Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response

In order to define extracellular localized epitopes of Hsp70 on human tumor cells which are accessible to the immune system, six commercially available Hsp70-specific monoclonal antibodies (mAb) with different recognition sites were examined by immunological approaches. The recognition pattern of these antibodies was analyzed on purified recombinant Hsp70 proteins (rHsp70, Hsc70, DnaK), on lysates of Hsp70-expressing colon carcinoma cells (CX+) and on lysates of M21 rat-1 cells that overexpress human Hsp70 or Hsp70 fragments: ΔBgl (del 120–428) consisting of the C-terminal part and ΔSma (del 438–618) consisting of the N-terminal part of human Hsp70. All antibodies reacted equally well with rHsp70 and cytoplasmic Hsp70 derived from human tumor cells or M21 rat-1 cells. Only one antibody (MA3–007; Hsp70, Hsc70) detects a region localized within the ATPase domain of Hsp70 (amino acid 122–264) and reacts positively with the C-terminal deletion mutant ΔSma. All other antibodies, including RPN1197 are directed against the C-terminal peptide binding domain of Hsp70 and react positively with the N-terminal deletion mutant ΔBgl. Although all six antibodies detect full-length Hsp70 protein, derived from plasma membrane fractions of CX+ tumor cells, cell surface expressed Hsp70 on viable CX+ tumor cells, as determined by flowcytometry, is only recognized with the antibodies MA3–006 (Hsp70, Hsc70; 504–617), MA3–009 (Hsp70; 504–617) and RPN1197 (Hsp70). An estimation of the ratio of membrane-bound to cytoplasmic Hsp70 molecules revealed that 15–20% of total Hsp70 molecules are expressed on the plasma membrane. This tumor-selective cell surface expression of Hsp70 correlates with an increased sensitivity to lysis mediated by non-MHC restricted natural killer (NK) cells. We demonstrate that only antibodies directed against membrane-bound Hsp70 (MA3–006, MA3–009, RPN1197) inhibit NK-killing activity against Hsp70-expressing tumor cells. Taken together our data indicate that at least the C-terminal region 504–617, that contains at least one single α-helix (amino acid 512–536), has to be localized extracellularly and might be of importance for an NK-mediated anti-tumor immune response.     

Heat shock protein 70/MAGE-3 fusion protein vaccine can enhance cellular and humoral immune responses to MAGE-3 in vivo

MAGE-3, a member of melanoma antigen (MAGE) gene family, is recognized as an ideal candidate for tumor vaccine because it is expressed in a significant proportion of tumors of various histological types and can induce antigen-specific immune response in vivo. There is now substantial evidence that heat shock proteins (HSPs) isolated from cancer cells and virus-infected cells can be used as vaccines to produce cancer-specific or virus-specific immunity. In this research, we investigated whether M. tuberculosis HSP70 can be used as vehicle to elicit immune response to its accompanying MAGE-3 protein. A recombinant protein expression vector was constructed that permitted the production of fusion protein linking amino acids 195–314 of MAGE-3 to the C terminus of HSP70. We found that HSP70-MAGE-3 fusion protein can elicit stronger cellular and humoral immune responses against MAGE-3 expressing murine tumor than those elicited by MAGE-3 protein in vivo, which resulted in potent antitumor immunity against MAGE-3-expressing tumors. Covalent linkage of HSP70 to MAGE-3 was necessary to elicit immune response to MAGE-3. These results indicate that linkage of HSP70 to MAGE-3 enhanced immune responses to MAGE-3 in vivo and HSP70 can be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens.     

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.     

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

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

Hsp70与汉滩病毒S基因融合蛋白的原核表达及免疫小鼠的初步研究

本文构建了hsp70与S基因的原核融合表达载体pGEX-4T-1/hsp70-S,在大肠杆菌中表达,并通过GSTrapFF柱进行了纯化。同时制备了NP和Hsp70两种纯化蛋白。分别用这三种纯化蛋白免疫BALB/c小鼠,结果表明纯化的NP和Hsp70-NP两种蛋白均可同时诱导产生抗汉滩病毒核蛋白(NP)抗体,且后者刺激产生的抗体效价明显高于前者。淋巴细胞增殖实验表明,两组免疫小鼠的脾细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP组免疫小鼠脾细胞对NP的增殖指数明显高于NP组免疫组。结果显示,与单独用NP免疫小鼠相比,Hsp70-NP纯化蛋白可以刺激机体产生更强的抗汉滩病毒体液免疫应答和特异性淋巴细胞增殖反应。     

Toll-like receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response

Toll-like receptors (TLRs) play important roles in initiation of innate and adaptive immune responses. Emerging evidence suggests that TLR agonists can serve as potential adjuvant for vaccination. Heat shock proteins (HSPs), functionally serving as TLR4 agonists, have been proposed to act as Th1 adjuvant. We have identified a novel Hsp70 family member, termed Hsp70-like protein 1 (Hsp70L1), shown that Hsp70L1 is a potent T helper cell (Th1) polarizing adjuvant that contributes to antitumor immune responses. However, the underlying mechanism for how Hsp70L1 exerts its Th1 adjuvant activity remains to be elucidated. In this study, we found that Hsp70L1 binds directly to TLR4 on the surface of DCs, activates MAPK and NF-κB pathways, up-regulates I-a(b), CD40, CD80, and CD86 expression and promotes production of TNF-α, IL-1β, and IL-12p70. Hsp70L1 failed to induce such phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating a role for TLR4 in mediating Hsp70L1-induced DC activation. Furthermore, more efficient induction of carcinoembryonic antigen (CEA)-specific Th1 immune response was observed in mice immunized by wild-type DCs pulsed with Hsp70L1-CEA(576-669) fusion protein as compared with TLR4-deficient DCs pulsed with same fusion protein. In addition, TLR4 antagonist impaired induction of CEA-specific human Th1 immune response in a co-culture system of peripheral blood lymphocytes (PBLs) from HLA-A2.1(+) healthy donors and autologous DCs pulsed with Hsp70L1-CEA(576-669) in vitro. Taken together, these results demonstrate that TLR4 is a key receptor mediating the interaction of Hsp70L1 with DCs and subsequently enhancing the induction of Th1 immune response by Hsp70L1/antigen fusion protein.     

Hsp70-NP融合蛋白增强诱导HTNV NP特异性CTL的实验研究

本文采用纯化蛋白Hsp70-NP,NP,Hsp70分别免疫C57/BL6小鼠,取各组小鼠脾淋巴细胞进行淋巴细胞增殖试验和细胞毒试验。此外,为了获得细胞毒实验的靶细胞,本文还采用脂质体介导质粒pcDNA3.1/S转染黑色素瘤细胞B16,通过G418筛选稳定克隆,并用RT-PCR,Westernblots以及免疫荧光染色证实N蛋白在胞浆中表达。淋巴细胞增殖实验表明,Hsp70-NP,NP组小鼠脾淋巴细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP组的增殖指数明显高于NP免疫组。细胞毒实验结果表明,LDH的释放具有效应细胞依赖性,Hsp70-NP,NP免疫组脾淋巴细胞均可以特异性杀伤靶细胞B16-N,而Hsp70-NP免疫组的杀伤率显著高于NP免疫组。实验结果显示,Hsp70可以增强NP诱导产生特异性CTL的能力。本研究结果为进一步设计基于NP的合成肽疫苗或基因疫苗提供了重要实验依据。     

Ubiquitin-Associated (UBA) Domain in Human Fas Associated Factor 1 Inhibits Tumor Formation by Promoting Hsp70 Degradation

Human Fas associated factor 1 (hFAF1) is a pro-apoptotic scaffolding protein containing ubiquitin-associating (UBA), ubiquitin like 1 and 2 (UBL1, UBL2), and ubiquitin regulatory X (UBX) domains. hFAF1 interacts with polyubiquitinated proteins via its N-terminal UBA domain and with valosin containing protein (VCP) via its C-terminal UBX domain. Overexpression of hFAF1 or its N-terminal UBA domain significantly increases cell death by increasing the degradation of polyubiquitinated proteins. In this study, we investigated whether hFAF1, whose expression level is reduced in cervical cancer, plays a role in tumor formation. We found that HeLa cells overexpressing full-length hFAF1 or the hFAF1 UBA domain alone, significantly suppressed the anchorage independent tumor growth in soft agar colony formation, increased cell death, and activated JNK and caspase 3. Employing UBA-specific tandem immunoprecipitation, we identified moieties specifically interacting with UBA domain of hFAF1, and found that polyubiquitinated Hsp70s are recruited to UBA domain. We also demonstrated that hFAF1 overexpression promotes Hsp70 degradation via the proteasome. We further found that mutating the UBA domain (I41N), as well as knocking down hFAF1 with specific RNAi, abolishs its ability to increase the proteasomal degradation of Hsp70. These findings suggest that hFAF1 inhibits tumor formation by increasing the degradation of Hsp70 mediated via its UBA domain.     

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.     

Inhibition of tumor growth in mice with severe combined immunodeficiency is mediated by heat shock protein 70 (Hsp70)-peptide-activated,CD94 positive natural killer cells

Previously, we reported that the major stress-inducible heat shock protein 70 (Hsp70) acts as a recognition structure for natural killer (NK) cells, if localized on the cell surface of tumor cells. Incubation of purified NK cells with low-dose interleukin (IL)-2 (100 IU/mL) plus recombinant Hsp70-protein or the immunogenic 14-mer Hsp70-peptide TKDNNLLGRFELSG450-463, termed TKD (2 microg/mL), enhances the cytolytic activity against Hsp70 membrane-positive (CX+) but not against Hsp70-negative (CX-) tumor cells. Here, we show that the cytolytic activity against Hsp70-positive tumor cells is inducible by incubation of unseparated peripheral blood mononuclear cells (PBMNC) with low-dose IL-2 plus TKD. Cell sorting experiments revealed that within the PBMNC population CD94(+)/CD3(-) NK cells, and not CD94(-)/CD3(+) T cells, mediate the cytotoxic activity against Hsp70-positive tumor cells. The antitumoral effect of PBMNC stimulated either with IL-2 plus TKD or with IL-2 alone was assessed in tumor-bearing severe combined immunodeficiency/beige mice. A single intravenous (iv) injection of 40 x 10(6) IL-2 plus TKD-stimulated PBMNC (containing 5.2 x 10(6) NK cells) on day 4 results in a 60% reduction in tumor size, from 3.89 g to 1.56 g. In contrast, the adoptive transfer of the identical amount PBMNC stimulated with low-dose IL-2 only (containing 4.4 x 10(8) NK cells) reduces the tumor size only less than 10% (3.64 g). A phenotypic characterization of the excised tumors revealed that predominantly Hsp70-positive tumor cells were eliminated by TKD-activated PBMNC. Kinetic studies demonstrate that the in vivo cytolytic capacity of TKD-stimulated PBMNC is dependent on the effector to target cell ratio. An iv injection of effector cells on day 1 or 2 after tumor cell inoculation results in significantly smaller tumors (0.77 g or 0.89 g) on day 21 as compared with mice that were immunoreconstituted on day 4 or 8 (1.39 g or 2.23 g). The tumor size of nonimmunoreconstituted control animals was 3.55 g.     

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.     

Hsp70 chaperone-based gel composition as a novel immunotherapeutic anti-tumor tool

The recent advances in designing Hsp70-based anti-cancer vaccines and the ability of the chaperone to penetrate inside a living cell prompted us to develop a non-invasive method for the treatment of surface tumors. We designed hydrogel-containing gel-forming substances and human recombinant Hsp70 and applied them on the surface of a 7-day-old B16F10 melanoma tumor. According to the results of histochemistry, Hsp70 diffused through skin layer inside the B16 tumor, and this transport was proved by biochemical data. The application of Hsp70 gel reduced the rate of tumor growth by 64 % and prolonged the life of animals by 46 %. Increased survival was correlated with the enhancement of B16-specific cytotoxicity and up-regulation of gamma–interferon production. Taken together, the data confirm the anti-tumor effect of pure recombinant Hsp70 delivered intratumorally and demonstrate the relevance of a novel non-invasive technology of Hsp70-based therapy.     

CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes

Tumor and viral antigens elicit a potent immune response by heat shock protein-dependent uptake of antigenic peptide with subsequent presentation by MHC I. Receptors on antigen-presenting cells that specifically bind and internalize a heat shock protein-peptide complex have not yet been identified. Here, we show that cells expressing CD40, a cell surface protein crucial for B cell function and autoimmunity, specifically bind and internalize human Hsp70 with bound peptide. Binding of Hsp70-peptide complex to the exoplasmic domain of CD40 is mediated by the NH(2)-terminal nucleotide-binding domain of Hsp70 in its ADP state. The Hsp70 cochaperone Hip, but not the bacterial Hsp70 homologue DnaK, competes formation of the Hsp70-CD40 complex. Binding of Hsp70-ADP to CD40 is strongly increased in the presence of Hsp70 peptide substrate, and induces signaling via p38. We suggest that CD40 is a cochaperone-like receptor mediating the uptake of exogenous Hsp70-peptide complexes by macrophages and dendritic cells.     

PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.     

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.     

Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.     

Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies

Cell Stress & Chaperones journal has become a major outlet for papers and review articles about anti-heat shock protein (HSP) antibodies. In the last decade, it became evident that apart from their intracellular localization, members of the heat shock protein 90 (Hsp90; HSPC) and Hsp70 (HSPA) family are also found on the cell surface. In this review, we will focus on Hsp70 (HSPA1A), the major stress-inducible member of the human Hsp70 family. Depending on the cell type, the membrane association of Hsp70 comes in two forms. In tumor cells, Hsp70 appears to be integrated within the plasma membrane, whereas in non-malignantly transformed (herein termed normal) cells, Hsp70 is associated with cell surface receptors. This observation raises the question whether or not these two surface forms of Hsp70 in tumor and normal cells can be distinguished using Hsp70 specific antibodies. Presently a number of Hsp70 specific antibodies are commercially available. These antibodies were generated by immunizing mice either with recombinant or HeLa-derived human Hsp70 protein, parts of the Hsp70 protein, or with synthetic peptides. This review aims to characterize the binding of different anti-human Hsp70 antibodies and their capacity to distinguish between integrated and receptor-bound Hsp70 in tumor and normal cells.     

EAE tolerance induction with Hsp70-peptide complexes depends on H60 and NKG2D activity

Inflammation leads to induction of tissue stress conditions that might contribute to the generation of mechanisms limiting ongoing immune responses. We have shown previously that peptides derived from brain tissue of mice with experimental autoimmune encephalomyelitis (EAE) complexed with the chaperone heat shock protein 70 (Hsp70-pc) induce an NK-cell-dependent tolerance for subsequent EAE sensitization. We now present data that showed that the MHC class I-related glycoprotein H60 determines Hsp70-pc-induced EAE inhibition. Hsp70-pc led to significant and selective up-regulation of H60 expression in SJL/J mice, and Ab-blocking of H60 expression led to loss of EAE tolerance. Similarly, blocking of the NK cell receptor for H60, NKG2D, also reversed the Hsp70-pc-induced EAE inhibition. In contrast, in C57BL/6 mice H60 was not expressed, and Hsp70-pc-induced tolerance was not detected. The NK cell mediated Hsp70-pc-induced tolerance to EAE was dependent on modulation of dendritic cells function leading to diminished T cell reactivity to PLP. As, no increase of H60 expression on T cells from EAE mice immunized with PLP was detected, and no enhanced loss of CD3+ H60+ over CD3+ H60- cells in Hsp70-pc-induced EAE tolerance was found direct killing of H60+ PLP-reactive cells seems not to be involved in the Hsp70-pc-induced tolerance induction. We have provided evidence that Hsp70-pc-induced tolerance for EAE, mediated by NK cells, involves induction of H60 ligand and its interaction with NKG2D receptor. NK cells tolerization of EAE depends on altered dendritic cells activity leading to enhanced death of Ag reactive cells.     

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c（Hsp70359–610） fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70（mHsp70） is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte（CTL） response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2（M2e）, was fused to the C-terminus of Mycobacterium tuberculosis Hsp70（Hsp70c）, to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c（Hsp70359–610）. And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.     

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL

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.