Extended Data Fig. 4: Gating strategy for T_H panel and naïve-like Mtb lysate-specific cell frequency in the validation cohort.

(a) Flow cytometry gating strategy for cells expressing memory markers, transcriptional factors and intracellular cytokines associated with T_H1 and T_H17 among the validation household contact cohort in response to stimulation with ESAT6/CFP10. A time gate was applied to exclude events affected by sample acquisition aberrations, followed by T cell identification by lymphocyte size, CD3, CD14, and CD19 markers. A singlet gate was then applied, followed by the identification of viable cells and a second lymphocyte gate. A secondary gate was applied to identify IL-17A⁺ cells due to spillover spreading. The same gating strategy was applied to samples stimulated with whole Mtb lysate (not shown). (b) Flow cytometry showing the median and interquartile range of frequencies of CD45RA⁺CCR7⁺ naïve-like cells in Mtb lysate-specific CD4⁺ T cells among validation household contact cohort RSTR (n = 17) and LTBI (n = 20). Whiskers represent minima and maxima. Statistical significance was determined by the two-sided Wilcoxon rank-sum test.