Method: Highly enriched T cells were adhered to pLL coated slides and activated for 5 and 15 min at 37°C with 4.5 mm superparamagnetic polystyrene beads coated with antibodies against CD3ε and CD28. Cells were fixed, permeabilized and stained with antibodies recognizing LAT, Grb2 and PLCγ1. The cell-bead complexes were evaluated by confocal microscopy and densitometries were obtained using ImageJ, 1.44, National Institutes of Health, USA.
Result: Analysis revealed that activation via CD3/CD28 during 15 minutes induced a significant decrease in the amount of LAT at the synapse in lupus T cells in comparisson with resting T cells (24.95 ± 4.48 vs. 31.51 ± 8.68; MFI ± SME, p=0.020, n= 8), a difference not found in healthy control cells. LAT levels out of the synapse were also diminished after activation of lupus T cells (18.39 ± 4.05 vs. 19.25 ± 6.28 ; MFI ± SME, p=0.022, n=8). PLCγ1 and Grb2 recruited to the synapse remained unchanged upon activation in both SLE and healthy T cells.
Conclusion: We conclude that activation via CD3/CD28 negatively regulates LAT expression both in and out of the synapse in SLE T cells. The mechanistic cues for the downregulation of LAT in response to T cell activation in lupus T cells are currently unclear. The diminished expression of LAT after TCR-CD3 activation may potentially disrupt downstream signaling events and impair activation of the MAPK cascade in human lupus T cells. Supported by FONACIT grants No.S1-200000440 and the program “Fortalecimiento al Postgrado de Desarrollo de Alto Nivel” No. 1220/OC-VE.
Disclosure: N. Abdoel, None; C. Bracho, None; M. A. Rodriguez, None; A. M. Blasini, None.