The regulatory role of heat shock protein 70 ... - Semantic Scholar

International Immunology, Vol. 10, No. 2, pp. 117–130

© 1998 Oxford University Press

The regulatory role of heat shock protein 70-reactive CD4F T cells during rat listeriosis Yuki Kimura1,6, Katsuo Yamada1,2, Tetsu Sakai3, Kenichi Mishima1, Hitoshi Nishimura1, Yoshihiro Matsumoto4, Mahavir Singh5 and Yasunobu Yoshikai1 1Laboratory

of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan 2Department of Thoracic Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan 3First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan 4Department of Internal Medicine, Nagoya University Branch Hospital, 1-1-20 Daiko-minami, Higashi-ku, Nagoya 461, Japan 5GBF-German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany 6Present

address: Departments of Medical Biophysics and Immunology, The Ontario Cancer Institute and The Amgen Institute, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2C1, Canada

Keywords: HSP70, IL-10, Listeria monocytogenes, rat, regulatory T lymphocytes, transforming growth factor-β

Abstract Protection against infection with Listeria monocytogenes depends primarily on Listeria-specific T cells. We show here that CD4F TCRαβF T cells are capable of recognizing the mycobacterial heat shock protein (HSP) 70, that appears in the peritoneal cavity of F344 rats infected i.p. with L. monocytogenes. The HSP70-reactive CD4F T cells recognized a peptide comprising 234–252 residues as present in the 70 kDa HSP of Mycobacterium tuberculosis in the context of RT1.B MHC class II molecules. Analysis of TCR Vβ gene expression with RT-PCR revealed that the HSP70reactive CD4F T cells predominantly used the Vβ16 gene segment, whereas the heat-killed Listeria (HKL)-specific T cells expressed a diverse set of Vβ gene segments. In contrast to the HKL-specific T cells producing IFN-γ, the HSP70-reactive CD4F T cells produced TGF-β1 and IL-10 but neither Th1- or Th2-type cytokines. Adoptive transfer with HSP70-reactive T cells rendered rats susceptible to listerial infection. Collectively, these results proposed that the HSP70-reactive CD4F T cells appearing during rat listeriosis may be involved in termination of Th1 cell-mediated excessive inflammation after the battle against L. monocytogenes has been won.

Introduction Heat shock proteins (HSP) are widely distributed in nature, and are highly conserved proteins among prokaryotes and eukaryotes (reviewed in 1). They are known to be involved in immune responses to many bacterial and parasitic pathogens (reviewed in 2–4), and in the pathogenesis of some types of autoimmune diseases (reviewed in 5). Among various HSP, mycobacterial 65 kDa HSP (HSP65) has been identified as an immune dominant target (6,7). TCRγδ1 T cells capable of

recognizing HSP65 are reported to respond predominantly to infection with various microbial pathogens (8). We have previously reported that the TCRγδ1 T cells appeared in the peritoneal cavity after i.p. infection with Listeria monocytogenes (9,10), Salmonella sp. (11,12) and Mycobacterium bovis Bacillus Calme´tte Gue´rin (BCG) (13) in mice. A significant fraction of TCRγδ1 T cells appeared to respond to HSP65 (14–16) and to play a protective role in bacterial infection via Th1-type

Correspondence to: Y. Yoshikai Transmitting editor: S. H. E. Kaufmann

Received 21 April 1997, accepted 15 October 1997

118 HSP70-reactive CD41 T cells during rat listeriosis cytokine production (10). TCRαβ1 T cells recognizing an epitope of HSP65 are reported to cause autoimmune diseases such as adjuvant arthritis (AA) (17) and non-AA in mice (18) and rats (7,19), while TCRαβ1 T cells recognizing a different epitope of HSP65 are suggested to modulate autoimmune diseases in rats (20). Thus, similar to T cells recognizing conventional antigens, HSP65-reactive T cells may be classified into several subsets in terms of functionality. The HSP70 family, one of the most abundant of various HSP (reviewed in 1 and 21), is also known to be associated with T cell-mediated immune responses. A member of the HSP70 family, heat shock cognate protein 70 (HSC70), which is constitutively expressed, plays an essential role as a molecular chaperone in the folding of newly synthesized or invaded proteins in cells (22). There have been several lines of evidence that HSC70 is involved in T cell recognition, acting as a presenting molecule complexed with cellular peptides to the T cells (23,24). Although anti-HSP70 antibodies are reported to be present in systemic lupus erythematosus (25), there are few reports concerning T cell recognition of HSP70 as target ligands. Thus, functions of such T cells remain unknown. L. monocytogenes, the intracellular pathogens, have been widely used for analyzing cell-mediated immune responses (26). A specific immune response is triggered by TCRαβ1CD41 Th1 cells, producing IFN-γ, which then leads to sterile eradication of Listeria (26–28). On the other hand, CD41 Th2 cells producing IL-4 are thought to mediate the opposite effect, leading to an impaired resistance to such a microbe. We have recently developed a rat model for listeriosis and found that TCRγδ1 T cells appeared in the peritoneal cavity of rats at the early stage after an i.p. inoculation of L. monocytogenes (29–31). In the present study, we found that HSP70-reactive CD41 T cells appeared in the peritoneal cavity during the course of rat listeriosis. The HSP70-reactive CD41 T cells produced significant amounts of transforming growth factor (TGF)-β1 and IL-10, and suppressed the resistance against listerial infection. The implications of these findings for the regulatory role of the HSP70-reactive CD41 T cells during rat listeriosis are discussed.

shown). CD31CD41 Vβ161 cells were sorted with Epics Elite ESP (Coulter, Miami, FL). Their purity was 99.3% as accessed by FACS analysis (data not shown).

Antibodies FITC-conjugated anti-TCRγδ mAb (V65), FITC-conjugated antiVβ16 mAb (HIS42), biotin-conjugated anti-CD3 mAb (G4.18), anti-RT1.A mAb (OX-18), anti-RT1.B mAb (OX-6) and antiRT1.D mAb (OX-17) were purchased from PharMingen (San Diego, CA). FITC-conjugated anti-NKR-P1 mAb (3.2.3), FITCconjugated anti-CD8 mAb (OX-8), FITC-conjugated anti-IL2Rα mAb (OX-39), phycoerythrin-conjugated anti-TCRαβ mAb (R73) and phycoerythrin-conjugated anti-CD4 mAb (W3/25) were purchased from Serotec (Oxford, UK). Anti-rat class I mAb (R4-8B1) was purchased from Seikagaku (Tokyo, Japan). Anti-nitrophenol mAb (H1-6-2) was obtained from the supernatants of H1-6-2 hybridoma cells as described previously (31). Anti-RT1.A mAb, anti-RT1.B mAb, anti-RT1.D mAb, anti-rat class I mAb and anti-nitrophenol mAb were dialyzed to remove sodium azide before culturing. The concentrations of all dialyzed mAb were determined by the Lowry method.

HSP and peptides

Methods

rHSP70 derived from M. tuberculosis was obtained from Escherichia coli K12 strain M1485, which carries plasmid Y3111/8 (32,33). rHSP65 derived from M. bovis BCG was kindly provided by Drs J. Kamerbeek and J. D. A. van Embden (National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands). Human rHSP70 and rHSP60 were purchased from StressGen (Victoria, Canada). All rHSP were passed through the Detoxi-Gel endotoxin removing gel (Pierce, Rockford, IL) to remove lipopolysaccharide (LPS). The LPS concentrations of the columnpassed rHSP70s, rHSP65 and rHSP60 were determined to be ,1 ng/mg of protein, according to the Limulus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, MD). The purities of these protein preparations were . 98% pure as determined by SDS–PAGE. The concentrations of the columnpassed rHSP were determined by the Lowry method. Synthetic peptides of the HSP70 of M. tuberculosis, which were synthesized using a method of simultaneous multiple peptide synthesis and were purchased from Kurabo (Osaka, Japan).

Animals and bacteria

Cell culture

Male F344 rats were purchased from Japan Clea (Shizuoka, Japan). L. monocytogenes strain EGD and heat-killed Listeria (HKL) were prepared as described previously (29).

Triplicate cultures of the enriched subset cells (7.53105 cells/ ml) were cultured with the same number of 30 Gy-irradiated normal syngeneic splenocytes as antigen-presenting cells (APC) in the presence or absence of antigens and/or mAb in 96-well flat-bottom plates (Falcon, Becton Dickinson, Lincoln Park, NJ) for proliferation assay, or in 24-well plates (Falcon) for flow cytometric analysis for 72 h. RPMI 1640 complete culture medium contained 10% fetal bovine serum (CSL Limited, Victoria, Australia), 100 U/ml penicillin, 100 mg/ml streptomycin and 10 mM HEPES. Antigen and mAb were added to the culture as follows: 25 µg/ml rHSP70 derived from M. tuberculosis, 25 µg/ml rHSP70 derived from human, 25 µg/ml rHSP65 derived from M. bovis, 25 µg/ml rHSP60 derived from human, 25 mg/ml each peptide, 53107 cells/ml HKL, 10 µg/ml anti-RT1.A mAb, 10 µg/ml anti-class I mAb, 10 µg/ml anti-RT1.B mAb, 10 µg/ml anti-RT1.D mAb and

Preparation of cells Rats were inoculated i.p. with 23107 viable L. monocytogenes (1/15 LD50) on day 0. Uninfected control rats were used in parallel. Non-adherent peritoneal exudate cells (PEC), liver lymphocytes and spleen cells were prepared from rats on day 0 and on day 10 after listerial infection as described previously (30). To purify each subset of T cells, the cells were passed over the Cellect rat T cell, CD8 cell and CD4 cell kit columns (Biotex, Alberta, Canada). After the cells were passed through T cells, CD8 cells and CD4 cells kit columns, it was found that there were .90% CD31, 87.3% CD81 and 85.6% CD41 cells as assessed by FACS analysis (data not

HSP70-reactive CD41 T cells during rat listeriosis 119 10 µg/ml isotype-matched anti-nitrophenol mAb as a control. For the last 18 h, triplicate cultures of cells were pulse-labeled with [3H]thymidine (1 µCi/well; Amersham Life Science, Amersham, UK) before harvesting on glass fiber filters. Incorporation of [3H]thymidine was determined by a liquid scintillation counter.

Flow cytometric (FCM) analysis For three-color FCM analysis, cells were stained with FITCconjugated anti-TCRγδ mAb, FITC-conjugated anti-NKR-P1 mAb, FITC-conjugated anti-CD8 mAb, FITC-conjugated anti-Vβ16 mAb, or FITC-conjugated anti-IL-2Rα mAb, phycoerythrin-conjugated anti-TCRαβ mAb or phycoerythrinconjugated anti-CD4 mAb, and biotin-conjugated anti-CD3 mAb followed by streptavidin–Red670 (Gibco/BRL, Gaithersburg, MD). In all cases, cells were preblocked with heat-inactivated normal syngeneic serum to block non-specific staining before the analysis. The stained cells were analyzed by a FACS flow cytometer (Becton Dickinson, Oxnard, MA).

RT-PCR and Southern blotting for cytokine and TCR Vβ gene expressions Cells were harvested after culture with antigens and thoroughly washed. Then, T cells were enriched from cultured cells using the Cellect rat T cell kit column. mRNAs were extracted from the enriched T cells by using the QuickPrep Micro mRNA purification kit (Pharmacia Biotech, Milwaukee, WI). cDNAs were synthesized with 1 µg of each mRNA in a total volume of 20 µl containing 10 mM Tris–HCl, 8 mM MgCl2, 50 mM KCl, 0.01% gelatin, 500 mM each dNTP, 1 mM dithiothreitol and 1.5 µg of random primers (BRL, Gaithersburg, MD). Then 1 µl of 200 U SuperScript II reverse transcriptase (Gibco/BRL) was added to this mixture and incubated at 37°C for 60 min. The reaction was stopped at 95°C for 5 min and then quenched at 4°C. Products were diluted to a final volume of 100 µl. An aliquot of the synthesized first-strand cDNA was amplified by means of PCR using 40 pmol of each primers with 2.5 U of AmpliTaq (Takara Shuzo, Kyoto, Japan) in a total volume of 100 µl reaction buffer consisting of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin and 200 µM dNTP. PCR cycles were run for 1 min at 94°C, followed by 2 min at 60°C and 2 min at 72°C with 22–30 cycles. For an IL-10 PCR reaction, an annealing temperature reaction was done at 70°C. Five microliters of PCR products were electrophoresed on a 1.8% agarose gel, and visualized with ethidium bromide and transferred to GeneScreen Plus (New England Nuclear, Boston, MA). Southern blot analyses were performed with cytokinespecific oligonucleotide probes, which were labeled with [γ-32P]ATP (Amersham Life Science) using the Megalabel 59 labeling kit (Takara Shuzo, Ohtsu, Japan), according to the manufacturer’s instructions. After hybridization for 18 h at 60°C in 1% SDS, 1 M NaCl, 10% dextran sulfate and 100 µg/ml heat-denatured salmon sperm DNA, the filters were washed in 23SSC (0.3 M NaCl, 0.03 M sodium citrate) and 1% SDS and exposed to X-ray film at –70°C with intensifying screens. To ensure that equivalent amounts of cDNA were used in each reaction, PCR was also performed for β-actin from each sample and the cDNA was adjusted to equivalent levels. All primers and probes were designed from published

sequence (34–39). The used primer sequences were as follows: β-actin sense, 59-AGAAGAGCTATGAGCTGCCTGACG-39, and antisense, 59-CTTCTGCATCCTGTCAGCCTACG-39, which produce a 236 bp fragment; IL-4 sense, 59-ACCTTGCTGTCACCCTGTTCTGC-39, and antisense, 59GTTGTGAGCGTGGACTCATTCACG-39, which produce a 352 bp fragment; IFN-γ sense, 59-ACACTCATTGAAAGCCTAGAAAGTCTG-39, and antisense, 59-ATTCTTCTTATTGGCACACTCTCTACC-39, which produce a 432 bp fragment; IL-2 sense, 59-AACAGCGCACCCACTTCAA-39, and antisense, 59TTGAGATGATGCTTTGACA-39, which produce a 400 bp fragment; IL-15 sense, 59-GTGATGTTCACCCCAGTTGC-39, and antisense, 59-TCACATTCTTTGCATCCAGA-39, which produce a 181 bp fragment; TGF-β1 sense, 59-CTTTAGGAAGGACCTGGGTT-39, and antisense, 59-CAGGAG-CGCACAATCATGTT-39, which produce a 257 bp fragment; IL-10 sense, 59-TGCCAACCCTTGTCAGAAATGATCAAG-39, and antisense, 59-GTATCCAGAGGGTCTTCAGCTTCTCTC-39, which produce a 127 bp fragment. The oligonucleotide probes were as follows: β-actin, 59-CTATCGGCAATGAGCGGTTC-39; IL-4, 59-ATGCACCGAGATGTTTGTACC-39; IFN-γ, 59-CTCCTTTTCCGCTTCCTTAG-39; IL-2, 59-CACTGAAGATGTTTC-39; IL-15, 59-GCAATGAACTGCTTTCTCCT-39; TGF-β1, 59-ACCTTGCTGTACTGTGTGTC-39; IL-10, 59-GGCCATTCCATTCCATCCGGGGTGACAATA-39. To determine Vβ usage, 2 µl of cDNA products was transferred to individual tubes that contained one of the Vβ oligonucleotide primers, Cβ1 primer (40), and other components that were the same as described above. PCR cycles were run for 1 min at 94°C, followed by 1.5 min at 55°C and 2 min at 72°C with 26–30 cycles. Electrophoresis and Southern blotting of PCR products were carried out as described above. The used oligonucleotide probe was designed based on the known sequence of the rat Cβ region (41) (at 59 side from the Cβ1 primer) using the OLIGO computer program (National Biosciences, Plymouth, MN): CβP, 59-CGGTGACTCCACCCAAGGTC-39.

Assay for cytokines Culture supernatants were harvested after 72 h incubation time. IFN-γ, IL-10 and TGF-β1 activities in the culture supernatants were measured by ELISA using the rat IFN-γ ELISA kit (Gibco/BRL), rat IL-10 ELISA kit (Bioscore International, Carmarillo, CA) and TGF-β1, human, ELISA system (Amersham Life Science) respectively. Adoptive transfer of T cell lines Peritoneal T cells, derived from rats on day 10 after listerial infection, were stimulated for 3 days with rHSP70 derived from M. tuberculosis, or HKL, or without antigens in the presence of irradiated normal syngeneic splenocytes as APC. Then T cell lines were passed over the Cellect rat T cell kits columns (Biotex). Enriched T cell lines (1.53106) in PBS or PBS alone were administered i.p. to the rats. Immediately following administration, the rats were injected i.p. with 13108 viable L. monocytogenes on day 0. Bacterial growth in the peritoneal cavities was examined on day 6 after listerial infection. Statistical analysis Data were analyzed by using Student’s t-test. Values of P , 0.05 were considered statistically significant. All experiments were performed at least twice.

120 HSP70-reactive CD41 T cells during rat listeriosis Results

HSP70-reactive T cells appear during rat listeriosis To determine whether HSP-reactive T cells can be primed during listerial infection in F344 rats, we examined the proliferative responses of the peritoneal T cells against mycobacterial rHSP70, rHSP65 or HKL. Enriched T cells (.90% CD31) in nonadherent PEC from uninfected rats (on day 0), and also from rats infected i.p. with L. monocytogenes 10 days previously (on day 10), were used as responder cells, and rHSP70, rHSP65 or HKL, while irradiated normal syngeneic spleen cells were used for antigen and APC respectively. As shown in Fig. 1(A), the peritoneal T cells of rats, which were infected with L. monocytogenes 10 days previously, showed significant response to rHSP70 (P , 0.001), and to a lesser extent, to rHSP65 (P , 0.05). The proliferative response of the peritoneal T cells to rHSP70 was detected at the maximum level after 72 h culture and in a dose-dependent manner (data not shown). The HSP70 reactivity was detected from day 3 to day 30 after listerial infection and the maximal response was observed in the peritoneal T cells on day 10 (data not shown). HSP are highly conserved proteins among prokaryotes and eukaryotes (reviewed in 1), and there is a report of crossreactive T cell recognition of mycobacterial HSP65 and self-HSP60 (20). To investigate whether mycobacterial HSPreactive T cells can cross-react to mammalian HSP, we then examined the proliferation of peritoneal T cells in response to human rHSP70 or rHSP60. As shown in Fig. 1(B), the peritoneal T cells showed significant proliferation in response to an optimal dose of human rHSP70 (P , 0.001), whereas they showed only two-fifths of the optimal response to mycobacterial rHSP70 (P , 0.05), while the T cells did not respond to human rHSP60. To investigate whether HSP70-reactive T cells were detected in the other organs, we attempted to detect such HSP70 reactivity of T cells from the mesenteric lymph nodes and the liver of rats, which were infected with Listeria 10 days previously. A lower but significant level of HSP70 reactivity was also detected in enriched T cells from the liver than compared with those in the peritoneal cavity (unpublished data). We focused on the HSP70-reactivity of the peritoneal T cells during rat listeriosis in subsequent experiments.

Cell surface phenotypes of HSP70-reactive T cells during listeriosis Using the same conditions as the proliferation assay, we next examined the cell surface phenotype on the proliferating T cells after in vitro culture with rHSP70. As shown in Fig. 2, the T cells bearing CD4 and TCRαβ chains but no NKR-P1 (rat NK marker) appeared to be the main cells expanding in response to rHSP70 in vitro. In contrast to HKL-specific T cells expressing IL-2Rα, most of T cells responding in vitro to rHSP70 did not bear IL-2Rα (Fig. 2). Each of the rHSP70and HKL-stimulating T cells were of the CD441CD45RC– phenotype, which indicated that both T cells are in an activated state (data not shown).

HSP70-reactive T cells require MHC class II for antigen recognition To assess which sub-population of T cells can recognize HSP70, we examined the proliferative responses of purified

CD41 and CD81 cells in the peritoneal cavities of infected rats to rHSP70 in vitro. CD41 cells (.85.6% CD41) and CD81 cells (.87.3% CD81) were purified from non-adherent PEC after listerial infection as described in Methods, and were used as responder cells. Consistent with earlier findings (44), both CD41 cells and CD81 cells proliferated in response to HKL, while only CD41 T cells did so in response to mycobacterial rHSP70 (Fig. 3). Next, we examined the effect of various anti-MHC class I and class II mAb on the HSP70 reactivity of the peritoneal CD41 cells from rats infected with L. monocytogenes 10 days previously. As shown in Fig. 4, the proliferative response of the peritoneal T cells to rHSP70 was significantly inhibited by addition of anti-RT1.B mAb (OX-6) (P , 0.001 versus without mAb), but not by anti-RT1.D mAb (OX-17), anti-RT1.A mAb (OX-18) or anti-class I mAb (R4-8B1) (not significant versus without mAb). Thus, these results suggest that the HSP70-reactive CD41 T cells appearing during rat listeriosis may recognize HSP70 in context of RT1.B (identical to mouse I-A) on APC.

TCR Vβ usage of HSP70-reactive CD41 T cells We next addressed the TCR Vβ usage of HSP70-reactive CD41 T cells by RT-PCR and Southern blotting. After in vitro culture with rHSP70 or HKL, the proliferating T cells were harvested and then mRNA was extracted as described in Methods. As shown in Fig. 5(A), the HSP70-reactive CD41 T cells used limited Vβ gene segments such as Vβ15 and Vβ16. In contrast, the repertoire of the HKL-specific T cells was diversified. Because only mAb against rat Vβ16 (HIS42) is available, we focused on Vβ161 T cells to confirm Vβ repertoire RT-PCR and Southern blotting analysis. Consistent with RT-PCR and Southern blotting analysis (Fig. 5A), FACS analysis revealed that the ratio of Vβ161 to Vβ16– of HSP70-reactive CD41 T cells increased after in vitro culture with rHSP70, while it did not change before and after culture in vitro with HKL (Fig. 5B). To further confirm that Vβ16 T cells are involved in HSP70reactivity, we sorted Vβ161 cells from non-adherent PEC of rats infected with Listeria 10 days previously by using an Epics Elite (Coulter) and examined their reactivity to rHSP70. As shown in Fig. 6(B), CD31CD41 Vβ161 (.99.3% Vβ161 in Fig. 6A) cells responded in vitro to rHSP70 but not HKL.

Identification of HSP70 T cell epitope peptide To identify the T cell epitope peptide in HSP70 of M. tuberculosis, we examined the reactivity of the peritoneal T cells of infected rats to the peptides as present in mycobacterial HSP70. Since MHC class II RT1.B, which is identical to the mouse I-A molecule, is involved in the recognition of the HSP70-reactive CD41 T cells (Fig. 4), we synthesized five peptides encompassing the residues which can bind to mouse I-Ad molecule as assessed by T Sites software (MedImmune by David C. Feller and Vidal F. de la Crutz). As shown in Fig. 7, the peritoneal T cells can proliferate to an optimal dose of a peptide representing the amino acid sequence 234–252, which is at a comparable level to the rHSP70. This peptide differs from the equivalent rat HSP70 by 8 amino acid substitution and shares 58% amino acid identity with rat HSP70. Thus, these results indicate that at

HSP70-reactive CD41 T cells during rat listeriosis 121

Fig. 1. Proliferative response of the peritoneal T cells in naive and Listeria-infected rats to rHSP70, rHSP65 or HKL. (A) Enriched peritoneal T cells (7.53105 cells/ml; .90% CD31), which were prepared from rats on day 0 (naive) and on day 10 after an i.p. infection with 23107 L. monocytogenes, were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis), rHSP65 (25 µg/ml; derived from M. bovis) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. (B) Cross-reactivity of peritoneal T cells in Listeria-infected rats to rHSP70s derived from bacteria or human. Enriched peritoneal T cells (7.53105 cells/ml; .90% CD31), which were prepared from rats on day 10 after an i.p. infection with 23107 L. monocytogenes, were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis or human), rHSP65 or rHSP60 (25 µg/ml; derived from M. bovis or human respectively), or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. The c.p.m. for [3H]thymidine incorporation was measured in triplicate cultures and the results are means 6 SD. The stimulation index was calculated as (c.p.m. after incubation with rHSP or HKL)/(c.p.m. medium only) (medium only range 1500 6 500 c.p.m.). *P , 0.05; **P , 0.001; NS, not significant.

122 HSP70-reactive CD41 T cells during rat listeriosis

Fig. 2. FACS analyses of surface phenotypes of peritoneal T cells stimulated with rHSP70 or HKL in vitro. Enriched T cells (7.53105 cells/ml; .90% CD31) from rats infected with L. monocytogenes 10 days previously were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. The pre- and post-cultured cells were incubated for 15 min on ice with heat-inactivated normal syngeneic serum, and then were stained with FITC-conjugated anti-TCRγδ mAb (V65), FITC-conjugated anti-CD8 mAb (OX-8), FITC-conjugated anti-NKR-P1 mAb (3.2.3), or FITC-conjugated anti-IL-2Rα mAb (OX-39), phycoerythrin-conjugated anti-TCRαβ mAb (R73) or phycoerythrin-conjugated anti-CD4 mAb (W3/25) and biotin-conjugated anti-CD3 mAb (G4.18) followed by streptavidin–Red670. The profiles of TCRγδ, CD8, NKR-P1 or IL-2Rα and TCRαβ or CD4 expressions are displayed after gating on the CD31 cells.

least a significant fraction of the HSP70-reactive T cells can be specialized to recognize an epitope peptide of the HSP70.

Cytokine expression by HSP70-reactive CD41 T cells To gain insight into the functions of HSP70-reactive CD41 T cells appearing during listeriosis, we analyzed the gene expression of IFN-γ, IL-4, IL-2, IL-15, TGF-β1 and IL-10 of the proliferating T cells after in vitro culture with rHSP70. The amount of cDNA was adjusted by PCR. The Southern hybridization analyses of serially diluted cDNA with β-actin primers, as described in Methods, and expression of mRNA specific for these cytokines was analyzed by RT-PCR. As shown in Fig. 8(A), the HKL-reactive T cells expressed mRNA specific for IFN-γ and IL-2, but not specific for IL-4, thereby suggesting

that these cells belong to Th1-type cells. On the other hand, HSP70-reactive T cells expressed no mRNA specific for either IFN-γ, IL-2 or IL-4. We confirmed that the IL-4 internal probe could hybridize to the PCR product derived from concanavalin A-stimulating T cells using IL-4-specific primers (data not shown). Notably, the HSP70-reactive CD41 T cells expressed IL-15, TGF-β1 and IL-10 mRNA, which were never detected in HKL-reactive T cells (Fig. 8A). To further examine the cytokine synthesis by HSP70-reactive CD41 T cells at the protein level, we measured the IFN-γ, IL10 and TGF-β1 levels in culture supernatants of T cells incubated with rHSP70 or HKL by ELISA. As shown in Fig. 8(B), the peritoneal T cells incubated with HKL secreted large amounts of IFN-γ but not TGF-β1 or IL-10, which was consistent


Fig. 3. Proliferative response of T cell subsets in rats infected with L. monocytogenes 10 days previously to rHSP70 or HKL. The peritoneal CD81 and CD41 cells were purified up to .87.3% CD81 and 85.6% CD41 respectively by using rat Cellect kit columns, and were used as responder cells. Enriched CD81 or CD41 T cells (7.53105 cells/ml) from rats infected with L. monocytogenes 10 days previously were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis and human) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. The c.p.m. for [3H]thymidine incorporation was measured in triplicate cultures and the results are means 6 SD. **P , 0.001 versus medium only; NS, not significant versus medium only

Fig. 4. Effects of mAb against the MHC class I and class II on the proliferative response of the peritoneal T cells to rHSP70. Peritoneal T cells (7.53105 cells/ml) from rats infected with L. monocytogenes 10 days previously were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) and with 10 µg/ml anti-RT1.B mAb (OX-6), 10 µg/ml anti-RT1.D mAb (OX-17), 10 µg/ml anti-nitrophenol mAb (H1-6-2), 10 µg/ml anti-RT1.A mAb (OX-18) or 10 µg/ml anti-class I mAb (R4-8B1) for 72 h. The c.p.m. for [3H]thymidine incorporation was measured in triplicate cultures and the results are means 6 SD. **P , 0.001; NS, not significant


Fig. 5. TCR Vβ usages by T cells responding to rHSP70 or HKL. (A) Peritoneal T cells (7.53105 cells/ml) from rats infected with L. monocytogenes 10 days previously were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. mRNAs were extracted from the enriched T cells. Vβ usage analyzed with RT-PCR and Southern blotting using the Cβ1 primer, 22 primers specific for various Vβ chains of rat TCR and Cβ-P oligonucleotide probe. (B) The pre- and post-cultured cells were stained with FITC-conjugated anti-Vβ16 mAb (HIS42), phycoerythrin-conjugated anti-CD4 mAb (W3/25) and biotin-conjugated anti-CD3 mAb (G4.18) followed by streptavidin–Red670. The analysis gate was set on CD31CD41 cells and Vβ16 expression was shown as a single-parameter histogram.

with the results by RT-PCR analysis. On the other hand, the peritoneal T cells responding to rHSP70 produced TGF-β1 and IL-10 but not IFN-γ in the culture supernatants (Fig. 8B).

Effects of adoptive transfer with the HSP70-reactive T cell line on eradication of bacteria in rats infected with L. monocytogenes Analysis of cytokine production supported the hypothesis that HSP70-reactive CD41 T cells might regulate inflammation and/

or an immune response via suppressive cytokine production. If this mechanism occurs during the middle phase of listeriosis, then it would modulate the eradication of bacteria in the infected local site of rats. To investigate the in vivo roles of HSP70-reactive CD41 T cells, we used HSP70-reactive T cell lines generated from rats at day 10 after listerial infection. As infection with viable L. monocytogenes for listeriosis was induced, these rats were transferred i.p. with T cells that had been stimulated in vitro with rHSP70. As shown in Fig. 9, the


Fig. 6. Proliferative response of purified Vβ16 peritoneal CD41 T cells to rHSP70 in vitro. (A) Non-adherent PEC of rats infected with L. monocytogenes 10 days previously were stained with FITC-conjugated anti-Vβ16 mAb (HIS42), phycoerythrin-conjugated anti-CD4 mAb (W3/25) and biotin-conjugated anti-CD3 mAb (G4.18), followed by streptavidin–Red670. The profiles of Vβ16 and CD4 expressions are displayed after gating on the CD31 cells. CD31CD41 Vβ161 cells, which are indicated in circle B, from non-adherent PEC, were sorted with Epics Elite cell sorting system. (B) CD31CD41 Vβ161 cells (5.03104 cells/ml; .99.3% Vβ161) were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (5.03104 cells/ml) for 72 h. For the last 18 h, triplicate cultures of cells were pulse labeled with [3H]thymidine before harvesting. The c.p.m. for [3H]thymidine incorporation was measured in triplicate cultures and the results are means 6 SD.

number of bacteria in the peritoneal cavity of PBS-treated control rats was 104 c.f.u. on day 6 after infection. A significant decrease in the number of Listeria was detected on day 6 in the peritoneal cavity of the HKL-stimulated T cell line- or of the non-stimulated T cell-transferred rats. However, on day 6 the number of Listeria in the peritoneal cavity of HSP70stimulated T cell line-transferred rats was comparable to that of the PBS-treated control rats. These results clearly indicated that HSP70-reactive T cells have a role in regulating the excessive Th1 response to infection. Discussion In this report, we present evidence for the appearance of HSP70-reactive CD41 T cells in the peritoneal cavity during the course of rat listeriosis. The HSP70-reactive CD41 T cells recognized a peptide comprising of residues 234–252 as present in the 70 kDa HSP of M. tuberculosis in the context of RT1.B MHC class II molecules. The HSP70-reactive CD41 T cells also expressed mRNA specific for TGF-β1, IL-10 and IL-15, and produced TGF-β1 and IL-10. Adoptive transfer of the HSP70-reactive T cell line failed to increase the antilisterial resistance on day 6 after infection, whereas the HKLstimulated T cell line- or non-stimulated T cell-transferred rats increased the resistance level at the same time point as the control rats (on day 6). This approach led to a striking suppressive effect in the elimination of Listeria. Collectively, these results imply that the HSP70-reactive CD41 T cells may play a role in the regulation of Th1 cell-mediated inflammation during the course of rat listeriosis.

The secretion of IL-2, IFN-γ and TNF-β for induction of cellmediated immunity, characterized by macrophage activation and cytotoxic T lymphocyte induction, is unique to CD41 Th1 cells (45,46). Meanwhile, the secretion of IL-4, IL-5 and IL-6 for assistance in antibody production is unique to Th2 cells (45,46). Th2-type cytokines do not only induce B cell development, but also inhibit activities of macrophages and NK cells by shutting down the synthesis of cytokines, such as IFN-γ, by Th1 cells. On the other hand, IFN-γ derived from Th1 cells inhibits proliferation of Th2. Thus, Th1 cells and Th2 cells directly regulate each other (45–49). L. monocytogenes, like other intracellular parasites such as M. tuberculosis, induce a Th1-dependent immune response. Our present study also confirmed that CD41 Th1 cells specific for Listeria can be induced in the peritoneal cavity of rats during primary listerial infection. Listeria-specific CD41 Th2 cells during the course of primary listerial infection were barely detectable. Instead, unique CD41 Th cells, which produce TGF-β1 and IL-10, were detected in the peritoneal cavity of rats infected with L. monocytogenes. Besides Th1 and Th2 cells, a recent study on oral tolerance suggests that unique CD41 Th cells, termed Th3 cells capable of producing TGF-β1 and IL-10, regulate a variety of immune responses, especially responses against food antigens (50). The unique CD41 Th cells in our study may correlate with such ‘CD41 Th3 cells’ and regulate an excessive immune response by inflammatory Th1 cells during the course of bacterial infection. Among the various kinds of HSP, HSP65 and HSP70 are identified as a target of T cells, which cause or regulate autoimmune diseases such as AA and autoimmune diabetes.


Fig. 7. Proliferative response of the peritoneal T cells in Listeria-infected rats to synthetic peptides of HSP70 from M. tuberculosis. (A) Enriched peritoneal T cells (7.53105 cells/ml; .90% CD31), which were prepared from rats infected with 23107 L. monocytogenes 10 days previously, were cultured with mycobacterial HSP70 peptides representing 84–103, 183–201, 234–252, 539–557, 574–592, a peptide representing 205– 221 of human and mouse HSP60 or mycobacterial rHSP70 in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. For the last 18 h, triplicate cultures of cells were pulse-labeled with [3H]thymidine before harvesting. The c.p.m. for [3H]thymidine incorporation was measured in triplicate cultures and the results are means 6 SD. (B) Synthetic peptides in this study which encompass the sequences of M. tuberculosis HSP70, and human and mouse HSP60. The amino acid sequence of M. tuberculosis HSP70 identical to rat HSP70 is indicated by an asterisk. The amino acid sequence of human and mouse HSP60 identical to M. bovis HSP65 is indicated as an asterisk.

Anderton et al. have reported that anrthrogeneic T cell clones bearing CD4 and TCRαβ derived from Lewis rats can recognize an epitope which is comprised of amino acid residues 180–188 in the mycobacterial HSP65 in the context of RT1.B MHC class II molecules (19,20). They also reported that the immunization with an epitope containing amino acid residues 256–270 in mycobacterial HSP65 protects against AA (19,20). Haque et al. have quite recently reported that oral administra-

tion of mycobacterial HSP65 suppresses the development of AA in rats (51). It has also been reported that diabetes in non-obese diabetics has been prevented by immunization with mycobacterial HSP65 (52). Kingston et al. have shown that preimmunization with mycobacterial HSP70 suppressed AA in rats (43). Our adoptive transfer data also revealed that HSP70-reactive CD41 T cells clearly inhibited the protective mechanisms against Listeria infection. Thus, we speculate


Fig. 8. Cytokine profile of HSP70-reactive T cells. (A) Expression of cytokine-specific mRNA of peritoneal T cells stimulated with rHSP70 or HKL in vitro. Peritoneal T cells (7.53105 cells/ml) from rats infected with L. monocytogenes 10 days previously were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ ml) for 72 h and then were washed 3 times with cold PBS. mRNAs extracted from these T cells were reverse-transcribed into cDNA and amplified by PCR using IFN-γ-, IL-4-, IL-2-, IL-15-, TGF-β1-, IL-10- or β-actin-specific primers. The PCR products were transferred to GeneScreen Plus membranes and hybridized with an internal probe specific for IFN-γ, IL-4, IL-2, IL-15, TGF-β1, IL-10 or β-actin respectively. (B) Detection of IFN-γ, IL-10 and TGF-β1 in the supernatants of peritoneal T cells responding to rHSP70 or HKL by using ELISA. Peritoneal T cells (7.53105 cells/ml) from rats infected with L. monocytogenes 10 days previously were cultured with rHSP70 (25 µg/ml; derived from M. tuberculosis) or HKL (53107 cells/ml) in the presence of irradiated normal syngeneic splenocytes (7.53105 cells/ml) for 72 h. The supernatants were harvested after 72 h and the cytokine activities in culture supernatants were examined for the presence of IFN-γ, IL-10 and active forms of TGF-β1 by ELISA. The data are representative of two separates experiments and are expressed as the means of triplicates 6 SD. *P , 0.05 versus medium only

that HSP70-reactive CD41 T cells, induced by immunization with rHSP70, play vital roles in the down-regulation of inflammation not only in AA, but also in listeriosis. We also found that the HSP70-reactive CD41 T cells were accumulated in the inflamed sites during the natural course of listerial infection and produced deactivating cytokines, such as TGF-β1 and IL-10. The HSP70-reactive CD41 T cells appear in the infected

site at the relatively late stage of listerial infection, when the bacteria were completely eliminated. Taken together, these results suggested that the HSP70-reactive CD41 T cells may be induced in terminating the inflammatory responses, once the battle against Listeria has been won. It is also notable that the HSP70-reactive CD41 T cells did not express IL-2Rα or IL-2 mRNA, but did express IL-15 at

128 HSP70-reactive CD41 T cells during rat listeriosis technical assistance in sorting lymphocytes, and J. M. Shields and M. L. Ng for reading and giving valuable comments to our manuscript. We would also like to thank anonymous reviewers for comments on the manuscript. rHSP65 and rHSP70 derived from M. tuberculosis received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Disease (TDR). This work was supported by grants from Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of the Japanese Government.

Abbreviations AA APC BCG FCM HKL HSC HSP LN LPS PEC Fig. 9. Effects of adoptive transfer of the HSP70-reactive T cell line on eradication of bacteria in rats infected with Listeria. On day 0, 1.53106 T cell lines in PBS or PBS alone were administered i.p. to rats. Immediately following administration, the rats were then injected with 13108 viable L. monocytogenes. Five rats were used in each group. The number of Listeria in the peritoneal cavities of rats, that were injected 6 days previously, was determined by a colony formation assay on tryptic soy agar. Values are means 6 SD for the group of five rats. **P , 0.001 versus PBS alone; NS, not significant versus PBS alone

the mRNA level. Despite a lack of structural homology between IL-2 and IL-15, these cytokines are involved in mainly immunological processed through binding to β and γc chains of IL2R (53). These processes include the stimulation of T, B and NK cell proliferation, cytotoxic T lymphocyte and NK cell activation, and the augmentation of Ig secretion by B cells (53–55). However, the HSP70-reactive CD41 T cells were neither NKR-P11(dim) CD4– CD8– nor NKR-P11(dim) CD41CD8– cells (56–58). Furthermore, we have recently found that TCRγδ1 T cells appearing during murine salmonellosis can proliferate in response to IL-15 (39). Similar to the TCRγδ1 T cells appearing during bacterial infection, the HSP70-reactive CD41 T cells may utilize IL-15 as a growth factor. IL-15 is known to be less active than IL-2 in the induction of lymphocyte proliferation (42). The fact that T cells show a weaker proliferative response to rHSP70 than to HKL may be a result of the different utilization of growth factors such as IL-15 and IL-12 by HSP70-reactive CD41 T cells and HKL-specific T cells. In conclusion, we have obtained the first evidence for the appearance of HSP70-reactive CD41 T cells during the natural course of listerial infection in rats. The HSP70-reactive CD41 T cells, which were of the NKR-P1– CD441CD45RC– IL-2Rα– phenotype, react to rHSP70 in the context of MHC class II (RT1.B) molecules. The HSP70-reactive CD41 T cells may play a regulatory role in inflammation during Listeria infection via the production of suppressive cytokines including TGFβ1 and IL-10. Acknowledgements We thank Drs J. Kamerbeek and J. D. A. van Embden for providing rHSP65 derived from M. bovis BCG, Y. Yamakawa for excellent

adjuvant arthritis antigen-presenting cells Bacillus Calme´tte Gue´rin flow cytometry heat-killed Listeria heat shock cognate protein heat shock protein lymph node lipopolysaccharide peritoneal exudate cells

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