Figure 3
LXR inhibition activates CD8+ T-cells. (A) The number of CD8+ effector T-cells. (CD3+ CD8+ Tbet+) produced in response to 10 μM SR9243 or 5 μM GW3965 or DMSO vehicle treatment for 24 h as determined by FACs. (B) Percentage of CD8 + effector T-cells (CD3+ CD8+ Tbet+) produced from differentiated CD8+ T-cells treated with SR9243, GW3965 or vehicle for 24 h. Naïve CD8+ T-cells were isolated from C57BL6J mouse splenocytes using negative selection column purification (Militenyi) and cultured under CD8+ differentiation conditions (αCD3, αCD28, IL2 and IL7) for 4 days and exposed to LXR ligands for 24 h starting on day 3. (C–E) Cytometric bead assay showing CD8+ T-cell production of (C) IFNγ (D) IL6 and (E) IL2 in response to 10 μM SR9243 or 5 μM GW3965 for 24 h. Differentiated CD8+ T-cells were re-stimulated using 50 ng/mL/1 µg/mL PMA/Ionomycin for 4 h prior to cytokine quantification. (F) Average percentage of IFNγ expressing CD8+ effector T-cells (CD3+ CD8+ Tbet+) produced from differentiated CD8+ T-cells exposed to vehicle, 10 μM SR9243 or 5 μM GW3965 in the presence or absence of TCM. Cells were treated with LXR ligands for 24 h prior to FACs analysis (G) FACs histogram showing PD-1 expression in CD8+ effector T-cells treated with vehicle, SR9243 or GW3965 in the presence of 50% E0771 TCM or relevant control media (RPMI1640) for 24 h (H) Average PD-1 expression in CD8+ effector T-cells treated with vehicle, SR9243 or GW3965 for 24 h as determined by FACs analysis. (I) Carboxyfluorescein succimidyl (CFSE) proliferation assay of differentiating CD8+ T-cells treated with SR9243 or DMSO. Column isolated CD8 T cells were labeled with 1 µM CellTraceTM CFSE (ThermoFisher) for 20 minutes at room temperature, then subjected to CD8+ T-cell differentiation conditions for 96 h, as described, in the presence of vehicle or 10 µM SR9243. Expansion, proliferation and replication indices were then determined via FACs analysis of CFSE dilution profiles using Flow-Jo. (J) Cell killing/CD8+ T-cell cytotoxicity assay showing CD8+ T-cell destruction of cultured E0771 cells in response to SR9243, with or without TCM co-treatment. CD8+ T-cells were differentiated for 4 days then exposed to 10 μM SR9243 along with 50% E0771 TCM or RPMI1640 for 24 h. CD8+ cells were exposed to CD3/CD28 activation in the presence of target cells; E0771 cells were pre-stained with 1 µM CFSE and 4 µg/mL 7-Aminoactinomycin-D (7-AAD). The percentage of E0771 cells (CFSE + 7-AAD+) were then quantified via FACs. (K) Mean percentage of IFNγ + GZMB+ human CD8+ T-cells (CD3+ CD8+ Tbet+) produced in response to SR9243, with or without PD-L1 immune-checkpoint suppression. CD8+ T-cells were isolated from PMBCs from healthy female volunteers via negative selection using the RosetteSep™ Human CD8+ T Cell Enrichment Cocktail. Cells were then exposed to 5 μg/mL purified human PD-L1 protein or vehicle (PBS) in the presence of 100 nM SR9243 for 24 h. *p < 0.05 as determined by 1-Way ANOVA.
