upregulate IL-33 production and induce T H 2-mediated allergic airway inflammation.

Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 antiviral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il-10 and Il-33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4-deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.

IPF has a heterogeneous course of disease in which some patients experience a slow or more stable course while others RATIONALE: experience rapid decline and early mortality. Currently we lack understanding of the mechanisms that underlie the pathogenesis responsible for the differences between stable IPF (S-IPF) and rapidly progressive IPF (RP-IPF). Previous studies examining whole genome expression arrays in PBMCs of IPF patients have demonstrated a signature that is predictive of progressive disease. The predominant pathways identified were altered gene expression in T cell activity and receptors. is to link these markers of The objective of our study progressive IPF to actual protein expression on peripheral lymphocytes, thereby determining potential mechanisms involved in disease progression. METHODS: We collected patient's peripheral blood mononuclear cells (PBMC) and classified patients as S-IPF or RP-IPF based on physiologic measures taken as part of their usual clinical care. We examined mRNA levels and cell surface protein expression by flow cytometry and correlated our findings with IPF severity, using the diffusing capacity of carbon monoxide (DLCO) as the physiological measurement. RESULTS: From our previous data sets, one of the T cell receptors with the highest alteration in gene expression patterns between S-IPF and RP-IPF was Band T-lymphocyte attenuator (BTLA), a surface co-inhibitory receptor on lymphocytes. A significant association was found between decreased BTLA mRNA levels and decreased predicted DLCO levels. Analysis of all samples demonstrated a significant and dramatically decreased BTLA surface expression on both peripheral CD4 and CD8 T cells, but not B cells, from RP-IPF patients compared to controls or S-IPF patients. CONCLUSION: Our data demonstrate that BTLA protein expression and mRNA levels are potential biomarkers and may have a predictive value for determining the patients most likely to rapidly progress. Decreased BTLA protein expression on peripheral blood T cells, may either be a cause of, or a result of, disease progression. Further, our data imply that the adaptive immune system, and T cells specifically, play a key role in the progression of IPF. Peripheral blood T cell biomarkers of rapid progression developed as a result of these efforts may be used in the development of personalized diagnostics. Furthermore, understanding these pathways may identify potential therapeutic targets and explain why the variability of disease course exists.

Our previous studies revealed that, in a murine model of asthma, mice that received Fas-deficient T cells developed a prolonged phase of airway inflammation, mucus production, and airway hyperreactivity that failed to resolve even 6 weeks after the last challenge. To investigate how Fas-Fas ligand (FasL) interaction occurs between T cells and other cells in vivo, Gld mice with abnormalities of the FasL signaling pathway were used. The reconstituted mice were made by transferring T cells from B6 or Gld mice to Rag(-/-) or FasL-deficient Rag(-/-) mice. We found that Rag(-/-) mice that received B6 T cells resolved the airway inflammation, whereas FasL-deficient Rag(-/-) mice that received Gld T cells developed a prolonged airway inflammation at Day 28, with decreased IFN-gamma production. Both FasL-deficient Rag(-/-) mice that received B6 T cells and Rag(-/-) mice that received Gld T cells also had completely resolved their airway inflammation by Day 28 after challenge. Interestingly, FasL-deficient Rag(-/-) mice that received Gld T cells eventually resolved airway inflammation at Day 42, with a similar level of IFN-gamma production to that of control group. These results demonstrate that FasL expression on either T cells only or non-T cells only was sufficient for the eventual resolution of airway inflammation, and the prolonged airway inflammation in FasL-deficient Rag(-/-) mice that received Gld T cells was correlated with decreased IFN-gamma production by Gld T cells.

The mucin-like protein CD43 is excluded from the immune synapse, and regulates T-cell proliferation as well as T-cell migration. While the CD43 cytoplasmic domain is necessary for regulation of T-cell activation and proliferation, the mechanism via which CD43 regulates trafficking is not well defined. To investigate whether CD43 phosphorylation regulates its function in T cells, we used tandem mass spectrometry and identified Ser76 in murine CD43 as a previously unidentified site of basal phosphorylation. Interestingly, mutation of this single serine to alanine greatly diminishes T-cell trafficking to the lymph node, while CD43 exclusion and CD43-mediated regulation of T-cell proliferation remain intact. Furthermore, the CD43 extracellular domain was also required for T-cell trafficking, providing a hitherto unknown function for the extracellular domain, and suggesting that the extracellular domain may be required to transduce signals via the cytoplasmic domain. These data reveal a novel mechanism by which CD43 regulates T-cell function, and suggest that CD43 functions as a signaling molecule, sensing extracellular cues and transducing intracellular signals that modulate T-cell function.