Fig. 4
Mechanisms of LA-induced activation of NK cells. a LA-induced NK cell activation through MICA/B-NKG2D signalling between DCs and NK cells. b IFN-γ secretion of different cells stimulated with YL or LA using an ELISPOT assay (n = 3). c Immune-related differentially expressed genes between LA-pulsed DCs and NP-pulsed DCs by RNA-seq. d The MICA/B expression level in LA-pulsed DCs was significantly increased (n = 3). Left, flow cytometry. Right, western blot. e IFN-γ expression and cytotoxicity of NK cells were significantly elevated with stimulation with LA/DCs. MICA/B antibody (Ab) could block LA/DC-mediated NK activation. (n = 3). f Phosphorylation level of p38 was significantly upregulated in LA-pulsed DCs, rather than the phosphorylation level of ERK1/2 or AKT (n = 3). g SB203580 (10 μM), an inhibitor of the p38 MAPK pathway, blocked LA-mediated upregulation of MICA/B on DCs (n = 3). h SB203580 blocked LA/DC-mediated IFN-γ expression and NK cell cytotoxicity (n = 3). i The RNA-seq results showed the molecular functions of immune-related differentially expressed genes between LA-pulsed DCs and NP-pulsed DCs. j Left panel, TLR4 on LA-pulsed DCs decreased among TLRs by RNA-seq. Right panel, upregulation of TLR4 downstream signals (MYD88 and TRAF6) and MICA/B in LA-pulsed DCs by RNA-seq. k TAK242 (DCs were pretreated for 1 h with TAK-242, 10 μM) inhibited LA-mediated upregulation of MYD88, TRAF6, p38 phosphorylation, and MICA/B in DCs (n = 3). l LA/DC&TAK242 could not promote proliferation of NK cells in PBMCs (n = 3). NC, negative control, water. NP, negative peptide, the peptide (PPGRPSPDN) derived from ECM1 with largest predicted IC50 of affinity with HLA-A2.1. d, e–h, l P values were obtained from independent-samples t-test; error bars denote standard deviation (SD). Compared with NC, *P < 0.05, **P < 0.01; compared with NP, #P < 0.05, ##P < 0.01, ###P < 0.001; compared with LA&MICA/B Ab, &&P < 0.01; compared with LA&SB203580, $P < 0.05, $$P < 0.01; compared with LA&TAK242, @P < 0.05