Detection and characterization of surface phenotype and TCR repertoire of βGL1-22– and LGL1-reactive T cells. (A) The chemical structure of glucosylceramide (top panel) and glucosylsphingosine (bottom panel) used in this study. (B) Panel showing staining of human PBMCs with CD1d tetramers loaded with α-GalCer, βGL1-22, or LGL1. Invariant TCR usage was monitored using anti-Vα24 and anti-Vβ11 antibodies. Numbers in the contour plots show percentages of cells in CD3⁺ tetrameter-negative (Tet⁻) and CD3⁺ tetrameter-positive (Tet⁺) gates, respectively. (C) Plot summarizing percentages of lymphocytes staining in freshly isolated PBMCs from healthy donors for CD1d tetramer loaded with βGL1-22, LGL1, or α-GalCer. Data are presented as mean ± standard error of the mean (SEM), with each dot indicating 1 donor (n = 20). (D) Compiled results from analyses of 5 different human PBMCs showing the mean percentages of CD4⁺, CD8⁺, and CD4⁻CD8⁻ (DN) expressing βGL1-22, LGL1, or α-GalCer tetramer-positive T cells. Error is presented as SEM among donors (***P < .0001; **P < .001). (E) Compiled results from analyses of 5 different human PBMCs showing mean percentages of CD45RA, CD45RO, CD62L, CD69, CD56, and CD161 expression by human βGL1-22, LGL1, or α-GalCer CD1d tetramer-positive T cells. Error is presented as SEM among donors (***P < .0001). (F) Intracellular flow cytometry with anti-PLZF monoclonal antibody (mAb) of α-GalCer tetramer-positive (gray shaded), βGL1-22 tetramer-positive (dashed line), or LGL1 tetramer-positive (solid line) and conventional T cells CD3⁺Tet⁻ (black shaded) from freshly isolated human PBMCs as indicated. The isotype control is the dotted line histogram. Data are representative of 4 experiments. (G) TCR sequencing analysis of in vitro expanded and sorted CD1d-α-GalCer, βGL1-22, or LGL1 tetramer-positive T cells. Pie charts representing the TCRVβ repertoire usage by α-GalCer–, βGL122–, and LGL1-specific T cells. Data are representative of 3 separate experiments.