CD69 and CD25 co-expression reveals a subset of memory CD4 ⁺ T cells that lack recognition of Mtb-infected macrophages. Related to Figs. 1 and 2. (A and B) (A) Flow cytometry plots comparing the co-expression of CD69 and CD25, gated on CD45RA^Lo CD4⁺ T cells after 16–18 h coculture with Mtb-infected (MOI 4–5) macrophages either alone or with addition of exogenous antigens (MTB300 or lysate) and (B) with and without α-MHC-II blocking antibodies. Plots are concatenated from three replicates from one experiment; data are representative of 10 independent experiments each with two to three replicates per condition. (C and D) (C) Summary bar graphs compare mean (± SEM) co-expression of CD69 and CD25 (10 participants), and (D) the difference in activation when MTB300 is added to Mtb-infected macrophages for samples from 10 LTBI and 6 non-LTBI participants. (E) Representative flow plots showing the gating of memory CD4⁺ T cells activated in response to Mtb-infected macrophages (or controls). After gating on lymphocytes (SSC Area [SSC-A] vs. FSC Area [FSC-A]) and single cells (FSC Height (FSC-H] vs. FSC-A), live CD4⁺ 7-AAD^Lo T cells were identified. Co-expression of CD69 and either CD25 or CD40L was used to identify activated T cells for sorting.