2351 - Linker for Activation of T Cells Is Displaced From the Immunological Synapse in Lupus T Cells After T Cell Activation

Tuesday, November 8, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Nursamaa Abdoel1, Carmen Bracho2, Martin A. Rodriguez1 and Ana M. Blasini1, 1Hospital Universitario de Caracas, Caracas, Venezuela, 2Instituto Venezolano de Investigaciones Científicas, Miranda, Venezuela
Presentation Number: 2351

Background/Purpose: Several abnormalities in T cell receptor (TCR)-mediated signaling have been demonstrated in lupus T cells. LAT, a critical protein localized in lipid rafts and intracellular cytoplasmic vesicles, provides docking sites for the assembly of the calcium activation complex and other molecules needed for the coordinated activation of the MAPK signaling pathway. We have shown impaired ERK activation in lupus T cells activated via TCR/CD3 complex. Abnormal assembly of supramolecular activation complexes may impair downstream MAPK activation in lupus T cells. We have previously observed that activation via TCR/CD3 induces a significant decrease in the amount of LAT in total cell lysates from lupus T cells stimulated for 5 minutes compared with healthy control T cells (13.25 ± 2.65 vs.  21.58 ± 2.78; MFI ± SME, p= 0.038,  n= 16). This finding was confirmed using confocal microscopy, a method that also revealed delocalization of LAT and GM1 at lipid rafts in SLE T cells stimulated for 5 min versus resting T cells (0.975 ± 0.002 vs. 0.978 ± 0.001; Pearson Rr  ± SME, p= 0.008, n= 14). In order to test whether diminished LAT levels induced by activation of SLE T cells could affect the trafficking pattern in and out of the immunological synapse, we examined the behaviour of LAT and two key signaling molecules (PLCγ1 and adaptor Grb2) in artificially in vitro simulated T-cell synapses in SLE patients and healthy controls. 

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.

Keywords: T cells, signal transduction and systemic lupus erythematosus (SLE)

Disclosure: N. Abdoel, None; C. Bracho, None; M. A. Rodriguez, None; A. M. Blasini, None.