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The Journal of Immunology, 2000, 164: 2362-2371.
Copyright © 2000 by The American Association of Immunologists

Heat Shock Protein 70 Is Able to Prevent Heat Shock-Induced Resistance of Target Cells to CTL1

Ralf Dressel, Leslie Elsner, Thomas Quentin, Lutz Walter and Eberhard Günther2

Division of Immunogenetics, University of Göttingen, Göttingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In several cell lines heat shock has been shown to induce resistance to cytotoxic immune mechanisms mediated by TNF-{alpha} (1, 2, 3, 4, 5), CTL (4, 6), or monocytes (1). Heat shock-induced resistance has been assigned to the expression of heat shock proteins (Hsps)3 and explained by their function as molecular chaperones. In the TNF-{alpha} model resistance could be mimicked by transfection with Hsp27 or Hsp70 genes (5, 7), and an anti-apoptotic function of Hsp70 has been localized downstream of caspase 3 activation (8). Suppression of glucose-regulated protein 78 (Grp78), a member of the Hsp70 family, has been demonstrated to eliminate stress-induced resistance of a fibrosarcoma cell line to CTL (9) and also to inhibit tumor progression in vivo (10). We have described heat shock-induced resistance to CTL in the rat myeloma cell line Y3 (6). Y3 cells are unable to express the major heat shock-inducible Hsp70 protein encoded by the MHC-linked Hsp70-1 and Hsp70-2 genes, indicating that Hsp70 is not indispensable for heat shock-induced resistance to occur. This cell model was used to further analyze the role of Hsp70 in heat shock-induced resistance to CTL. Two experimental approaches were followed: 1) the use of somatic cell hybrids between Y3 cells and lymphocytes, and 2) loading of Y3 cells with recombinant Hsp70. It will be shown that Hsp70, instead of being protective, is able to prevent heat shock-induced resistance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Lymphocytes, cell lines, hybrid cell clones, and heat shock conditions

The rat myeloma cell line 210-RCY3-Ag1.2.3 (Y3) derived from LOU/C rats (RT1u) (11) and hybrid cell clones generated therefrom (see below) were maintained in NaHCO3-buffered DMEM (Biochrom, Berlin, Germany) supplemented with 10% FCS (Biochrom), pyruvic acid (110 mg/L), penicillin (100000 U/L), and streptomycin (100 mg/L) in petri dishes (Sarstedt, Nümbrecht, Germany) at 37°C in a 10% CO2 atmosphere. Rat lymphocytes were prepared from lymph nodes as described (12). For mitogenic stimulation 5 µg/ml Con A (Amersham Pharmacia Biotech, Freiburg, Germany) were added to the cultures. Lymphocytes were used as targets in cytotoxicity experiments after 4–5 days of mitogenic stimulation. Somatic cell hybrids between Y3 cells and normal syngeneic (LOU/CGun, RT1u) or allogeneic (BUF/Gun, RT1b) lymph node lymphocytes were generated by cell fusion with polyethylene glycol according to common fusion protocols used for hybridoma production (13), selection in HAT medium (DMEM containing 1 x 10-4 M hypoxanthine, 4 x 10-7 M aminopterin, 1.6 x 10-5 M thymidine), and cloning by limiting dilution. For induction of a heat shock response, 5 x 106 cells in 5 ml medium were incubated in 13-ml polypropylene tubes in a fine-regulated water bath (Julabo, Schütt, Göttingen, Germany) usually for 1 h at 42°C or 30 min at 44°C. For recovery, cells were cultured as described above.

Recombinant Hsp70

The rat Hsp70-1 gene (14) was amplified from LEW.1W rat (RT1u) DNA using specific primers including BamHI or HindIII restriction sites, respectively, in the 5' regions (5'-ATTGAATCCGCCAAGAAAACAGCGATCGGCAT-3' and 5'-CCAAAGCTTCTAATCCACCTCCTCGATGGTGG-3'). The PCR product was cloned in the pQE30 vector (Qiagen, Hilden, Germany) making use of the BamHI and HindIII restriction sites. The open reading frame starts with 6 N-terminal His codons. Correctness of the construct was confirmed by sequencing. Escherichia coli bacteria (strain M15, Qiagen) were transformed with this construct and cultured until an OD of 0.6–0.8 was reached, when the expression of the recombinant Hsp70 was induced by adding 1 mM isopropyl ß-D-thiogalactopyranoside (IPTG) (Biomol, Hamburg, Germany) for 5 h. Recombinant Hsp70 was present in inclusion bodies and purified by using a Ni-NTA-agarose column (Qiagen) according to the manufacturer’s instructions. Fractions containing recombinant Hsp70 were dialyzed against PBS at 4°C for 14–16 h. For certain experiments recombinant Hsp70 was FITC-labeled using the FluoReporter FITC labeling kit (F-6434, Molecular Probes, Mo Bi Tec, Göttingen, Germany) according to the manufacturer’s instructions. Bacteria containing recombinant His-tagged dynein were obtained from Dr. J. Neesen (Division of Human Genetics, University of Göttingen) and purified as above. Recombinant Hsp70 of human origin was purchased from StressGen (SPP-755, StressGen, Biomol). ß-Galactosidase (ß-gal) from E. coli (G-6008, Sigma, Deisenhofen, Germany) was used as further control protein.

Loading of cells with proteins by electroporation

For protein loading (15), 0.5 ml of HEPES-buffered DMEM/1% FCS containing 5 x 106 cells were pipetted into 0.4-cm gap electroporation chambers (peqlab, Erlangen, Germany) together with 50 µg protein dissolved in PBS (1 µg/µl) and exposed to electric pulses of 750 V/cm for 1.5 ms using an electroporation impulse generator (EPI 2500, Dr. L. Fischer, Heidelberg, Germany). Cells were then cultured as described above.

Cytotoxic cells

Allospecific CTL against Y3 cells or hybrid cell clones were generated by immunizing inbred BUF/Gun (RT1b) rats into the footpads with 15 x 106 LEW.1W/Gun (RT1u) spleen cells. Ten to 14 days later lymphocytes from regional lymph nodes were restimulated in vitro for 5 days by coculturing 0.75 x 106 responder cells/well of round-bottom microtiter plates (Nunc, Wiesbaden, Germany) with 0.75 x 106 irradiated (30 Gy) lymph node cells from LEW.1W/Gun rats in 200 µl of NaHCO3-buffered DMEM, supplemented with 10% FCS, pyruvic acid, penicillin, streptomycin, 10-5 M 2-ME, and 50 µl of supernatant from Con A-stimulated rat lymphocytes.

Chromium release assay

Target cells were labeled by incubating 1 x 106 cells in 350 µl HEPES-buffered DMEM containing 50 µl FCS and 50 µCi Na251CrO4 (ICN, Eschwege, Germany) for 1 h at 37°C and washed three times with HEPES-buffered DMEM. Effector cells were added to 104 51Cr-labeled target cells in triplicate at ratios of 100:1 to 1:1 in round-bottom microtiter plates in 200 µl HEPES-buffered DMEM/10% FCS per well, centrifuged for 5 min at 40 x g, and incubated at 37°C for 4 h. After centrifugation for 5 min, radioactivity was determined in supernatant and sediment separately for each well using a MicroBeta Trilux counter (Wallac, Freiburg, Germany). Percentage of specific lysis was determined by subtracting percent spontaneous 51Cr release, which was between 10 and 15%. The SD of specific lysis in triplicate cultures was usually below 5%. In inhibition experiments anti-CD8b mAb (clone 341, mouse IgG1) was added to the cytotoxicity test, with anti-{gamma}{delta}TCR mAb (clone V65, mouse IgG1) serving as isotype control. Both mAb were used as 1:20 or 1:100 diluted hybridoma supernatants (Dr. T. Herrmann, Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany). In some experiments 2 mM EGTA and 4 mM MgCl2 were added to inhibit calcium-dependent killing. Furthermore, the following inhibitors of caspases were used: Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-CH2F; inhibitor of caspase-1-like proteases, 40 µM), Ac-YVAD-CHO (acetyl-Tyr-Val-Ala-Asp-aldehyde; caspase-1 inhibitor, 100 µM), and Z-DEVD-FMK (Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK; caspase-3 inhibitor, 100 µM) (Calbiochem, Bad Soden, Germany).

Flow cytometry

Intracellular Hsp70 was determined by flow cytometry after cytoplasmic staining as described previously (16) and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using Lysis II or CellQuest software.

MHC class I cell surface expression was determined with anti-RT1.Au mAb (clone NR3/31, rat IgG2a, MCA12, Serotec, Biozol, Eching, Germany) at a dilution of 1:50 and anti-RT1.Ab mAb (clone B5, rat IgM, 22371D, PharMingen, Hamburg, Germany) at a concentration of 1 µg/1 x 106 cells. Cell surface expression of adhesion molecules CD54 (ICAM-1) and CD11a (LFA-1) was determined by mAbs 1A29 (mouse IgG, MCA773, Serotec, Biozol) and WT.1 (mouse IgG, MCA774, Serotec, Biozol), respectively, at a concentration of 1 µg/1 x 106 cells. Cells were washed twice with PBS in 5-ml polystyrene tubes, resuspended in 200 µl PBS containing the primary Ab, and incubated for 1 h at 4°C. After washing with PBS, cells were resuspended in 200 µl PBS containing as secondary reagents 2 µl of FITC-conjugated goat anti-mouse IgG (115-095-062, The Jackson Laboratory, Bar Harbor, ME; Dianova, Hamburg, Germany) or FITC-conjugated goat anti-rat IgG + IgM Igs (112-095-068, The Jackson Laboratory; Dianova). After incubation at 4°C for 1 h in the dark cells were washed twice with PBS, resuspended for 15 min in 500 µl PBS containing 10 µg/ml propidium iodide (PI) and analyzed. Cells not treated with Abs or with secondary reagent only served as controls. PI-positive dead cells were excluded from analysis.

The DNA content of hybrid cell clones and the rate of apoptotic cells appearing in the sub G1 peak of DNA histograms were determined as described (17). In some experiments the TUNEL test was perfomed in parallel to assess DNA fragmentation (APO-BrdU, PharMingen) according to the manufacturer’s instructions. A very good correlation of the TUNEL technique and sub-G1 peak determination was observed.

Exposure of phosphatidylserine as a membrane parameter of apoptosis was determined by staining cells in binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) with 5 µl annexin V-FITC or annexin V-PE (PharMingen) in combination with PI or 7-amino-actinomycin D to distinguish apoptotic from already dead cells.

Sorting of Hsp70-loaded cells

Cells loaded with FITC-labeled Hsp70 were separated into Hsp70-positive and a Hsp70-negative fractions by FACS (FACSVantage, Becton Dickinson).

Separation of apoptotic and nonapoptotic cells

Apoptotic cells were separated magnetically from nonapoptotic cells after staining with annexin V microbeads using the apoptotic cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Positively selected apoptotic cells and the negative fractions containing nonapoptotic cells were used as targets in the chromium release assay. Efficiency of selection was tested by flow cytometry as described above.

Immunoblot

Supernatant of cell lysates were prepared and separated by SDS-PAGE as described (16). Proteins were transferred to nitrocellulose (Schleicher & Schüll, Dassel, Germany) and stained with one of the following mAbs diluted 1:2000 in PBS/0.05% Tween 20: anti-Hsp70 (clone C92F3A-5, mouse IgG1, SAP-810, StressGen, Biomol), anti-Hsp70/Hsc70 (clone N27F3-3, mouse IgG1, SAP-820, StressGen, Biomol), anti-Grp75 (clone 30A5, mouse IgG1, SPA-825, StressGen, Biomol), anti-Hsp60 (clone LK-1, mouse IgG1, SPA-806, StressGen, Biomol), anti-RGS-His (mouse IgG1, 34610, Qiagen), and anti-ß-actin (clone AC-15, mouse IgG1, A-5441, Sigma). Subsequently blots were incubated with goat anti-mouse IgG Ig (115-005-003, The Jackson Laboratory; Dianova) and peroxidase-conjugated rabbit anti-goat IgG Ig (305-035-045, The Jackson Laboratory; Dianova) at dilutions of 1:10000. The substrate reaction was conducted with 0.05% 3,3'-diaminobenzidine/0.003% H2O2 in PBS/0.05% Tween 20.

Gene probes

Hybridization probes specific for MHC-linked rat Hsp70-1 (positions +2875 to +3070; GenBank accession no. X77207) and Hsp70-2 (positions +3174 to +3322; GenBank accession no. X77208) were derived from the 3' untranslated region of the respective genes by genomic PCR amplification. A probe containing the coding part of a human Hsp70 gene (18) and a Hsc70-specific probe (19) have been described previously. The human ß-actin cDNA was purchased from Clontech (Heidelberg, Germany).

Northern blot analysis

RNA was prepared according to Chomczynski and Sacchi (20), separated electrophoretically on denaturating 1.6% agarose gels, transferred onto nitrocellulose (Schleicher & Schüll), and cross-linked by UV irradiation. Blots were hybridized under stringent conditions with [32P]dCTP-labeled probes (16) and exposed to Hyperfilm MP (Amersham Pharmacia) at -70°C. Before rehybridization, blots were washed with 0.1% SDS at 95°C until complete removal of the probe.

Histochemical detection of ß-gal

Cells were fixed in 2% paraformaldehyde/PBS for 10 min, washed with PBS, and incubated for 14–18 h with 1 mg/ml 5-brom-4-chloro-3-indolyl ß-galactosidase (X-Gal), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl2 in PBS (21). At least 200 cells were counted microscopically to determine the percentage of blue-stained ß-gal-positive cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heat shock-induced resistance to CTL in Hsp70-defective Y3 cells

The effect of heat shock on the susceptibility of Y3 myeloma cells to allospecific CTL was tested in a 51Cr release assay (Fig. 1GoA). A representative experiment is shown in Fig. 1GoB. Heat shock of 1 h at 42°C or of 30 min at 44°C followed by a 6-h recovery period induced resistance to lysis, confirming data reported earlier (6). Y3 cells are Hsp70 defective and do not express Hsp70-1 and Hsp70-2 transcripts or protein after both heat shock protocols, whereas the activity of the constitutively expressed Hsc70 gene can be detected at the RNA and protein level (Fig. 1Go, C and D). Lysis of Y3 target cells by cytotoxic cells could be inhibited by anti-CD8b mAb (Fig. 2GoA). Thus, the predominant cytotoxic effector cells are CD8+ CTL. Because cytotoxic activity could also be inhibited almost completely by EGTA (Fig. 2GoB), killing is calcium dependent. Caspase inhibitors Z-VAD-FMK, Ac-YVAD-CHO, and Z-DEVD-FMK had no effect (Fig. 2GoB). Thus, the granzyme/perforine, but not the Fas, pathway seems to be predominant in killing Y3 cells.



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  		FIGURE 1. Resistance of Hsp70-defective Y3 rat myeloma cells to allospecific CTL after heat shock. A, Y3 cells were heat shocked (hs) for 1 h at 42°C or 30 min at 44°C and allowed to recover at 37°C for 6 h before being used as 51Cr-labeled target cells in a standard 4-h 51Cr release assay. Control cells were maintained at 37°C. B, Individual experiment that is representative for more than 30 independent tests. Mean and SD of triplicates are given. C, Northern blot analysis of Hsp70-1 and Hsp70-2 gene expression in Y3 cells before (37°C) or 6 h after heat shock (1 h at 42°C or 30 min at 44°C). Mitogen-stimulated lymphocytes (Con A-Ly) that were heat shocked for 1 h at 42°C are included as positive control and ß-actin hybridization as loading control. D, Immunoblot analysis of Hsp70 and Hsc70 expression in Y3 cells using mAb C92, which is specific for the inducible Hsp70, and mAb N27 detecting both Hsc70 and Hsp70. Hsc70 is characterized by a slightly higher m.w. so that two bands appear in heat-shocked lymphocytes but not in the Hsp70-defective Y3 cells.

 


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  		FIGURE 2. Characterization of the allospecific CTL used in 51Cr release assays. A, Specific lysis of Y3 target cells is inhibited by anti-CD8b mAb compared with the isotype control (Co). Both mAbs were used as 1:20 diluted hybridoma supernatants. Mean and SD of triplicates are given. B, Effect of 2 mM EGTA plus 4 mM MgCl2 (EGTA) and caspase inhibitors Z-VAD-FMK (VAD, 40 µM), Ac-YVAD-CHO (YVAD, 100 µM), and Z-DEVD-FMK (DEVD, 100 µM) on specific lysis of Y3 cells. Mean and SD of triplicates are given. The experiment shown was repeated three times with essentially the same results.

 
A strong non-Hsp70-inducing heat shock leads to resistance of Con A-stimulated lymphocytes

Normal and Con A-stimulated rat lymphocytes express Hsp70 after a heat shock of 1 h at 42°C, of 30 min at 43°C, or of 10 min at 44°C when assayed after recovery periods of 4–10 h by immunoblot analysis with mAb C92 specific for inducible Hsp70 (data not shown). Surprisingly, rat lymphocytes did not express Hsp70 following a heat shock of 30 min at 44°C (Fig. 3GoA). Therefore, lysability of Con A-stimulated lymphocytes by CTL was compared after a Hsp70-inducing heat shock (1 h at 42°C) and a non-Hsp70-inducing heat shock (30 min at 44°C). Resistance to CTL occurred after the non-Hsp70-inducing heat shock (Fig. 3GoB).



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  		FIGURE 3. Hsp70 expression and killing of mitogen-stimulated lymphocytes by allospecific CTL after heat shock. For the experimental scheme see Fig. 1GoA. A, Immunoblot analysis of Hsp70 expression with mAb C92. Lymphocytes, stimulated for 4 days with Con A, were heat shocked for 1 h at 42°C or 30 min at 44°C and allowed to recover at 37°C for 6 h before protein lysates were prepared. Control cells were maintained at 37°C. Loading of equivalent amounts of protein per lane has been controlled by immunoblot analysis with a ß-actin-specific mAb (not shown). B, 51Cr release assay with cells analyzed in A. Mean and SD of triplicates are given. The experiment was repeated four times with essentially the same results.

 
A non-Hsp70-inducing heat shock results in resistance of Y3 x lymphocyte somatic cell hybrids

To study further the effect of Hsp70 expression on heat shock-induced resistance, somatic cell hybrid clones between Y3 cells and syngeneic (LOU/CGun, RT1u) or allogeneic (BUF/Gun, RT1b) lymphocytes were established. The hybrid nature of the clones was proven by HAT resistance, increased DNA content, and, in the case of allogeneic hybrids, by the expression of partner-derived MHC Ags. The DNA content of hybrid cell clones was usually higher compared with parental Y3 cells (Table IGo). The presence of the MHC of the fusion partner is especially informative, because the two major heat-inducible Hsp70 genes Hsp70-1 and Hsp70-2 are localized in the MHC (22).


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  		Table I. Analysis of Y3 x rat lymphocyte hybrid cell clones for hybrid status (HAT resistance, DNA content, RT1.A cell surface expression), heat-induced Hsp70 RNA and protein expression, and heat-induced resistance to CTL

 
Two types of hybrids could be distinguished: hybrids that fail to express Hsp70 in general, and hybrids that express Hsp70 protein following a heat shock of 1 h at 42°C, but not after a heat shock of 30 min at 44°C (Table IGo). It is noteworthy that most hybrids showed Hsp70 expression at the mRNA level also after severe heat shock at 44°C (Table IGo). Thus, translation of Hsp70 mRNA appears to be more sensitive to severe heat shock than transcription.

The hybrid cell clones were tested for their susceptibility to allospecific CTL directed against the RT1u gene products. Each hybrid clone was susceptible to lysis by CTL and became resistant to CTL-mediated killing after a non-Hsp70-inducing heat shock (Table IGo). The most informative result was obtained with hybrid cell clones that express Hsp70 after a 42°C heat shock, but not after a 44°C heat shock. These cells developed no or only weak resistance after the Hsp70-inducing 42°C heat shock, but were clearly less susceptible to CTL after a heat shock of 44°C that did not induce Hsp70 (Table IGo). Hybrids that failed to express Hsp70 after a 42°C heat shock became resistant like Y3 cells (Table IGo). Thus, heat shock-induced resistance to CTL-mediated lysis appears to be inversely correlated with the presence of Hsp70 in the target cells. Other heat shock proteins such as Hsc70, Grp75, or Hsp60 were present to a similar degree in controls as well as after both types of heat shock treatment (data not shown).

Hsp70 loading abolishes resistance after heat shock

To analyze the effect of Hsp70 on heat shock-induced resistance to CTL directly, Y3 cells were loaded with recombinant rat Hsp70 protein, the gene product of the MHC-linked Hsp70-1 gene. Cells were loaded with proteins by electroporation, allowed to recover for 1.5 h and then heat shocked at 44°C for 30 min. After 2 h recovery cells were labeled with 51Cr for 1 h, washed, and exposed to CTL for 4 h (Fig. 4GoA). Y3 cells loaded with the control proteins ß-gal (Fig. 4Go, B and D) or His-tagged dynein (Fig. 4GoC) became resistant to CTL, whereas Y3 cells loaded with rat Hsp70 were almost as susceptible as non-heat-shocked cells (Fig. 4Go, B and C). The same effect was obtained with human instead of rat recombinant Hsp70 (Fig. 4GoD). Thus, prevention of heat shock-induced resistance is a specific effect of Hsp70. Efficient loading of Y3 cells with Hsp70 was proven by flow cytometry after intracellular staining (Fig. 5GoA) and immunoblot with Hsp70 (Fig. 5Go, B and C) or His-tag specific mAb (Fig. 5GoB). Loading of dynein protein was detected by immunoblot with anti-His-tag mAb (Fig. 5GoB), and ß-gal loading was monitored histochemically by cleavage of the substrate X-Gal (Fig. 5GoD).



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  		FIGURE 4. Effect of Hsp70 loading on the lysability of Y3 cells by allospecific CTL after heat shock. A, Protocol of the experiments. Y3 cells were loaded with Hsp70 or control proteins by electroporation. After a recovery of 1.5 h at 37°C, cells were heat shocked (hs) at 44°C for 30 min or kept at 37°C. Following a second recovery of 2 h cells were labeled with 51Cr for 1 h, washed, and used as targets in a standard 4-h 51Cr release assay. B, Effect of heat shock on specific lysis of Y3 cells loaded with ß-gal or rat Hsp70. C, Effect of heat shock on specific lysis of Y3 cells loaded with dynein or rat Hsp70. D, Effect of heat shock on specific lysis of Y3 cells loaded with ß-gal or human Hsp70. Each experiment is representative of at least four independent tests.

 


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  		FIGURE 5. Monitoring of loading Y3 cells with Hsp70 and control proteins by electroporation. A, Hsp70-loading of the Y3 cells used for the 51Cr release assay shown in Fig. 4GoB. Flow cytometric analysis after intracellular staining with mAb C92 (bold line); the fluorescence of control cells not loaded with Hsp70 (Co) is in the range of cells stained with the secondary Ab only (fine line). The specific increase of the mean fluorescence channel due to Hsp70 transfer was 297. B, Protein loading of cells used in the experiment of Fig. 4GoC as determined by immunoblot using Hsp70 (anti-Hsp70) or His-tag specific mAb (anti-His). Recombinant Hsp70 and dynein proteins were analyzed in parallel with cell lysates obtained after protein loading. C, Loading of Y3 cells used for the experiment shown in Fig. 4GoD with human Hsp70 (StressGen) as analyzed by immunoblot with Hsp70-specific mAb C92. D, Quantitative evaluation of ß-gal-positive cells used in the experiment of Fig. 4GoD. Efficiency of protein transfer was monitored in each cytotoxicity test and was always in the range of the experiments shown.

 
Indirect intracellular staining of Hsp70 (Fig. 5GoA) indicated that not all cells electroporated with Hsp70 might be loaded sufficiently. To separate loaded from unloaded cells by FACS sorting, FITC-labeled recombinant Hsp70 was used for loading. The fraction of positive cells was between 61 and 92% in six experiments. Positive and negative cells were sorted (Fig. 6GoA) in two independent experiments. Hsp70-negative cells developed resistance whereas Hsp70-postitive cells remained fully susceptible to CTL after heat shock (Fig. 6GoB). The use of FITC-labeled Hsp70 appeared to result in a higher percentage of loaded cells (61–92%) than the use of unlabeled Hsp70 (42–59%, see Fig. 5GoA). This discrepancy is most likely only apparent and due to the fact that in experiments where Hsp70 was detected indirectly the secondary Ab reacts strongly with Y3 cells. Therefore, the number of loaded cells is underestimated and in fact presumably in these experiments in the range of 80%. This assumption is supported by the efficiency of ß-gal loading being in the 80% range (Fig. 5GoD).



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  		FIGURE 6. Analysis of lysability by allospecific CTL after loading with FITC-labeled Hsp70 and cell sorting. A, Y3 cells were loaded with FITC-labeled recombinant Hsp70 and stained with PI. Control cells were loaded with unlabeled Hsp70. Hsp70-FITC loaded cells were FACS sorted using gates R1 and R2. These gates were defined by loading with unlabeled Hsp70 (left). About 10% of the cells stained with PI, although weaker than usually found for definitely dead cells. Nevertheless, this fraction was excluded from the two sorted populations. Because in this individual experiment the percentage of Hsp70-positive cells was rather low (61%), sufficient numbers of Hsp70-negative cells could be obtained for subsequent experiments. B, Both fractions were split and maintained at 37°C or heat shocked at 44°C for 30 min followed by a recovery period of 4 h before being used as targets of allospecific CTL in a 4-h 51Cr release assay. Mean and SD of triplicates are given.

 
Hsp70 loading does not affect expression of MHC class I and adhesion molecules

Heat shock for 30 min at 44°C lead to a slight decrease of MHC class I molecules (Table IIGo). Expression of adhesion molecules CD11a (LFA-1) and CD54 (ICAM-1) remained unaltered after heat shock (Table IIGo). Loading with Hsp70 or ß-gal did not affect MHC class I, CD54, and CD11a expression. Notably, the slight decrease of MHC class I cell surface expression occurring after severe heat shock (30 min at 44°C) was not reversed by Hsp70 loading (Table IIGo).


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  		Table II. Analysis of cell surface molecule expression and apoptosis in Y3 cells after heat shock and protein loading

 
Hsp70 loading does not protect against heat shock-induced apoptosis

The percentage of dead cells monitored by PI or trypan blue staining was low after both types of heat shock treatment (Table IIGo). Heat shock of 30 min at 44°C leads to more apoptotic cells than heat shock of 1 h at 42°C, as is evident from annexin V staining (Table IIGo), sub-G1 analysis (Table IIGo), and TUNEL test (data not shown). Loading of Y3 cells with Hsp70 or ß-gal did not affect the occurrence of heat shock-induced apoptosis when tested 6 h after protein loading and 4 h after heat shock. Thus, the recombinant Hsp70 does not confer resistance to heat shock-induced apoptosis in Y3 cells.

Heat shock-induced resistance occurs also in nonapoptotic Y3 cells

Annexin V-positive (apoptotic) and annexin V-negative (nonapoptotic) Y3 cells were separated magnetically after heat shock (Fig. 7Go, A and B) and used as target cells. Heat shock-induced resistance to CTL clearly occurred in Y3 cells depleted of apoptotic cells (Fig. 7GoC). Heat-shocked cells that were enriched for apoptotic cells became also more resistant to CTL than annexin V-positive, non-heat-shocked cells, but these experiments were less reliable due a high spontaneous 51Cr release of up to 35% (data not shown). Thus, heat shock-induced apoptosis and subsequent selective survival of nonapoptotic cells was not responsible for the heat shock-induced resistance to CTL.



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  		FIGURE 7. Susceptibility of heat-shocked and non-heat-shocked Y3 cells to CTL after depletion of apoptotic cells. For the experimental scheme, see Fig. 1GoA. A, Percentage of live (annexin V-negative/PI-negative, An-/PI-), apoptotic (An+/PI-), and dead (An+/PI+ or An-/PI+) Y3 cells kept at 37°C or exposed to 44°C for 30 min and determined at the beginning of the 51Cr release assay, i.e., 6 h after heat shock. Unseparated cells and cells depleted of annexin-positive cells 3.5 h after heat shock (An. neg.) were further analyzed. B, Flow cytometric analysis 10 h after heat shock, i.e., at the end of the 51Cr release assay. C, 51Cr release assay with unseparated Y3 cells and cells depleted of apoptotic cells (An. neg.) as target and allospecific CTL as effector cells. Mean and SD of triplicates is given. This experiment is representative of three independent tests.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cells of the rat myeloma line Y3 are defective in the expression of the major heat shock-induced Hsp70 genes that are encoded in the MHC. After heat shock, these cells become resistant to the cytotoxic effect of alloreactive CTL (6). We now show that Hsp70 is able to prevent the induction of the resistant phenotype. The evidence for this conclusion is threefold. 1) In Con A-stimulated lymphocytes and 2) in somatic cell hybrids between Y3 cells and lymphocytes heat shock-induced resistance is associated with the absence of Hsp70 expression. 3) Loading of Y3 cells with Hsp70 abrogates the development of resistance.

The failure of rat lymphocytes to express Hsp70 after severe heat shock (30 min at 44°C) has not been detected before. When carefully monitoring the actual temperature of the cell suspensions it turned out that a heat shock of 44°C did not induce Hsp70 any longer when applied for 30 min instead of 10 min. Transcription and translation are known to be reduced after heat shock except for heat shock genes (23). It appears that expression of Hsp70 is also lost when severe heat shock is applied. The same association between severity of heat shock and Hsp70 expression was found in somatic cell hybrids between Y3 cells and lymphocytes. In both systems, lymphocytes and somatic cell hybrids, inducibility of resistance to CTL by heat shock, and absence of Hsp70 expression were reproducibly correlated. It is noteworthy that in human lymphocytes Hsp70 induction could be observed under each of the heat shock conditions tested for rat lymphocytes including 30 min at 44°C (our unpublished data). However, after a more severe heat shock, e.g., 1 h at 44°C or 45°C, also in human lymphocytes Hsp70 induction was not observed any longer when assayed by flow cytometry (24).

To investigate whether indeed Hsp70 prevents the development of resistance to CTL Y3 cells were loaded with recombinant Hsp70 by electroporation before heat shock treatment. Furthermore, Y3 cells were sorted into Hsp70-positive and Hsp70-negative fractions after loading with FITC-labeled Hsp70. Also, in these experiments the presence of Hsp70 abrogated the development of heat shock-induced resistance to CTL. The recombinant Hsp70 used for supplementation is encoded by the MHC-linked Hsp70-1 and Hsp70-2 genes that are not expressed in Y3 cells. The supplementation effect is not due to loading with proteins in general and is not caused by the His-tag of the recombinant Hsp70 or by contaminating proteins, because control proteins used for loading by electroporation did not affect the development of heat shock-induced resistance. Recombinant His-tagged dynein, which was prepared by the same procedure as recombinant His-tagged Hsp70 and ß-gal protein did not affect the resistance phenotype. On the other hand, human Hsp70 that is 97% identical to rat Hsp70 at the amino acid level (22) and does not contain a His-tag had the same effect as rat Hsp70. Thus, it can be concluded that the products of the MHC-linked heat inducible Hsp70 genes are involved in regulating lysability of target cells by CTL.

It is unlikely that the slight decrease of MHC class I expression on Y3 cells after severe heat shock (30 min at 44°C) is responsible for the resistance phenomenon. Hsp70 loading prevented resistance after heat shock without reverting the slightly decreased MHC class I expression. In addition, in cold target inhibition experiments Y3 cells made resistant by heat shock were as effective as nonresistant cells (Ref. 6 and our unpublished observations). A decrease of MHC class I molecules has been described to occur in some human melanoma cell lines after long and severe heat shock treatment without effect on susceptibility to NK cell cytotoxicity (25). Furthermore, adhesion molecules CD11a and CD54 are not responsible for resistance of Y3 cells after heat shock, because their cell surface expression was not changed.

The lysis-promoting effect reported here for Hsp70 is at variance with the commonly described protective effect of heat shock proteins in general and of Hsp70 in particular when cells are exposed to cytotoxic mechanisms (8, 26, 27, 28). However, some reports show that heat shock can also increase susceptibility to lysis by NK or LAK cells (29, 30). A death-promoting effect of Hsp70 itself has been described after TCR/CD3 or CD95 activation in Hsp70-transfected Jurkat cells (31). In acute myeloid leukemia cells apoptosis correlated with the intracellular Hsp70 level (32). Thus, the finding reported here is not without precedent. Together, these data challenge the view that Hsp70 is generally a cell death-preventing protein (8). A particular mechanism of how Hsp70 can increase target cell lysis has been described for some tumor cell lines that express Hsp70 on their cell surface. Recognition and lysis by NK cells is then improved (33). The target cells studied here did not express Hsp70 on the cell surface and were not susceptible to NK or LAK cell cytotoxicity (our unpublished data).

The allospecific cytotoxic cells that mediate lysis of the Y3 cells and somatic cell hybrids used in this study are CTL as is evident from inhibition by anti-CD8 mAb. The main mechanism of killing appears to be mediated by the granule exocytosis pathway, because it is calcium dependent and not affected by caspase inhibitors. Exocytosis of CTL granules is generally thought to induce apoptosis in the target cell (34). Granzymes are assumed to activate late phases of apoptosis signaling. The pathway that actually leads to cell death appears to be independent of caspases, but the proteins involved are not yet known (35). Granzyme B has been shown to cleave directly several downstream caspase substrates (36), and granzyme A induces a distinct pathway of apoptosis in target cells (37, 38).

We hypothesize that Hsp70 interferes with apoptotic pathways that are induced in the target cells by CTL due to granule exocytosis. Functioning as a molecular chaperone Hsp70 might "protect" certain proteins that are necessary for granule-mediated killing of the target cell. If Hsp70 is lacking, the respective protein might loose its function due to heat shock-mediated denaturation. Killing by CTL is then impaired, so that the resistant phenotype occurs. It is of interest that granzyme A has been shown to be bound by Hsp27 and Hsp70 (39), indicating that Hsp70 indeed is involved in CTL-induced cell death. In Y3 cells Hsp70 does not protect against apoptosis that is induced by heat shock, as is shown in our experiments (Table IIGo), but only against apoptosis that is additionally elicited by CTL. This observation implies that the pathway of CTL-induced cell death should differ from that of heat shock-induced apoptosis. In additional experiments relevant target proteins of Hsp70 during CTL-induced cell death will have to be identified.

Our findings may be relevant for the interpretation of some experimental tumor models. Because of its protective role, Hsp70 is expected to favor tumor growth, as has indeed been shown (40). On the other hand, Hsp70 expression was found associated with enhanced tumor immunogenicity and regression in vivo (41, 42, 43). Furthermore, Hsp70, among other chaperones, has been shown to bind antigenic peptides and to elicit tumor immunity after vaccination (44). The chaperoned peptides can be channeled into the endogenous class I presentation pathway of APC (45), which are then able to prime CD8+ CTL. Thus, tumors might elicit a specific immune response by Hsps released from dying tumor cells. One might expect that a cytoprotective effect of Hsp70 would counteract the effectiveness of the CTL response. This apparent contradiction my be resolved by the data reported here, showing that Hsp70 need not confer protection against the CTL-mediated cytotoxicity but instead can improve lysability of target cells after stress.


   Acknowledgments
 
We thank Dr. J. Neesen (Division of Human Genetics, University of Göttingen, Göttingen, Germany) for bacteria producing the recombinant dynein, Dr. T. Herrmann (Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany) for providing the anti-CD8b and anti-{gamma}{delta}TCR mAb, Dr. K.-P. Hermann and colleagues (Division of Medical Physics and Biophysics, University of Göttingen) for irradiating cells, and M. Podleschny and Dr. F. Griesinger (Division of Hematology and Oncology, University of Göttingen) for FACS sorting. The expert assistance of E. Munk during the animal experiments is gratefully acknowledged.


   Footnotes
 
1 This work was supported by Grant SFB 500 from the Deutsche Forschungsgemeinschaft. T.Q. was suppported by a Lower Saxony/Israel joint project. Back

2 Address correspondence and reprint requests to Dr. Eberhard Günther, Division of Immunogenetics, University of Göttingen, D-37073 Göttingen, Germany. E-mail address: Back

3 Abbreviations used in this paper: Hsp, heat shock protein; ß-gal, ß-galactosidase; Grp, glucose-regulated protein; Hsc, heat shock cognate protein; HAT; hypoxanthine, aminopterin, thymidine; MIF, mean intensity of fluorescence; PI, propidium iodide; X-Gal, 5-brom-4-chloro-3-indolyl ß-galactosidase; Z-VAD-FMK, Z-Val-Ala-Asp (OMe)-CH2F; Ac-YVAD-CHO, acetyl-Tyr-Val-Ala-Asp-aldehyde; Z-DEVD-FMK, Z-Asp (OCH3)-Glu (OCH3)-Val-Asp(OCH3)-FMK Back

Received for publication August 2, 1999. Accepted for publication December 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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