Effects of soluble programmed death ligand 1 on regulating the proliferation of T lymphocytes and its mechanism

Abstract

To explore the effects of soluble programmed death ligand 1 (sPD-L1) on the proliferation of T lymphocytes and its mechanism. T lymphocytes were isolated from healthy human peripheral blood and activated by phytohemagglutinin (PHA). The experiment had group A: resting T lymphocytes, group B: activated T lymphocytes, group C: activated T lymphocytes+sPD-L1Ig, group D: activated T lymphocytes+sPD-L1Ig+membrance-bound immunoglobulin (mIgG) and group E: activated T lymphocytes+sPD-L1Ig+anti-PD-L1 antibody (2H11). The absorbance value (A) of T lymphocytes in each group was measured by cell counting kit (CCK-8). The cell cycle and apoptosis of T lymphocytes induced by sPD-L1 were measured by flow cytometry. And the phosphorylation level of programmed death 1 (PD-1) signaling motif tyrosine was measured by Western blot. Furthermore, the amounts of signal adaptor molecule Src homology 2 domain-containing tyrosine phosphatase (SHP)-1 and SHP-2 were quantified by immunoprecipitation. And the exciting mechanism of sPD-L1 was explored for PD-1 inhibitory signals. CCK-8 study showed that A values in each group were 0.42 ± 0.03, 1.20 ± 0.06, 0.87 ± 0.05, 0.78 ± 0.05 and 1.11 ± 0.09 respectively when the concentration of sPD-L1Ig was 250 ng/ml. The proliferation of T lymphocytes in group C significantly decreased compared with group B (t = 3.946, P = 0.017) while group E significantly increased compared with group D (t = 3.139, P = 0.035). The percentage of cell number in G1 phase of the above-mentioned 5 groups were (94.49 ± 0.50)%, (79.22 ± 0.50)%, (89.62 ± 0.33)%, (92.89 ± 0.80)% and (87.94 ± 0.87)% respectively and group C significantly increased compared with group B (t = 17.310, P < 0.001). The apoptotic rate of the above-mentioned five groups were (35.77 ± 1.82)%, (35.20 ± 2.70)%, (62.77 ± 0.24)%, (64.47 ± 0.44)% and (36.80 ± 3.53)% respectively. And apoptotic rate in group C significantly increased compared with group B (t = 10.160, P = 0.001) while group E significantly decreased compared with group D (t = 7.790, P = 0.002). The expressions of SHP-1 and SHP-2 showed no inter-group difference (all P > 0.05). However, the expressions of p-SHP-1 and p-SHP-2 in group C was higher than those in group B (t = 10.790, P < 0.001; t = 13.051, P < 0.001) while the expression of p-SHP-1 decreased in group E compared with group D (t = 3.361, P = 0.028). Soluble PD-L1 can effectively inhibit the proliferation of T lymphocytes. The phosphorylation of SHP-1 and SHP-2 contributes to the inhibitory signaling of PD-1/sPD-L1 pathway. And anti-PD-L1 blocking antibody may partially restore the proliferation of T lymphocytes through a down-regulated expression of p-SHP-1..