The immunophenotyping of MAIT, iNKT, and γδ T cells was performed as previously described
8 (link) on cryopreserved PBMCs from P1 prepared from a sample collected at the age of three years; and as previously described
10 (link) on cryopreserved PBMCs from P2 prepared from a sample collected at the age of six years. Both patients were receiving broad-spectrum antimycobacterial drugs. Briefly, staining was performed in the presence of
Fcblock (Miltenyi Biotec), with
Zombie-NIR live-dead exclusion dye (#423105, BioLegend), anti-CD3-Alexa532 (Clone UCHT1, # 58-0038-42, Thermo Fisher Scientific),
anti-γδTCR-FITC (#11-9959-42, Thermo Fisher Scientific),
anti-Vδ2-APC-Fire750 (#331420, BioLegend), anti-CD56-BV605 (clone 5.1H11, #362538, BioLegend),
anti-CD4-BV750 (#5663656, BD Biosciences),
anti-CD8a-BV510 (clone RPA-T8, #301047, BioLegend), anti-Vα7.2-BV711 (clone 3C10, #351731, BioLegend), anti-Vα24-Jα18-PE-Cy7 (clone 6B11, #342912, BioLegend),
anti-Vδ1-Vioblue (#30-100-555, Miltenyi Biotec), anti-CD161-PE (clone HP-3G10, #339938, BioLegend) and
anti-Vβ11-APC (Miltenyi Biotec) antibodies. Cells were analyzed with an
Aurora cytometer (Cytek). The gating strategy for MAIT cells, iNKT cells, γδ1
+ T cells, and γδ2
+ T cells has been described elsewhere
8 (link),69 (link).
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