GL7 ligand expression defines a novel subset of CD4+ TRM cells in lungs recovered from pneumococcus.

Streptococcus pneumoniae is the most common etiology of bacterial pneumonia, one of the leading causes of death in children and the elderly worldwide. During non-lethal infections with S. pneumoniae, lymphocytes accumulate in the lungs and protect against reinfection with serotype-mismatched strains. Cluster of differentiation CD4+ resident memory T (TRM) cells are known to be crucial for this protection, but the diversity of lung CD4+ TRM cells has yet to be fully delineated. We aimed to identify unique subsets and their contributions to lung immunity. After recovery from pneumococcal infections, we identified a distinct subset of CD4+ T cells defined by the phenotype CD11ahiCD69+GL7+ in mouse lungs. Phenotypic analyses for markers of lymphocyte memory and residence demonstrated that GL7+ T cells are a subset of CD4+ TRM cells. Functional studies revealed that unlike GL7- TRM subsets that were mostly (RAR-related Orphan Receptor gamma T) RORγT+, GL7+ TRM cells exhibited higher levels of (T-box expressed in T cells) T-bet and Gata-3, corresponding with increased synthesis of interferon-γ, interleukin-13, and interleukin-5, inherent to both T helper 1 (TH1) and TH2 functions. Thus, we propose that these cells provide novel contributions during pneumococcal pneumonia, serving as important determinants of lung immunity.

Recruitment and training of alveolar macrophages after pneumococcal pneumonia.

Recovery from pneumococcal pneumonia remodels the pool of alveolar macrophages so that they exhibit new surface marker profiles, transcriptomes, metabolomes, and responses to infection. Mechanisms mediating alveolar macrophage phenotypes after pneumococcal pneumonia have not been delineated. IFN-γ and its receptor on alveolar macrophages were essential for certain, but not all, aspects of the remodeled alveolar macrophage phenotype. IFN-γ was produced by CD4+ T cells plus other cells, and CD4+ cell depletion did not prevent alveolar macrophage remodeling. In mice infected or recovering from pneumococcus, monocytes were recruited to the lungs, and the monocyte-derived macrophages developed characteristics of alveolar macrophages. CCR2 mediated the early monocyte recruitment but was not essential to the development of the remodeled alveolar macrophage phenotype. Lineage tracing demonstrated that recovery from pneumococcal pneumonias converted the pool of alveolar macrophages from being primarily of embryonic origin to being primarily of adult hematopoietic stem cell origin. Alveolar macrophages of either origin demonstrated similar remodeled phenotypes, suggesting that ontogeny did not dictate phenotype. Our data reveal that the remodeled alveolar macrophage phenotype in lungs recovered from pneumococcal pneumonia results from a combination of new recruitment plus training of both the original cells and the new recruits.

CPHEN-011: Comprehensive phenotyping of murine lung resident lymphocytes after recovery from pneumococcal pneumonia.

Recovery from pneumococcal (Spn) pneumonia induces development of tissue resident memory CD4+ TRM cells, BRM cells, and antibody secreting plasma cells in experienced lungs. These tissue resident lymphocytes confer protection against subsequent lethal challenge by serotype mismatched Spn (termed as heterotypic immunity). While traditional flow cytometry and gating strategies support premeditated identification of cells using a limited set of markers, discovery of novel tissue resident lymphocytes necessitates stable platforms that can handle larger sets of phenotypic markers and lends itself to unbiased clustering approaches. In this report, we leverage the power of full spectrum flow cytometry (FSFC) to develop a comprehensive panel of phenotypic markers that allows identification of multiple subsets of tissue resident lymphocytes in Spn-experienced murine lungs. Using Phenograph algorithm on this multidimensional data, we identify unforeseen heterogeneity in lung resident adaptive immune landscape which includes unexpected subsets of TRM and BRM cells. Further, using conventional gating strategy informed by our unsupervised clustering data, we confirm their presence exquisitely in Spn-experienced lungs as potentially relevant to heterotypic immunity and define CD73 as a highly expressed marker on TRM cells. Thus, our study emphasizes the utility of FSFC for confirmatory and discovery studies relating to tissue resident adaptive immunity.

Antigen presentation by lung epithelial cells directs CD4+ TRM cell function and regulates barrier immunity.

Barrier tissues are populated by functionally plastic CD4+ resident memory T (TRM) cells. Whether the barrier epithelium regulates CD4+ TRM cell locations, plasticity and activities remains unclear. Here we report that lung epithelial cells, including distinct surfactant protein C (SPC)lowMHChigh epithelial cells, function as anatomically-segregated and temporally-dynamic antigen presenting cells. In vivo ablation of lung epithelial MHC-II results in altered localization of CD4+ TRM cells. Recurrent encounters with cognate antigen in the absence of epithelial MHC-II leads CD4+ TRM cells to co-express several classically antagonistic lineage-defining transcription factors, changes their cytokine profiles, and results in dysregulated barrier immunity. In addition, lung epithelial MHC-II is needed for surface expression of PD-L1, which engages its ligand PD-1 to constrain lung CD4+ TRM cell phenotypes. Thus, we establish epithelial antigen presentation as a critical regulator of CD4+ TRM cell function and identify epithelial-CD4+ TRM cell immune interactions as core elements of barrier immunity.

Lung-resident memory B cells protect against bacterial pneumonia.

Lung-resident memory B cells (BRM cells) are elicited after influenza infections of mice, but connections to other pathogens and hosts - as well as their functional significance - have yet to be determined. We postulate that BRM cells are core components of lung immunity. To test this, we examined whether lung BRM cells are elicited by the respiratory pathogen pneumococcus, are present in humans, and are important in pneumonia defense. Lungs of mice that had recovered from pneumococcal infections did not contain organized tertiary lymphoid organs, but did have plasma cells and noncirculating memory B cells. The latter expressed distinctive surface markers (including CD69, PD-L2, CD80, and CD73) and were poised to secrete antibodies upon stimulation. Human lungs also contained B cells with a resident memory phenotype. In mice recovered from pneumococcal pneumonia, depletion of PD-L2+ B cells, including lung BRM cells, diminished bacterial clearance and the level of pneumococcus-reactive antibodies in the lung. These data define lung BRM cells as a common feature of pathogen-experienced lungs and provide direct evidence of a role for these cells in pulmonary antibacterial immunity.

Pneumonia recovery reprograms the alveolar macrophage pool.

Community-acquired pneumonia is a widespread disease with significant morbidity and mortality. Alveolar macrophages are tissue-resident lung cells that play a crucial role in innate immunity against bacteria that cause pneumonia. We hypothesized that alveolar macrophages display adaptive characteristics after resolution of bacterial pneumonia. We studied mice 1 to 6 months after self-limiting lung infections with Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Alveolar macrophages, but not other myeloid cells, recovered from the lung showed long-term modifications of their surface marker phenotype. The remodeling of alveolar macrophages was (a) long-lasting (still observed 6 months after infection), (b) regionally localized (observed only in the affected lobe after lobar pneumonia), and (c) associated with macrophage-dependent enhanced protection against another pneumococcal serotype. Metabolomic and transcriptomic profiling revealed that alveolar macrophages of mice that recovered from pneumonia had new baseline activities and altered responses to infection that better resembled those of adult humans. The enhanced lung protection after mild and self-limiting bacterial respiratory infections includes a profound remodeling of the alveolar macrophage pool that is long-lasting; compartmentalized; and manifest across surface receptors, metabolites, and both resting and stimulated transcriptomes.

Lack of Ikaros Deregulates Inflammatory Gene Programs in T Cells.

CD4 Th cells are organizers of the immune response, directing other immune cells to initiate and maintain effective humoral and cellular immunity. CD4 T cells differentiate into distinct Th effector or regulatory subsets in response to signals delivered to them during the course of infection. Ikaros is a transcription factor that is expressed in blood cells from the level of the hematopoietic stem cell. It is required for normal thymic T cell development and serves as a tumor suppressor, as lack of Ikaros in developing lymphoid cells results in leukemia. To study the role of Ikaros in CD4 T cell differentiation and function, an Ikaros conditional knockout mouse was developed such that Ikaros expression was deleted specifically in mature T cells, thus avoiding defects observed in germline Ikaros mutant mice. Using this model system, we have shown that in the absence of Ikaros, CD4 T cells are able to attain Th1, Th2, and Th17, but not inducible regulatory T, cell fates. However, they show enhanced expression of a cohort of proinflammatory cytokines, resulting in differentiation of Th17 cells with a phenotype that has been associated with autoimmunity and pathological inflammation. In addition, we define Ikaros as a repressor of the gene program associated with the response to type I IFNs, another key pathway whose deregulation is linked to autoimmunity. Taken together, these data definitively define Ikaros as a critical regulator at the center of the inflammatory response in T cells and highlight a potential role in suppressing autoimmunity.