Autogramin-2, a synthetic sterol, inhibits T cell adhesion and effector function

Abstract

Plasma membrane resident cholesterol is a key regulator of T cell immunity as it stabilises immune synapses, thus supporting effector T cell function. Recent publications suggest that increasing plasma membrane cholesterol content boosts anti-viral and anti-cancer immunity in T cells. Inhibition of cholesterol transport from the plasma membrane to the endoplasmic reticulum in adoptively transferred T cells therefore constitutes a promising therapeutical avenue for improving cancer therapy. I hypothesised that inhibition of the cholesterol transport protein Aster A using the synthetic sterol autogramin-2 enhances T cell effector function by increasing plasma membrane cholesterol content. Treatment of effector T cells with autogramin-2 inhibited, rather than boosted, effector T cell function: proliferation and activation induced increase of activation marker CD69 were impaired. Production of pro-inflammatory cytokines (TNF, IFN-), degranulation, and cytotoxic capacity, as measured by a co-culture killing assay with Raji cells and bi-specific antibody blinatumomab, were decreased upon treatment. This was accompanied by a reduction in cell-to-cell contact. Furthermore, LFA-1/integrin dependent adhesion to ICAM-1 and extracellular matrix components was inhibited within 30min of treatment with 3.75M autogramin-2 in an Aster A independent manner. Autogramin-2 did not significantly inhibit LFA-1/TCR downstream signalling in PHA blasts, nor affect activation induced conformational change of LFA-1. Instead, plasma membrane architecture was altered upon treatment: membrane asymmetry decreased independently of phosphatidyl-serine, and membrane fluidity increased in a sphingomyelin-complexed cholesterol independent manner. Plasma membrane lipid raft enriched fractions isolated from autogramin-2 treated samples showed a near significant reduction in LFA-1 -chain (ITGAL) staining (p=0.0535) compared to vehicle control (ctrl.) treated fractions. Concomitantly, relative abundance of lipolysis substrates triacyl-glyceride and diacyl-glyceride decreased significantly in Jurkat T cells treated with autogramin-2, accompanied by a trend towards decrease in monoacyl-glycerides (p=0.0711). Furthermore, relative abundance of acyl-carnitines increased significantly, with a similar trend observed for free fatty acids (p=0.0511). Based on the data presented here, I propose the following working model: autogramin-2 inhibits T cell integrins, including LFA-1, by rapidly stimulating lipolysis. Fatty acids released during lipolysis react with carnitine to form acyl-carnitine. Both acyl-carnitines and free fatty acids cause plasma membrane remodelling resulting in the expulsion of LFA-1 from stabilising lipid rafts, thus inhibiting T cell adhesion and effector function. Further investigation is needed to refine this model and elucidate the underlying mechanism.