Pneumonia represents the leading infectious cause of death in the United States. Foxp3+ regulatory T cells promote recovery from severe pneumonia in mice, but T cell responses in patients with pneumonia remain incompletely characterized because of the limited ability to serially sample the distal airspaces and perform multidimensional molecular assessments on the small numbers of recovered cells. As T cell function is governed by their transcriptional and epigenetic landscape, we developed a method to safely perform high-resolution transcriptional and DNA methylation profiling of T cell subsets from the alveoli of critically ill patients. Our method involves nonbronchoscopic bronchoalveolar lavage combined with multiparameter fluorescence-activated cell sorting, unsupervised low-input RNA-sequencing, and a modified reduced-representation bisulfite sequencing protocol. Here, we demonstrate the safety and feasibility of our method and use it to validate functional genomic elements that were predicted by mouse models. Because of its potential for widespread application, our techniques allow unprecedented insights into the biology of human pneumonia.
James M. Walter, Kathryn A. Helmin, Hiam Abdala-Valencia, Richard G. Wunderink, Benjamin D. Singer
This file is in Adobe Acrobat (PDF) format. If you have not installed and configured the Adobe Acrobat Reader on your system.
PDFs are designed to be printed out and read, but if you prefer to read them online, you may find it easier if you increase the view size to 125%.
Many versions of the free Acrobat Reader do not allow Save. You must instead save the PDF from the JCI Online page you downloaded it from. PC users: Right-click on the Download link and choose the option that says something like "Save Link As...". Mac users should hold the mouse button down on the link to get these same options.