Synaptic interface, degranulation pattern and kinetics of degranulation by LN and blood CD4+ T cells.

Freshly isolated CD4+ T cells were exposed to planar lipid bilayers containing fluorescent labeled ICAM-1 and anti-CD3 antibodies and the structure of the T cell/bilayer interface and pattern and kinetics of degranulation were analyzed by TIRF microscopy. (A) Representative images of T cell/bilayer interface demonstrating patterns of accumulation and segregation of TCR and integrin molecules and the appearance of CD107a proteins at the interface are shown. The cells fall into 4 different groups: 1) T cells demonstrating the formation of classical cytolytic synapse containing central (cSMAC) domain, peripheral ring junction (pSMAC), and centrally located CD107a indicating granule release (solid red); 2) T cells showing the formation of cytolytic synapse without detectable granule release (solid blue); 3) T cells that are characterized by aggregation of TCR and integrin molecules without formation of mature cytolytic synapse, but with detectable granule release (red stripes); 4) T cells that display overlapping aggregates of TCR and integrin molecules without granule release (blue stripes). (B) Diagrams showing representation of LN- and PB-derived T cells of HIV-infected ART- and uninfected individuals with different structure of synaptic interfaces and patterns of granule release displayed in panel (A); HIV-specific cloned CD4+ T cells AC25 are shown for comparison reasons. (C) Time-dependent changes of the structure of T cell/bilayer interfaces and appearance of the released granules for representative T cells derived from LN and PB are shown. (D) Quantitation of the kinetics of granule release by LN (closed circles) and PB (open circles) CD4+ T cells isolated from HIV-infected ART- (red circles) and uninfected (black circles) individuals. The kinetics of granule release by HIV-specific T-cell clone AC25 is shown for comparison reasons (depicted by open blue circles). Each individual circle designates first appearance of detectable granule release by individual cells. Median and IQR are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between indicated groups of T cells; *P < 0.05, **P < 0.01.