TCR Sequencing of M. tuberculosis-Specific CD8+ T Cells

Single-cell TCR sequencing was performed as described previously (36 (link), 37 (link)). Briefly, cryopreserved PBMCs from 12 healthy adolescents with evidence of M. tuberculosis infection (determined by QuantiFERON-Plus and/or TST positive test results) were thawed, rested for 6 h, and stimulated for 12 h with M. tuberculosis lysate (10 μg/ml; BEI Resources) in the presence of anti-CD49d Ab (1 μg/ml), and anti-CD154-PE (10 μl/ml). Cells were stained with LIVE/DEAD Fixable Aqua Stain (Thermo Fisher Scientific) and then with Abs (Supplemental Table I). Single, activated (i.e., CD69⁺CD137⁺ and/or CD69⁺CD154⁺) TCRαβ⁺ CD8⁺ cells were sorted by FACS (BD FACSAria II) into 96-well plates containing OneStep RT-PCR buffer (QIAGEN). TCRαβ sequences were amplified using a panel of TCRαβ primers and further amplified in a nested PCR before sequencing on a MiSeq (Illumina) instrument, as described previously (36 (link), 37 (link)). TCR sequences from six of these 12 adolescents were published recently (38 (link)), as indicated in Supplemental Table II.