To minimize background staining and ensure that we were only detecting MTB-specific CD4+ T cells, a dump gate to exclude dead cells, CD14+ monocytes, and CD19+ B cells was used. Singlet cells were then gated on CD3+ CD4+ cells and analyzed for CCR7 and CD45RA expression to determine T cell subsets. CD4+ T cell subsets were defined as naïve CCR7+ CD45RA+, central memory CCR7+ CD45RA−, effector memory CCR7− CD45RA−, and effector as CCR7− CD45RA+.