Effects of phosphatidylinositol 3-kinase specific inhibitor LY294002 on activation of γδΤcells by low molecular weight peptide antigen of mycobacterium tuberculosis

Objective To observe the effects of phosphatidylinositol 3-kinase (PI3K) specific inhibitor LY294002 on CD3 monoclonal antibody (CD3 mAb) activated T cells and γδΤ cells activated by low molecular peptide antigen of mycobacterium tuberculosis (Mtb-Ag) , and to investigate the role of PI3K in TCR signal transduction pathway in CD3 mAb activated T cells and Mtb-Ag activated γδΤ cells. Methods Human peripheral blood mononuclear cells (PBMC) were isolated from healthy subjects, and stimulated with CD3 mAb, phorbol myristate acetate+ ionomycin (PMA+ IM) , and Mtb-Ag, respectively. Flow cytometry was used to measure expression of CD69 molecules in the CD3+ T cells and γδΤ cells at 0, 6, 12, 24, 48 h and 72 h of stimulation. The stimulation culture was repeated after treatment of PBMC with various levels of LY294002 (0, 0.4, 2, and 10 μmol/L). EIA kit was used to measure the interleukin (IL)-2 levels in CD3 T cells in the CD3 mAb and PMA + IM stimulation groups. The effects of LY294002 on proliferation CD3 mAb activated T cells and Mtb-Ag activated γδΤ cells were determined after CD3PE/CD69FITC and γδPE/CD69FITC double staining. The number of these cells was calculated after 10 days of cultivation. Results The CD69 expression peaked in CD3+ T cells and γδΤ cells stimulated with CD3 mAb for 24 h (both around 56%) , in those stimulated with PMA+IM for 6 h (both around 99%) , and in those stimulated with Mtb-Ag stimulation for 24 h [(16.0 ± 0.0)% in CD3+ T cells vs (75.2 ± 0.7)% in γδΤ cells) ]. At any time point, the CD69 expression level in CD3+ T cells was statistically significant among groups with different stimulating agents (P 0.05). After treatment with 0, 0.4, 2 and 10 μmol/L (final concentration) LY294002, the CD69 expression in CD3 mAb-stimulated CD3+ T cells were (52.0 ± 0.5)% , (46.3 ± 0.4)% , (33.2 ± 0.4 )% and (19.1 ± 0.0)% , respectively; and in Mtb-Ag stimulated γδΤ cells, (66.2±0.6)%, (58.4±0.5)%, (38.1±0.3)% and (19.3± 0.1)%, respectively. LY294002 did not inhibit the CD69 expression in CD3+ T cells or γδΤ cells stimulated with PMA+IM, with the expression level > 99% in both cells. After treatment with 0, 0.4, 2 and 10 μmol/L (final concentration) LY294002, the IL-2 level in CD3 mAb-stimulated CD3+ T cells were (561.1±86.2) , (477.0±75.5) , (280.3±53.9) and (36.0±8.7) ng/L, respectively; and in PMA+IM stimulated CD3+ T cells, (1 922.2±371.3) , (1 928.3±381.7) , (1 938.1±262.1) , and (1 915.4±201.6) ng/L, respectively. Moreover, the total counts of CD3+ T cells increased from 1.5×106 before culture to (35.6±8.4) ×106, (31.1±7.3) ×106, (18.7±5.0) ×106 and (7.0±2.0) ×106, respectively; those of γδΤ cells from 1.5×106 before culture to (7.0± 1.1) ×106, (4.7±1.0) ×106, (1.3±0.8) ×106 and (0.2±0.1) ×106, respectively. Conclusion Mtb-Ag may specifically activate γδΤ cells by using the TCR pathway for transduction of the activation signals. LY294002 exhibits obviously inhibitory effects on CD3 mAb activated T cells and Mtb-Ag activated γδΤ cells. Key words: Receptors, antigen, T-cell, gamma-delta; Low molecular weight peptide antigen of mycobacterium tuberculosis; Phosphatidylinositol 3-kinases; Enzyme inhibitors