Modulation of gamma/delta T cell activation by neutrophil elastasa

TOWSTYKA, NADIA YASMÍN; SABBIONE, FLORENCIA; KEITELMAN, IRENE; GEFFNER, JORGE R.; TREVANI, ANALÍA S.; JANCIC, CAROLINA CRISTINA

Congreso; 64 Reunión Anual de la Sociedad Argentina de Inmunología; 2016

Gamma/delta T cells play a critical role in the recruitment and activation of neutrophils at the sites of infection. In addition, neutrophils can regulate gamma/delta T cell activity. We have previously reported that elastase secreted by neutrophils enhances gamma/delta T cell activation triggered by anti-CD3 antibodies. Now, we aim to investigate the mechanism by which elastase modulates gamma/delta T cell activation. Gamma/delta T cells were purifiedfrom human peripheral blood mononuclear cells, by using an anti-TCR gamma/delta MicroBead isolation kit. Neutrophils were isolated by dextran sedimentation. After purification, gamma/delta T cells were stimulated with anti-CD3 antibodies (250 ng/ml), and cultured with or without neutrophils. After 24 h, we analyzed in gamma/delta T cells, the CD69 expression by flow cytometry; and the IFN-gamma and TNF-alpha production by ELISA. We evaluated cell conjugates formation and elastase expression by confocalmicroscopy. Finally, we analyzed the expression of the proteases activated receptor 1 (PAR1) by flow cytometry and the functionality of PAR1 by using the agonist thrombin (10 U/ml), and the antagonist RWJ56110 (10 uM). Gamma/delta T cells and neutrophils formed cell conjugates, 69±1% of these conjugates recruited elastase at the cell-to-cell contact (p<0.05, n=4). Gamma/delta T cells expressed PAR1 on cell surface (p<0.001, n=12). PAR1 was activated by thrombin treatment, measured as an increased in CD69 expression (p<0.05, n=13) and IFN-gamma (p<0.05, n=7) and TNF-alpha (p<0.05, n=7) production. Moreover, in presence of PAR1 antagonist, RWJ56110, neutrophils did not potentiate the activation of gamma/delta T cells induced by anti-CD3 antibodies, measured through the expression of CD69 (p<0.05, n=6) and the production of IFN-gamma (p<0.05, n=4) and TNF-alpha (p<0.05, n=8). Our results indicate that PAR1 mediates the effect of neutrophils elastase on gamma/delta T cells activated through CD3.