Antigen specificity, gene expression, and cytokine profile of βGL1-22– and LGL1-specific type II NKT cells. (A) IFN-γ production by intracellular staining of freshly isolated PBMCs cultured with α-GalCer (1 μg/mL) or βGL1-22 and LGL1 (20 µg/mL) for 12 hours is shown. Live and dead staining was performed using the live/dead fixable dead cell stain kit. IFN-γ expression is represented in terms of mean fluorescence intensity (MFI) following stimulation in tetramer-positive cells vs tetramer-negative cells as a control. (B) Bar graphs depict the IFN-γ production by CD3⁺ α-GalCer, βGL1-22, or LGL1 tetramer-positive T cells in human PBMCs stimulated with α-GalCer (1 μg/mL) or βGL1-22 and LGL1 (20 µg/mL) in the presence of either CD1d blocking (CD1d42) or isotype control mAb. Data are representative of 3 experiments. (C) Bar graphs showing IFN-γ production by sorted βGL1-22, LGL1, or α-GalCer tetramer-positive T-cell cultures in response to CD1d-transfected (C1rD-CD1d) or untransfected (C1rD-Mock) APCs with indicated antigens. Vehicle-pulsed CD1d-transfected APCs served as control. IFN-γ secretion from the culture supernatants collected after 48 hours was monitored by Luminex. Data are representative of 3 experiments (* indicates saturating IFN-γ levels). (D) Representative fluorescence-activated cell sorter (FACS) analysis showing expansion of βGL1-22, LGL1, or α-GalCer tetramer-positive T-cell cultures in response to monocyte-derived DCs pulsed with either vehicle or βGL1-22, LGL1, and α-GalCer. Expansion of iTCR was monitored using anti-Vα24 and anti-Vβ11 antibodies. Numbers in the contour plots show percentages of cells in CD3⁺ tetramer-positive and CD3⁺ tetramer-negative gates, respectively. (E) Principal component (PC) analysis of global gene expression profiles of sorted βGL1-22/LGL1–specific type II NKT and α-GalCer–specific type I NKT cells, either before or after anti-CD3/28 stimulation. Each data point/shape represents a microarray, with all 6 arrays presented on PC analysis plot either before or after stimulation. The α-GalCer array shows some separation from βGL1-22/LGL1 arrays in both stimulated and unstimulated conditions. Given PC1 describes the largest amount of data variance (40.0%), the aggregation of the arrays is mostly accounted for by the sample location. The percentage values in parentheses indicate the proportion of the total variance described in each PC: PC1 (x-axis), PC2 (y-axis), and PC3 (z-axis). (F) Differentially expressed genes in sorted βGL1-22/LGL1–specific type II NKT compared with α-GalCer–specific type I NKT. Bar graphs showing fold change in expression levels of selected genes related to transcription, differentiation, and cytokine and chemokine signaling compared with type I NKT cells. (G-H) Cytokine expression by flow-sorted CD1d-βGL1-22⁺, CD1d-LGL1⁺, or α-GalCer⁺ T-cell populations in response to stimulation (G) stimulation with anti-CD3/CD28 beads and (H) stimulation with CD1d or mock-transfected lipid-loaded APCs. Culture supernatants collected after 48 hours were analyzed for 39 different analytes by Luminex. Analytes that showed significant levels when compared with unstimulated control are shown. Data are shown as mean (±SEM) of 3 similar experiments. DMSO, dimethylsulfoxide; MDC; macrophage-derived chemokine; MIP-1β, macrophage inflammatory protein 1β.