CXCR3 signaling is required for restricted homing of parenteral TB vaccine-induced T cells to both the lung parenchyma and airway

While most novel tuberculosis (TB) vaccines are designed for delivery via the muscle or skin for enhanced protection in the lung, it has remained poorly understood whether systemic vaccine-induced memory T cells can readily home to the lung mucosa prior to and shortly after pathogen exposure. We have investigated this issue by using a model of parenteral TB immunization and intravascular immunostaining. We find that systemically induced memory T cells are restricted to the blood vessels in the lung, unable to populate either the lung parenchymal tissue or the airway under homeostatic conditions. We further find that after pulmonary TB infection, it still takes many days before such T cells can enter the lung parenchymal tissue and airway. We have identified the acquisition of CXCR3 expression by circulating T cells to be critical for their entry to these lung mucosal compartments. Our findings offer new insights into mucosal T cell biology and have important implications in vaccine strategies against pulmonary TB and other intracellular infections in the lung. To identify the potential molecular determinants of lung mucosal homing of systemically induced T cells, difference in transcriptional signature of parenteral TB immunization-induced Ag-specific CD8 T cells collected when they were largely in lung vasculature and after they homed to lung mucosal tissue following pulmonary Mycobacterium tuberculosis infection were analyzed by using Affymetrix Mouse Gene 2.0ST microarray. We elected to focus on days 0, 7 and 21 timepoints post-infection to compare the T cells located in lung vasculature (d0 & d7- largely resting memory T cells) with those in lung mucosal tissue (d21-largely secondary effector T cells. RNA from purified CD8 T cells was isolated using RNeasy® Mini Kit (Qiagen, Germantown, MD, USA). Quality of RNA and subsequent microarray was carried out by Center for Applied Genomics of The Hospital for Sick Children, Toronto, Ontario, Canada. Quality of RNA samples were analyzed using the Agilent 2100 bioanalyzer, which uses RNA 6000 Nano LabChip platform (Agilent Technologies Canada Inc., Mississauga, ON, Canada). On average RNA integrity number (RIN) for all the samples included in the study were above 7. RNA samples were then subjected to RNA Microarray expression analysis using Mouse Gene 2.0ST Affymetrix array. This array contains protein-coding regions. Triplicate sets of lung cell-derived RNA samples per timepoint was generated (8 mice/set; 24 mice/timepoint)