Study On the Molecular Mechanisms of Cytotoxicity of NK Cells against RPMI 8226 Cells.
Authors: Wang Xiuju , Yin Songmei , Ma Liping , Nie Danian , Xie Shangfeng , Li Yingqing 
Published: October 2019

Abstract
Abstract 4745

To observe how did the allogeneic NK cells kill human multiple myeloma cell RPMI-8266 and the immune mechanisms why RPMI-8266 cells escape from the NK cytotoxicity.

Methods
Detect the cytotoxicity of NK cells against RPMI 8226 cells by LDH release assay at different effect-to-target cell ratios in vitro, K562, high-sensitive with NK cells,was control cell. Test the expression of MHC-± chain-related molecules (MICA/B), human cytomegalovirus glycoprotein UL16-binding protein (ULBP1-3) and HLA -± MHC molecules on K562 and RPMI-8266 cell by flow cytometry. Detect mRNA level of MICA / B and ULBP1 ∼ 3 in K562, RPMI-266, and KIR genotyping of NK cell from 9 cases of healthy volunteers' by PCR method. As the E:T was 20:1, we used AMO-1, BMO-1, M295, M310, M551 and W6/32 mAb to block the effect of cell-surface protein MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA-± in K562 and RPMI-8266 cell respectively, then observe the change of cytotoxicity of NK cells on K562 and RPMI-8266 cell.

Results
When the E:T was 5:1, cytotoxicity of NK cells on K562 and RPMI-8266 were (29.52 ± 0.27)% and (2.15 ± 0.32)% respectively; As the E:T was 10:1, the cytotoxicity were (36.37 ± 0.78) % and (5.26 ± 0.84)%; As the E:T was 20:1, they were (59.57 ± 1.05)% and (7.63 ± 1.05)%; As the E:T was 40:1, they were (70.64 ± 1.34)% and (10.18 ± 1.53)%. There wre significantly difference of the cytotoxicity of NK cell between on RPMI-8266 and K562 at all E:T ratio(P <0.05); As for different E:T ratio, the cytotoxicity of NK cell on 8266 cells were statistic difference among them (P <0.05). In K562 cell, we detect MICA / B and ULBP 1 ∼ 3 mRNA and protein, but no HLA -± molecules; RPMI-8266 cell expressed MICA / B and ULBP 1 ∼ 3 and HLA -± molecule, but no MICA / B and ULBP 1 ∼ 3. The genotype of KIR were mismatch between RPMI-8266 cell and NK cells from 9 cases of healthy volunteers. The E:T set as 20:1, we used monoclonal antibodies AMO-1, BMO-1, M295, M310 and M551, respectively, to block the effect of MICA, MICB, ULBP1, ULBP2 and ULBP3, killing activity of NK cell on K562 cells significantly decreased, respectively, (40.82 ± 1.47)%, (43.26 ± 2.41)%, (45.42 ± 1.58)%, (50.74 ± 2.16)%, (41.72 ± 1.66)%, compared with the previous block, they were significantly different (P <0.05); The killing activity of NK cell on RPMI-8266 did not significant change after bloking by all monoclonal antibody, they were (5.81 ± 0.72)%, (7.83 ± 0.91)%, (6.77 ± 0.82)%, (8.25 ± 1.46)%, (6.42 ± 0.87)% respectively (P> 0.05); But as we bloked HLA -± molecules with W6/32 monoclonal antibody, the cytotoxicity of NK cell on RPMI-8266 increased to (48.77 ± 4.61)%, there were significant differences between before and after bloking (P <0.05).

Conclusion
For 8266 cell, the mechanisms of immune escape from NK killing effect may be related to high expression of HLA -± molecule, and did not express the NKG2D ligands MICA / B and ULBP1 ∼ 3 molecules.

Disclosures:
No relevant conflicts of interest to declare.