A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity

Abstract

Compared with normal cells, tumor cell lines exhibit an unusual plasma membrane localization of heat shock protein 70 (Hsp70). This tumor-selective Hsp70 membrane expression has been found to correlate with an increased sensitivity to lysis mediated by human natural killer (NK) cells that transiently adhere to plastic following cytokine stimulation. A human Hsp70-specific monoclonal antibody (mAb) detects membrane-bound Hsp70 on viable tumor cells and blocks the immune response of NK cells against Hsp70-expressing tumor cells. By peptide scanning (pep-scan) analysis, the epitope of this mAb was mapped as the C-terminal-localized 8-mer NLLGRFEL (NLL, amino acids [aa] 454-461). Most interestingly, similar to full-length Hsp70 protein, the N-terminal-extended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450-463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp70 protein. Blocking studies revealed that an excess of the 14-mer peptide TKDNNLLGRFELSG inhibits the cytolytic activity of NK cells similar to that of Hsp70 protein. In comparison, other TKD-related peptides, including the 8-mer antibody epitope NLLGRFEL (aa 454-461), the 12-mer TKDNNLLGRFEL (aa 450-461), the 13-mer C-terminal-extended peptide NLLGRFELSGIPP (aa 454-466), the 14-mer TKD-equivalent sequences of Hsp70hom TKDNNLLGRFELTG (aa 450-463), Hsc70 TKDNNLLGKFELTG (aa 450-463), and DnaK AADNKSLGQFNLDG (aa 447-460) failed to activate NK activity.

Fig. 2.

Comparison of the proliferative response of NK cells against different Hsp70 peptides. NK cells were stimulated either with IL-2 (100 IU/mL) alone or with IL-2 plus peptides GIPP (13-mer), NLL (8-mer), TKD (14-mer), TKD12 (12-mer), HSC70 (14-mer), HOM (14-mer), and DNAK (14 mer) at a concentration of 2 μg/mL. The proliferative response of NK cells was measured 48 hours after peptide incubation and an 18-hour incubation period with ³H thymidine (0.5 μCi/mL). Values are given as the means of 2 independent experiments ± SD

Fig. 3.

Cytolytic activity of NK cells either stimulated with low-dose IL-2 (100 IU/mL) alone or with IL-2 plus the 14-mer peptide TKD (2 μg/mL) or with IL-2 plus the 8-mer peptide NLL (2 μg/mL) for 4 days. As target cells, ⁵¹Cr-labeled CX+ and CX− tumor cells, which differ with respect to their capability to express Hsp70 on the plasma membrane, were used. The results are expressed as the percentage of specific lysis at varying E:T ratios ranging from 1:1 to 40:1. The percentage of spontaneous release for each tumor target cell line was less than 20%. A phenotypic characterization of the NK cells reveals the following: NK cells plus TKD: CD3, 4%; CD16/56, 83%; NK cells plus NLL: CD3, 6%; CD16/56, 80%; NK cells: CD3, 3%; CD16/56, 84%. The data represent 1 representative experiment of 4

Fig. 4.

Blocking of the cytolytic activity of low-dose IL-2 (100 IU/mL) stimulated NK cells (CD16/56, 49%) against CX+ and CX− tumor cells either by incubation of the target cells with Hsp70 mAb (10 μg/1 × 10⁶ cells) or by incubation of NK cells with TKD peptide (10 μg/mL) for 30 minutes at room temperature, directly before the assay. The data represent 1 representative experiment of 3

Fig. 1.

(A) Comparison of the proliferative response of NK cells against Hsp70 peptides. NK cells were stimulated either with IL-2 (100 IU/mL) alone or with IL-2 plus peptides NLL (8-mer), TKD (14-mer), and GIPP (13-mer) at concentrations of 0.02, 0..2, 2, 4, and 8 μg/mL. NK cells were also stimulated with rHsp70 protein at a concentration of 10 and 200 μg/mL, respectively. The proliferative response of NK cells was measured 72 hours after peptide incubation and an 18-hour incubation period with ³H thymidine (0.5 μCi/mL). Values are given as the means of 5 independent experiments ± SD. (B) To evaluate the optimal stimulatory dose of TKD (14-mer), an extended concentration range was tested: NK cells were stimulated either with IL-2 (100 IU/mL) alone or with IL-2 plus peptides TKD (14-mer) at the following concentrations 0.1, 0.2, 0.5, 1, 1.5, 2.5, and 3 μg/mL. The proliferative response of NK cells was measured 48 hours after peptide incubation and an 18-hour incubation period with ³H thymidine (0.5 μCi/mL). Values are given as the means of 3 independent experiments ± SD

Fig. 1.

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