Influence of Hsp70 and HLA-E on the killing of leukemic blasts by cytokine/Hsp70 peptide-activated human natural killer (NK) cells

Abstract

This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30, NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte antigen (HLA)-E membrane-negative (Hsp70(+)/HLA-E(-)) CX(+) and K562 cells. However, it had no effect on the responsiveness to Hsp70(-)/HLA-E(-) CX(-) cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear cells against Hsp70(+)/HLA-E(+) leukemic blasts was weaker than that against Hsp70(+)/HLA-E(-) K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK cell-mediated lysis of K562 cell populations that had been transfected with HLA-E(R) or HLA-E(G) alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule) and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities of cytokine/TKD-activated NK cells.

Fig. 1

Correlation of the granzyme B ELISPOT and ⁵¹Cr-release assays for the determination of NK cell-mediated cytotoxicity at effector to target cell ratios of 5:1, 2:1, and 1:1. Effector cells were human CD3⁻ NK cells that had been stimulated with low-dose IL-2 (100 IU/mL) plus Hsp70 peptide TKD (2 μg/mL). Hsp70 membrane-positive K562 erythroleukemic cells were used as targets. The experiment summarizes the results of three independent experiments. The correlation between ELISPOT dots and percentage lysis is highly significant (P < 0.001)

Fig. 2

Granzyme B release by IL-2/TKD- (100 IU/mL/2 μg/mL), IL-2- (100 IU/mL), and TKD- (2 μg/mL) stimulated PBLs in response to CX⁺ (Hsp70⁺/HLA-E⁻) and CX⁻ (Hsp70⁻/HLA-E⁻) colon carcinoma sublines and erythroleukemic K562 (Hsp70⁺/HLA-E⁻) cells. Specific lysis (as reflected by granzyme B release) was determined using the ELISPOT assay at E/T ratios ranging from 10:1 to 5:1. Data are means ± SD from four independent experiments. Asterisk, Compared to IL-2 only, granzyme B release by PBLs in response to CX⁺ and K562 cells was significantly greater following stimulation with IL-2/TKD (P < 0.05)

Fig. 3

Granzyme B release (as an indicator of cytotoxic activity) by PBLs from healthy donors (a), CD3⁻ NK cells (purity >95%, b), and CD3⁺ T cells (purity >98%, c) in response to K562 cells and leukemic blasts from two patients (patient no. 19, center panel; patient no. 6, right panel) as target cells. Blasts from patient no. 19 comprised 76% Hsp70 membrane-positive cells and those from patient no. 6 comprised 50% Hsp70 membrane-positive cells (right panel). Effector cells were either unstimulated or stimulated either with IL-2/TKD (100 IU/mL/2 μg/mL), IL-15/TKD-treated (10 IU/mL/2 μg/mL) or IL-2 (100 IU/mL). Effector to target cell ratios of PBL and T cells ranged between 2:1 and 10:1; that of NK cells between 5:1 and 1:1. The phenotypic characteristics of the target cells are provided in Table 2. Asterisk, Compared to IL-2/TKD and IL-15/TKD, granzyme B release by PBLs and NK cells in response to K562 cells and leukemic blasts (no. 19) was significantly lower following stimulation with IL-2 only (P < 0.05)

Fig. 4

Effect of the anti-Hsp70 mAb (cmHsp70.2, 10 μg/mL) and the inhibition of Hsp70 expression using siRNA (mut3) on the cytolysis of K562 target cells by IL-2/TKD-treated (100 IU/mL/2 μg/mL) PBL effector cells. Specific lysis was determined using the 4-h ⁵¹Cr-release assay at E/T ratios ranging from 5:1 to 0.5:1. Data show one representative experiment out of three independent assays

Fig. 5

Cytolytic activity of CD3⁻ NK effector cells against K562 cells (Hsp70⁺/HLA-E⁻) into which HLA-E^GB5 (73.2%), HLA-E^GC6 (32.5%), HLA-E^RA2 (20.7%), and HLA-E^RA4 (11.2%) alleles had been transfected. The Hsp70 positivity was comparably high in all transfectants (47 ± 19, 38 ± 11, 47 ± 18, and 36 ± 17 vs 40 ± 9% in untransfected K562 cells, respectively). Specific lysis was determined using the 4-h ⁵¹Cr-release assay at E/T ratios ranging from 20:1 to 0.5:1. Data show one representative experiment out of three