Population analysis of CD4+ T cell chemokine receptor transcript expression during in vivo typeâ 1 (mycobacterial) and typeâ 2 (schistosomal) immune responses

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141990/1/jlb0363.pd

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)

Background Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. Methods The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Results Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Conclusions Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. Trial registration This study was registered with ClinicalTrials.gov as NCT01969344

Cysteine-Cysteinyl Chemokine Receptor 6 Mediates Invariant Natural Killer T Cell Airway Recruitment and Innate Stage Resistance during Mycobacterial Infection

This study examined the contribution of cysteine-cysteinyl chemokine receptor 6 (CCR6) to the innate pulmonary antimycobacterial immune response. Using a mouse model of Mycobacterium bovis BCG airway infection, we detected maximal induction of the CCR6 agonist CCL20 in lungs at 1 week after infection. Infected CCR6 knockout (CCR6–/–) mice displayed an early impairment of bacterial clearance, but ultimately eliminated the attenuated organisms with the onset of adaptive immunity. Flow-cytometric analyses of bronchoalveolar lavages and dispersed lungs revealed a 60% reduction in TCR-α/β+ T cells in airways but no compromise of TCR-γ/δ+ T cells. The subset of CD1d-restricted, CD8-TCR-α/β+ natural killer cells, which mediate innate mycobacterial resistance, was profoundly reduced (90%). Analysis of the adaptive response using ovalbumin-specific transgenic TCR T cell (OT-II) transfer combined with infection with recombinant M. bovis BCG producing ovalbumin peptide indicated no impairment of adaptive T cell activation in CCR6–/– mice. There was also no impairment of the induction of cytokine-producing cells in draining lymphoid tissue of CCR6–/– mice. Taken together, our findings indicate that CCR6 is not required for induction of the adaptive antimycobacterial response, but is likely critical to airway compartment mobilization of TCR-α/β+CCR6+ innate and adaptive effector T cells