Session Type: Abstract Submissions (ACR)
We previously showed an increased metabolic rate of transmembrane adaptor protein LAT in TCR/CD3 stimulated lupus T cells, associated with delocalization of this adaptor molecule from lipid rafts and from the immunological synapse after TCR/CD3-CD28 stimulation of lupus T cells. Additionally, unstimulated T cells from systemic lupus erythematosus (SLE) patients seem to be in a preactivated state showing augmented phosphorylation of signaling proteins, among other evidences, compared to non-SLE patients and healthy controls. In this study we evaluated the presence of key signaling molecules in lipid rafts of lupus T cells.
SLE patients were diagnosed according to the American College of Rheumatology criteria. Healthy donors were from the blood bank of Hospital Universitario de Caracas. All individuals signed an informed consent previously approved by the Bioethics Committee. Highly enriched T cells, obtained from peripheral blood samples and subjected to RossetteSepTM isolation, were adhered to pLL coated slides and activated for 5 and 15 min at 37°C, with 4.5 μm superparamagnetic polystyrene beads coated with antibodies against CD3ε and CD28. The cells were fixed, permeabilized and stained with antibodies recognizing Grb2 and PLCγ1, and CT-B as a lipid raft marker. The cell-bead complexes were evaluated by confocal microscopy and densitometries were obtained using ImageJ, 1.44, National Institutes of Health, USA.
Results: We observed increased localization of adaptor protein Grb2 at the immunological synapse in unstimulated T cells from lupus patients compared to healthy controls (23.46 ± 3.57 vs. 20.67 ± 2.28, MFI ± SME, n=10). PLCγ1 showed the same pattern of increased localization at the immunological synapse in these cells (23.14 ± 4.10 vs. 17.22 ± 2.14, MFI ± SME, n=10). The colocalization of either Grb2 or PLCγ1 with GM1, as well as colocalization of Grb2 and PLCγ1 themselves was similar between SLE patients and healthy controls, suggesting normal coupling of signaling molecules in lipid rafts in lymphocytes from lupus patients.
Since partition of PLCγ1 and Grb2 in lipid rafts occurs after ligation of the TCR, we conclude that our findings suggest an in vivo dysregulated activation state in SLE T cells, which may contribute to known downstream signaling abnormalities such as MAPK activation.
A. M. Blasini,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/localization-at-the-immunological-synapse-of-adaptor-protein-grb2-and-plc-gamma-1-in-non-stimulated-peripheral-blood-t-lymphocytes-from-patients-with-systemic-lupus-erythematosus-suggests-an-in-vivo/