In activated T cells, signalling molecules
such as Syk associate with the MRs. The lateral diffusion of MRs by decreasing receptor proximity allows protein interactions, initiating cell signalling . A similar role of CD28 co-stimulatory molecule has been suggested for MRs during T cell activation . In this study, we show that in human CD4+ T cells, ICs and late complement pathway plays a role in the activation of Syk via recruitment of FcRγ chain with the membrane FcγRIIIA. Blood from normal and SLE patients was collected with informed consent in the Saint Louis University Rheumatology clinics. The normal group consisted of female volunteers in the 24–35-year age group. The SLE patients were in the 18–45-year age group, with disease duration ranging from 3 to 10 years. The patients fulfilled the 1982 revised criteria for diagnosis of Pexidartinib price SLE . The blood was collected
in heparinized tubes and cells were isolated within 4 h of sample collection. Affinity-purified antibodies against FcγRIIIB/CD16, FcγRI/CD64 and a monoclonal recognizing FcγRIIIA/B were purchased from R&D Systems (Minneapolis, MN, USA). Anti-FcRγ antibody was from Upstate Cell Signaling Solutions (Beverley, MA, USA) and anti-pSyk was from Cell Signaling Technology. Cholera toxin-B (CTB)–fluorescein isothiocyanate (FITC) was purchased from Sigma Chemicals (St Louis, MO, USA). Other common reagents and cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA) and Sigma Chemicals. The reagents to purify the human naive CD4+ T cells were procured from Miltenyi
selleck chemicals llc Biotec (Bergisch Gladbach, Germany). Anti-CD3 and anti-CD 28 antibodies were purchased from eBiosciences (San Diego, CA, USA). Human CD4+ cells were purified from peripheral blood mononuclear cells (PBMC) isolated from normal or SLE patients using Histopaque CYTH4 gradient. The monocytes were removed by plating the cells for 6 h in Nunc culture dishes; thereafter, CD4+ cells were purified by positive selection using magnetic beads and human naive CD4+ T cells by negative selection, using magnetic bead cell isolation kits (Miltenyi Biotec). The purified CD4+ T cells were maintained in interleukin (IL)-2 (20 ng/ml) supplemented complete RPMI-1640 medium. The purity of these cells was analysed by staining for CD4+, CD3+ and CD45RA+. The purified cells were 94–96% positive for these three markers. The cell viability was more than 97%, as indicated by staining with vital dye trypan blue. These purified cells were expanded using plates coated with 0·5 µg/ml of anti-CD3 and 0·5 µg/ml of soluble anti-CD28. Thereafter, cells were maintained in culture for 10 days after stimulation in the presence of IL-2 (20 IU/ml); such cells are referred as ‘expanded cells’. AHG was prepared as described previously . One mg of the AHG protein was labelled with AlexaFluor® 488 using the protein labelling kit, as per the manufacturer’s protocol (Invitrogen).