[New perspectives in the management of non-small-cell lung carcinoma (NSCLC)].
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Biomarkers, Tumor↗
Biomedical subjects
Gabriele Multhoff
Publications and source records attributed to Gabriele Multhoff.
Control of metastasized pancreatic carcinomas in SCID/beige mice with human IL-2/TKD-activated NK cells.
Pancreatic carcinoma, the fifth leading cause of cancer-related mortality, frequently presents the stress-inducible heat shock protein 70 (Hsp70) on the cell membrane. Therefore, we explored an immunological approach exploiting the efficacy of NK cells activated either with low dose IL-2 plus Hsp70-peptide TKDNNLLGRFELSG (TKD; IL-2/TKD) or with IL-2 alone in a xenograft pancreatic carcinoma model. An orthotopic injection of either 2.5 x 10(6) or 1 x 10(6) Colo357 cells in SCID/beige mice resulted in rapidly growing primary tumors and the development of hepatic metastases on days 5 and 10, respectively. In line with results of in vitro migration assays, these NK cells also had the capacity to infiltrate pancreatic tumors and liver metastases in tumor-bearing mice. In vitro, a combined treatment of NK cells with IL-2/TKD but neither of the two treatments alone causes a profound increase in the lytic capacity against Hsp70 membrane-positive Colo357 cells. In vivo, a single i.v. injection of these NK cells on day 15 post-tumor inoculation resulted in a significant reduction in tumor weights, a delayed onset of hepatic metastases, and a prolonged life expectancy. In contrast, identically treated T cells and NK cells treated with IL-2 alone were significantly less efficient in controlling pancreatic tumors and metastases. Most importantly, four repeated i.v. infusions of IL-2/TKD-activated NK cells eradicated primary tumors and prevented hepatic metastases. In summary, our mouse data have implicated that NK cells preactivated with IL-2/TKD might provide a novel therapeutic tool for the treatment of aggressive, Hsp70-positive pancreatic carcinoma.
Adenocarcinoma↗
Stress down south: meeting report of the fifth International Workshop on the Molecular Biology of Stress Responses.
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Animals↗
Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72.
IFN-gamma exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-gamma enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-gamma triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-gamma-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl beta-cyclodextrin completely abrogated IFN-gamma-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-gamma promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.
Antigens, CD↗
Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.
Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.
Adaptor Proteins, Signal Transducing↗
Nucleofection of non-B cells with mini-Epstein-Barr virus DNA.
A tumor-specific cell surface localization of heat shock protein 70 (Hsp70) on CX+ colon carcinoma cells provides a recognition structure for NK cells but not for NKT and T cells. Incubation with low-dose IL-2 plus Hsp70-peptide TKD enhances production and release of granzyme B by NK cells and thus renders Hsp70-positive tumors more sensitive to their cytolytic attack. To provide the experimental basis for the generation of Hsp70-reactive NK cell lines we established a modified nucleofection technique as a rapid and efficient method for gene transfer into non-B cells. Therefore, TKD-stimulated, CD3/CD19-depleted effector cells, consisting of 85% CD3- CD16/56+ NK cells, 1.4% CD3+ CD16/56+ NKT cells, and 0.3% CD3+ CD16/56- T cells were nucleofected with the green fluorescent protein (GFP)-containing mini-Epstein-Barr virus (mini-EBV) plasmid p2667 (1478.A d2GFP). GFP, a marker for the expression of EBV-associated genes, became visible for the first time on day 18 after transfection. On day 28 mini-EBV-transfected cells consisted of 49% NKT, 38% T cells, and 13% NK cells; no contaminating B cells were detectable. Even 1.5 years after transfection GFP and CD94 were found to be co-expressed on transfectants. These data indicated that mini-EBV provides a useful tool for the nucleofection of non-B cells. The cytolytic activity of NK-transfectants towards Hsp70 membrane-positive CX+ tumor cells was comparable to that of non-transfected effector cells. In summary, our results might provide the basis for the generation of non-B effector cell lines including NK cells with conserved Hsp70-reactivity.
Cell Line, Tumor↗
Retinoid- and sodium-butyrate-induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology.
Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.
Butyrates↗
Immunostimulatory functions of membrane-bound and exported heat shock protein 70.
In the search for tumor-specific antigens, microbial and eukaryotic heat shock proteins (HSP) have been identified. Intracellularly, HSPs function as molecular chaperones supporting folding and transport of a great variety of polypeptides and proteins under normal physiological conditions and following stress stimuli. Furthermore, interferon-gamma and elevated body temperature induced by exercise have been found to increase serum levels of HSPs in humans. Extracellularly localized or plasma membrane-bound HSPs elicit a potent anti-cancer immune response mediated either by the adaptive or innate immune system. Following uptake of HSP (HSP70 and gp96)-peptide complexes by antigen presenting cells (APCs) and "cross-presentation" of HSP-chaperoned peptides on MHC class I molecules, a CD8-specific T cell response is induced. Apart from chaperoning tumor-specific peptides, HSPs per se provide activatory signals for the innate immune system. Binding of peptide-free HSP70 to APCs via Toll-like receptors (TLRs) initiates the secretion of pro-inflammatory cytokines and thus results in a broad non-specific immunostimulation. An unusual membrane localization of Hsp70, the major heat-inducible member of the HSP70 family, on tumor cells but not on corresponding normal tissues was found to act as a tumor-specific recognition structure for natural killer (NK) cells. Soluble as well as cell membrane-bound HSP70 can directly activate the cytolytic and migratory capacity of NK cells. APCs and tumor cells actively release HSP70s in lipid vesicles with biophysical properties of exosomes. These HSP70-presenting exosomes are thought to stimulate the adaptive and innate immune system in vivo. Taken together, depending on their intra/extracellular localization, peptide loading status, origin and route of application, HSPs either exert immune activation as danger signals in cancer immunity or protect cells from lethal damage induced by exogenous stress stimuli.
CD8-Positive T-Lymphocytes↗
Natural killer cells activity and neuroimmunological treatment of cancer.
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Colonic Neoplasms↗
Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.
Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c-induced, apoptosome-dependent caspase activation in vitro and Fas ligand-induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.
Animals↗
Treatment of colon and lung cancer patients with ex vivo heat shock protein 70-peptide-activated, autologous natural killer cells: a clinical phase i trial.
PURPOSE: The 14 amino acid sequence (aa(450-463)) TKDNNLLGRFELSG (TKD) of heat shock protein 70 (Hsp70) was identified as a tumor-selective recognition structure for natural killer (NK) cells. Incubation of peripheral blood lymphocyte cells with TKD plus low-dose interleukin 2 (IL-2) enhances the cytolytic activity of NK cells against Hsp70 membrane-positive tumors, in vitro and in vivo. These data encouraged us to test tolerability, feasibility, and safety of TKD-activated NK cells in a clinical Phase I trial. EXPERIMENTAL DESIGN: Patients with metastatic colorectal cancer (n = 11) and non-small cell lung cancer (n = 1) who had failed standard therapies were enrolled. After ex vivo stimulation of autologous peripheral blood lymphocytes with Hsp70-peptide TKD (2 microg/ml) plus low-dose IL-2 (100 units/ml), TKD was removed by extensive washing, and activated cells were reinfused i.v. The procedure was repeated for up to six cycles, applying a dose escalation schedule in 4 patients. RESULTS: The percentage of activated NK cells in the reinfused leukapheresis products ranged between 8 and 20% of total lymphocytes, corresponding to total NK cell counts of 0.1 up to 1.5 x 10(9). Apart from restless feeling in 1 patient and itching in 2 patients, no negative side effects were observed. Concomitant with an enhanced CD94 cell surface density, the cytolytic activity of NK cells against Hsp70 membrane-positive colon carcinoma cells was enhanced after TKD/IL-2 stimulation in 10 of 12 patients. Concerning tumor response, 1 patient was in stable disease during therapy by formal staging criteria and another patient showed stable disease in one metastases and progression in another. CONCLUSIONS: Reinfusion of Hsp70-activated autologous NK cells is safe. Immunological results warrant additional studies in patients with lower tumor burden.
Adult↗
Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by anti-inflammatory drugs.
PURPOSE: Modulation of the heat shock protein (HSP) response affects sensitivity to therapeutic agents in cancer. Here, drugs with anti-inflammatory potential (cyclooxygenase 1/2 inhibitors) and peroxidase proliferator-activated receptor-gamma agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern. EXPERIMENTAL DESIGN: In dose kinetics, the nonlethal concentration of acetyl-salicyl acid, celecoxib, rofecoxib, and the insulin-sensitizer pioglitazone was identified for the human adenocarcinoma cell line CX-. With the exception of CLX, which was diluted in DMSO, all reagents were dissolved in water. After treatment with the different compounds at nontoxic concentrations for 6 h, followed by a 1-h recovery period, the cytosolic Hsp70 levels were measured in CX-2 and CX- tumor cells by Western blot analysis. Fold increase was calculated in relation to the housekeeping protein tubulin. Membrane-bound Hsp70 was analyzed by flow cytometry using a FITC-labeled Hsp70-specific monoclonal antibody. Untreated cells and cells incubated with equivalent amounts of the diluting agents served as controls. The immunological function was tested in granzyme B apoptosis assays, standard (51)Cr release assays, and antibody blocking studies. RESULTS: Compared with aqua dest, the cytoplasmic amount of Hsp70 was equally enhanced in CX-2 and CX- cells by all compounds. An increase in membrane-bound Hsp70, detected selectively in CX- cells, corresponded to an enhanced sensitivity to granzyme B- and natural killer cell-mediated kill that was blockable by using a Hsp70-specific antibody. CONCLUSIONS: Although increase in cytosolic Hsp70 levels conferred resistance to further stress, membrane-bound Hsp70 rendered tumor cells more sensitive to the immunological attack mediated by granzyme B and natural killer cells. Our data provide a biological rational for combining anti-inflammatory drugs with immunotherapy in cancer therapy.
Anti-Inflammatory Agents↗
The cell surface-localized heat shock protein 70 epitope TKD induces migration and cytolytic activity selectively in human NK cells.
Profiling of surface-bound proteins uncovers a tumor-selective heat shock protein 70 (Hsp70) membrane expression that provides a target structure for human NK cells. Hsp70 peptide TKD (TKDNNLLGRFELSG; aa 450-463) was found to enhance the cytolytic activity of NK cells. In this study, we demonstrate that TKD-activated CD3-CD56+CD94+ NK cells are selectively attracted by Hsp70 membrane-positive tumor cells, and supernatants derived thereof. Hsp70 membrane-negative tumors failed to attract these NK cells. The capacity to migrate was associated with a substantial lytic activity against Hsp70-positive tumor cells. Because NK cell migration was independent of cell-to-cell contact, the involvement of a soluble factor was assumed. Interestingly, synthetic Hsp70 protein and Hsp70 peptide TKD, mimicking surface-bound Hsp70, initiates migration of NK cells in a concentration-dependent (1-5 microg/ml), highly selective, and chemokine-independent manner. In summary, our results indicate that Hsp70 peptide TKD not only stimulates cytolysis but also chemotaxis in CD3-CD56+CD94+ NK cells.
Adjuvants, Immunologic↗
Heat shock protein 70 (Hsp70) membrane expression on head-and-neck cancer biopsy-a target for natural killer (NK) cells.
PURPOSE: Heat shock protein 70 (Hsp70) was detected on the cell membrane of human tumor cell lines, but not on normal cells. Here we studied Hsp70 membrane expression as a target for natural killer (NK) cells on tumor material and control tissues of head-and-neck cancer patients. METHODS AND MATERIALS: Membrane-bound Hsp70 was determined by flow cytometry on single-cell suspensions of tumors and the corresponding normal tissues of head-and-neck cancer patients. The cytolytic activity of NK cells against Hsp70-positive tumor cells was measured in a standard cytotoxicity assay. RESULTS: In total, 54 of 74 primary tumors were found to be Hsp70 membrane-positive (73%); tongue/mouth, 21 of 24 (88%); oropharynx, 13 of 20 (65%); hypopharynx, 3 of 6 (50%); larynx, 8 of 11 (73%); trachea 1 of 2 (50%); esophagus, 4 of 5 (80%); lymph node metastases, 4 of 6 (67%). The corresponding control tissue was negative for membrane-bound Hsp70. Biopsies (6 of 6) of patients after in vivo gamma-irradiation (fractionated 5 x 2 Gy) were strongly Hsp70 membrane-positive. Irradiated, Hsp70-positive tumor cells are targets for Hsp70-peptide stimulated NK cells. CONCLUSION: An irradiation-inducible, tumor-selective Hsp70 membrane localization provides a target structure for Hsp70-peptide stimulated human NK cells.
Biopsy↗
Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B.
Cell surface-bound heat shock protein 70 (Hsp70) renders tumor cells more sensitive to the cytolytic attack mediated by natural killer (NK) cells. A 14-amino acid Hsp70 sequence, termed TKD (TKDNNLLGRFELSG, aa450-463) could be identified as the extracellular localized recognition site for NK cells. Here, we show by affinity chromatography that both, full-length Hsp70-protein and Hsp70-peptide TKD, specifically bind a 32-kDa protein derived from NK cell lysates. The serine protease granzyme B was uncovered as the 32-kDa Hsp70-interacting protein using matrix-assisted laser desorption ionization time-of-flight mass peptide fingerprinting. Incubation of tumor cells with increasing concentrations of perforin-free, isolated granzyme B shows specific binding and uptake in a dose-dependent manner and results in initiation of apoptosis selectively in tumor cells presenting Hsp70 on the cell surface. Remarkably, Hsp70 cation channel activity was also determined selectively in purified phospholipid membranes of Hsp70 membrane-positive but not in membrane-negative tumor cells. The physiological role of our findings was demonstrated in primary NK cells showing elevated cytoplasmic granzyme B levels following contact with TKD. Furthermore, an increased lytic activity of Hsp70 membrane-positive tumor cells could be associated with granzyme B release by NK cells. Taken together we propose a novel perforin-independent, granzyme B-mediated apoptosis pathway for Hsp70 membrane-positive tumor cells.
Apoptosis↗
Heat shock protein 70 membrane expression and melanoma-associated marker phenotype in primary and metastatic melanoma.
Cell membrane localization of the 72 kDa heat shock protein 70 (Hsp70) has been found on different tumour cell lines, on biopsy material from solid tumours and metastases and on leukaemic blasts from acute myelogenous leukaemia patients, but not on the corresponding normal tissues, as determined by flow cytometry using the Hsp70-specific monoclonal antibody C92F3B1. In the present study Hsp70 membrane expression was studied on primary malignant melanomas, melanoma metastases, melanocytes, human skin fibroblasts and peripheral blood lymphocytes, together with expression of the melanoma-associated markers Mel-1, Mel-2 and Mel-5, major histocompatibility complex class I and the fibroblast-specific marker ASO2. As previously shown, fibroblasts and peripheral blood lymphocytes from healthy human volunteers were found to be negative for Hsp70 and for the melanoma-associated markers Mel-1, Mel-2 and Mel-5. Human melanocytes from healthy human donors were also negative for Hsp70, but were positive for Mel-1 and Mel-5. Independent of the Clark's level, all the malignant melanomas (n = 9) and metastases (n = 11) exhibited were positive for both Mel-1 and Mel-2. The primary melanomas could be divided into two groups according to their Hsp70 and Mel-5 expression pattern: those with an Hsp70-negative and a Mel-5-positive phenotype (-/+) (five out of nine), and those with an Hsp70-positive and a Mel-5-negative phenotype (+/-) (four out of nine). All the melanoma metastases (n = 11) had an Hsp70-positive, Mel-5-negative phenotype (+/-). These data provide the first hint that the marker combination Hsp70 positive/Mel-5 negative might be useful in estimating the metastatic potential of a melanoma. Investigations on changes in the marker combination Hsp70/Mel-5 during onset of melanoma disease and progression will clarify its potential as a prognostic risk factor.
Adult↗
Heat shock protein 70-reactivity is associated with increased cell surface density of CD94/CD56 on primary natural killer cells.
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.
Antigens, CD↗
Interaction of heat shock protein 70 peptide with NK cells involves the NK receptor CD94.
Full-length Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) both stimulate the cytolytic activity of naive natural killer (NK) cells against Hsp70-positive tumor target cells. Here, we describe the characterization of Hsp70-NK cell interaction with binding studies using the human NK cell line YT. Binding of recombinant Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) to YT cells is demonstrated by immunofluorescence studies. A phenotypic characterization revealed that none of the recently described HSP-receptors (alpha2-macroglobulin receptor CD91, Toll-like receptors 2, 4, 9, CD14) are expressed on YT cells. Only the C-type lectin receptor CD94 is commonly expressed by YT cells and Hsp70 reactive NK cells. A correlation of the cell density-dependent, variable CD94 expression and the binding capacity of Hsp70 was detected. Furthermore, Hsp70 binding could be completely abrogated by preincubation of YT cells with a CD94-specific antibody. Competition assays using either unlabeled Hsp70 protein or an unrelated protein (GST) in 20-fold excess and binding studies with escalating doses of Hsp70 protein provide evidence for a specific and concentration-dependent interaction of Hsp70 with YT cells. In addition to Hsp70 and Hsp70C, a 14-mer Hsp70 peptide termed TKD is known to exhibit comparable stimulatory properties on NK cells. Similar to full-length Hsp70 protein (10 microg/ml-50 microg/ml), a specific binding of this peptide to YT cells was observed at 4 degrees C, at equivalent concentrations (2.0 microg/ml-8.0 microg/ml). Following a 30 min incubation period at 37 degrees C, membrane-bound Hsp70 protein and Hsp70 peptide TKD were completely taken up into the cytoplasm.
Antibodies↗