Subset of memory CD4 ⁺ T cells lack recognition for Mtb-infected macrophages. (A) Schematic of experimental workflow to coculture infected macrophages with autologous memory CD4⁺ T cells for flow cytometry or sorting. Created in BioRender. Carpenter, S. (2025) https://BioRender.com/v53j172. (B and C) Flow cytometry plots from a representative experiment comparing activation marker co-expression of CD69 with CD40L (top row) or IFNγ (bottom row), (B) gated on CD45RA^Lo CD4⁺ T cells after 16–18 h coculture with Mtb-infected macrophages ± treatment with MTB300 or lysate, and (C) in the presence of α-MHC-II blocking antibodies. Data are representative of 10 (CD69 vs. CD40L) and 6 (CD69 vs. IFNγ) experiments and participants. (D and E) Summary bar graphs compare (D) median (and IQR) co-expression of CD69 and CD40L, and (E) the difference in activation when MTB300 is added to infected macrophages (10 LTBI and 7 non-LTBI participants). (F and G) Summary bar graphs compare (F) median (and IQR) CD69 and IFNγ co-expression, and (G) change in activation when MTB300 is added (6 LTBI and 6 non-LTBI participants). Each symbol represents the mean of one to three replicates from independent experiments. Statistical significance was determined by the Wilcoxon matched-pairs signed rank test.