Heat shock protein 70-reactivity is associated with increased cell surface density of CD94/CD56 on primary natural killer cells

Abstract

Previously we described an involvement of the C-type lectin receptor CD94 and the neuronal adhesion molecule CD56 in the interaction of natural killer (NK) cells with Hsp70-protein and Hsp70-peptide TKD. Therefore, differences in the cell surface density of these NK cell-specific markers were investigated comparatively in CD94-sorted, primary NK cells and in established NK cell lines NK-92, NKL, and YT after TKD stimulation. Initially, all NK cell types were positive for CD94; the CD56 expression varied. After stimulation with TKD, the mean fluorescence intensity (mfi) of CD94 and CD56 was upregulated selectively in primary NK cells but not in NK cell lines. Other cell surface markers including natural cytotoxicity receptors remained unaffected in all cell types. CD3-enriched T cells neither expressing CD94 nor CD56 served as a negative control. High receptor densities of CD94/CD56 were associated with an increased cytolytic response against Hsp70 membrane-positive tumor target cells. The major histocompatibility complex (MHC) class I-negative, Hsp70-positive target cell line K562 was efficiently lysed by primary NK cells and to a lower extent by NK lines NK-92 and NKL. YT and CD3-positive T cells were unable to kill K562 cells. MHC class-I and Hsp70-positive, Cx + tumor target cells were efficiently lysed only by CD94-sorted, TKD-stimulated NK cells with high CD94/CD56 mfi values. Hsp70-specificity was demonstrated by antibody blocking assays, comparative phenotyping of the tumor target cells, and by correlating the amount of membrane-bound Hsp70 with the sensitivity to lysis. Remarkably, a 14-mer peptide (LKD), exhibiting only 1 amino acid exchange at position 1 (T to L), neither stimulated Hsp70-reactivity nor resulted in an upregulated CD94 expression on primary NK cells. Taken together our findings indicate that an MHC class I-independent, Hsp70 reactivity could be associated with elevated cell surface densities of CD94 and CD56 after TKD stimulation.

Fig 1.

CD94-positive NK cell and CD3-positive T cell selection. CD94-positive NK cells were selected from peripheral blood mononuclear cells (PBMNC) derived from 8 healthy human donors using anti-CD94 biotin monoclonal antibody and anti-biotin magnetic microbeads. The phenotype of unseparated PBMNC (A) and CD94-positive NK cells (B) after stimulation with low-dose interleukin (IL)-2 (100 IU/mL) plus TKD (2 μg/mL) for 4 days was determined by flow cytometry using the following antibodies: CD3 fluorescein isothiocyanate (FITC), CD16/CD56 phycoerythrin (PE), CD56 FITC and CD94 PE. Dot-blot diagrams of 1 representative flow-cytometric analysis of CD94-positive NK cells on day 4 is shown. Mean values of the percentages of 8 experiments ± standard deviation are summarized on the right-hand side of each graph. CD3-positive T cells (C) were selected from PBMNC derived from 3 healthy human donors using anti-CD3 magnetic microbeads. Dot-blot analysis of 1 representative flow cytometry of CD3-positive cells after 4 days stimulation with low-dose IL-2 (100 IU/mL) plus TKD (2 μg/mL) and mean values of 3 experiments are shown.

Fig 2.

CD94 and CD56 expression pattern on CD94-sorted, primary natural (NK) cells after long-term (5 weeks) stimulation with TKD. CD94-positive NK cells were cultured in medium containing low-dose interleukin (IL)-2 (100 IU/mL) plus TKD (2 μg/mL). Medium was completely renewed once a week. Cell density was kept at 2 × 10⁶ cells/mL. Phenotypic characterization was performed by flow cytometry on days 14, 21, 28, and 35 using fluorescence-conjugated CD56 fluorescein isothiocyanate and CD94 phycoerythrin antibodies. The data represent 1 representative profile of 3 independent experiments

Fig 3.

Comparative cytolytic activity of CD94-positive, primary natural killer (NK) cells, NK-92, NKL, YT cell lines and CD3-positive, primary T cells. K562, Hsp70 membrane–positive Cx+ and negative Cx− cells were used as target cells in a 4-hours ⁵¹Cr cytotoxicity assay. The spontaneous release for each target cell was less than 20%. It is important to note that effector to target cell ratios ranged from 10:1 to 2:1 in case of CD94-positive, primary NK cells and from 40:1 to 10:1 in case of NK cell lines. Mean values of 3 independent experiments are shown

Fig 4.

Hsp70-specific antibody inhibits the increased lysis of Cx+ tumor cells. After labeling, Cx+ and Cx− tumor target cells were incubated for 15 minutes with Hsp70-specific monoclonal antibody (cmHsp70.1, 5 μg/mL) and used as targets for CD94-sorted natural killer cells. Lysis of Cx− cells remained unaffected after antibody treatment (data not shown). The spontaneous release for each target cell was less than 20%. Effector to target cell ratios were 10:1, 5:1, and 2:1

Fig 5.

Increased Hsp70 membrane expression after treatment with paclitaxel (P) increases lysis of Cx− tumor target cells. Cx+ and Cx− cells were either heat shocked at the nonlethal temperature of 42°C for 1 hour (hs) or incubated with a nonlethal dose of paclitaxel (1 μM) for 1 hour. The major histocompatibility complex class I, class II, and Hsp70 phenotype of the tumor target cells before and after treatment are summarized in Table 4. Cx− tumor cells exhibited an upregulated Hsp70 expression after P treatment that correlates with an increased sensitivity to lysis. Nonlethal heat shock neither increases Hsp70 membrane expression nor lysis of Cx− cells. The spontaneous release for each target cell was less than 20%. Effector to target cell ratios were 10:1, 5:1, and 2:1

Fig 6.

Comparative cytolytic activity of CD94 high (+++) and CD94 low (++) expressing natural killer (NK) cells. Peripheral blood mononuclear cells were separated in CD94 high (+++) and CD94 low (++) expressing NK cells by flow cytometry cell sorting. Both subpopulations were stimulated with low dose interleukin-2 (100 IU/mL) and TKD (2 μg/mL) for 4 days and used as effector cells in a 4-hours ⁵¹Cr cytotoxicity assay. Major histocompatibility complex class I- and Hsp70-positive tumor cells were used as target cells. The spontaneous release for each target cell was less than 20%. Effector to target cell ratios were only 2:1, 1:1, and 0.5:1

Fig 7.

The 14-mer Hsp70-peptide TKD, but not the 14-mer LKD, exhibiting 1 amino acid exchange at position 1 (T to L), stimulates Hsp70 reactivity concomitant with an increase in the mean fluorescence intensity of CD94 on natural killer cells. Lysis of Cx+ tumor cells was significantly enhanced as compared with that of Cx− cells. The spontaneous release for each target cell was less than 20%. Effector to target cell ratios were 10:1, 5:1, and 2:1