Abstract
Intracellular heat shock proteins (HSP) function as molecular chaperones, they support folding and transport mechanisms of other proteins under physiological conditions and following physical or chemical stress. More recently, extracellular localized HSP have been found to play key roles in the induction of a cellular immune response. Either they act as carrier molecules for immunogenic peptides that are presented on Antigen Presenting Cells (APC) to cytotoxic T-cells or they themselves act as activatory molecules for the innate immune system. Binding of uncomplexed HSP to HSP-receptors on APC has been found to induce the secretion of inflammatory cytokines. Furthermore, an unusual tumor-selective membrane-localization of non-conserved regions of the 72 000 Da HSP (Hsp70) has been found to act as a recognition structure for natural killer (NK) cells. In this review the interaction of NK cells with Hsp70 or peptides derived thereof will be eluciated in more detail.
Abbreviations
| APC: | = | antigen presenting cells; |
| Hsp70: | = | 72 000 Da heat shock protein; |
| LPS: | = | lipopolysaccharide; |
| mAb: | = | monoclonal antibody; |
| NK: | = | natural killer cells; |
| eradicate Pepscan: | = | peptide scanning; |
| ADCC: | = | antibody dependent cytotoxicity; CD, cluster of differentiation; |
| ER: | = | endoplasmic reticulum; |
| Ig: | = | immunoglobulin; ILT, Ig-like transcript receptors; |
| ITIM/ITAM: | = | immunoreceptor tyrosine-based inhibition/activation motif; |
| KIR: | = | killer cell Ig-like receptor; |
| KLR: | = | killer cell lectin-like receptor; |
| NCR: | = | natural cytotoxicity receptor; |
| TAP: | = | Transporeter-associated Processing; |
| TLR: | = | Toll-like receptor |
Introduction
Heat Shock Proteins (HSP) are highly conserved proteins that are inducible by a variety of stressful stimuli and by physiological processes including cell growth, differentiation and development Citation1. Their major aim is to protect cells from lethal damage induced by physical (i.e. heat) as well as chemical (i.e. heavy metals, reactive oxygen species, cytostatic drugs) stress. Intracellular HSP function as molecular chaperones, they are involved in protein or polypeptide folding/unfolding processes, transport mechanisms, antigen processing and presentation Citation2,Citation3. The search for tumour-selective antigens resulted in the identification of HSP that were isolated from tumours Citation4,Citation5. HSP have been found to play important roles in eliciting potent anti-cancer immune responses mediated by T-cells, Antigen Presenting Cells (APC) and Natural Killer (NK) cells. In order to explain the paradoxical situation of how highly conserved HSP mediate cancer immunity, Srivastava Citation5 proposed the following four paradigms: (a) due to short non-conserved immunogenic regions, (b) because of a tissue specific expression pattern HSP can act as classical foreign antigens, (c) HSP could mimick classical T-cell epitopes, or (d) act as carrier molecules for immunogenic peptides. Presently, experimental evidence exists for possibilities (a) and (c). Tumour-derived HSP with a molecular weight of ∼70 000 (Hsp70, Hsc70) and 96 000 Da (gp96) have been shown to chaperone immunogenic peptides Citation6,Citation7 to MHC molecules that elicit T-cell responses against primary tumours and metastases Citation8,Citation9. Antigen presenting cells have been found to be key for mediating this specific immune response Citation10. A hint for the existence of HSP specific receptors on APC was shown by cell surface binding and uptake studies of HSP Citation11. Presently, several receptors including the α-2 macroglobulin receptor CD91 Citation11–13, members of the Toll-Like Receptor family including TLR-2/TLR-4, together with the Lipopolysaccharide (LPS) receptor CD14 could be identified as potential interacting partners for HSP Citation7,Citation14. An involvement of LPS contaminations for the induction of an HSP mediated immune stimulation was ruled out by co-incubation experiments and by inactivating LPS during the assay (R. Issels, personnel communication). Following receptor-mediated uptake of HSP-peptide complexes by APC, the immunogenic peptides are re-presented on MHC class I molecules either via an endosomal Transporter-associated Antigen Processing (TAP) and proteasome-independent or a classical TAP and proteasome-dependent ER-golgi route Citation15 and, thereby, stimulate a CD8 cytotoxic T cell response Citation8,Citation16. This specific topic will be illuminated in more detail by Hans-Jorg Schild and Valeria Milani Citation5. Although HSP without peptides were found to be non-immunogenie for T-cells, binding of peptide-free HSP70 to TLR-2/TLR-4 on APC has been found to induce the secretion of immunostimula-tory cytokines, including GM-CSF, IL-12 or TNF-α Citation14. This might indicate that non-complexed Hsp70 and peptide-associated Hsp70 exhibit different immunostimula-tory capacities.
A tumour-selective plasma membrane localization of HSP with a molecular weight of 70 000 and 90 000 Da was reported by several groups, including this one Citation17–21. This group was the first to demonstrate that membrane-bound Hsp70 provides a target recognition structure for transiently plastic adherent NK cells Citation22–24. A correlation of the cytolytic activity of NK cells with the amount of plasma membrane-bound Hsp70 has been determined Citation25,Citation26. Although an Hsp70-NK cell interaction could be demonstrated in binding studies (unpublished data) the mechanism of how NK cells lyse Hsp70 positive tumour target cells remains to be elucidated.
NK cells
NK cells, also termed large granular lymphocytes, comprise between 5–20% of peripheral blood mononuclear cells Citation27. They exhibit a cytolytic response against tumour cells and play important roles in the immunological control of bacteria, parasites and viruses. As compared to T-lymphocytes, NK cell-mediated cellular cytotoxicity and cytokine secretion (IFN-γ, IL-2, IL-12, TNF and GM-CSF) does not require primary stimulation. Also, in contrast to T-lymphocytes, Major Histocompatibility Complex (MHC) class I molecules confer resistance to an NK-mediated lysis Citation28. Therefore, the question arises as to which molecules are responsible for the interaction of NK cells with MHC class I antigens. It is well known that NK cells do not express T-cell receptors together with the CD3 complex; nearly all NK cells exhibit a positive phenotype for the sheep red blood cell receptor Citation29 and the low affinity Fc-γreceptor CD16 Citation30, that mediates antibody dependent cellular cytotoxicity (ADCC). According to the intensity of the CD16 expression, a CD16 ‘dim’ and a CD16 ‘bright’ sub-population is distinguishable that differ with respect to the cytolytic capacity. More recently, the molecular nature of so-called Killer cell inhibitory/activating Immunoglobulin (Ig)-like Receptors (KIR), and Ig-Like Transcript receptors (ILT), also known as Leukocyte Ig-like Receptors (LIR) has been elucidated. In comparison to KIR, the expression of ILT/LIR is not restricted to NK and T-cells. Monocytes and macrophages also have been found to express MIR. The majority of NK cell receptors have been shown to interact with MHC class Ia and Ib alleles or with groups of alleles Citation31,Citation32. According to their receptor repertoire, NK cells can be sub-divided into groups with distinct reactivity patterns. A complex set of signalling events that is mediated either by a long Immunoreceptor Tyrosine-based Inhibition Motif (ITIM), that recruits and activates protein tyrosine phosphatases such as the Src Homology 2 domain-containing Phosphatases SHP1 and SHP2 to inactive effector function, or by a short Immuno receptor Tyrosine-based Activating Motif (ITAM), that contains a positively charged amino acid residue, including arginine or lysine, within the transmembrane domain. This domain is necessary for the interaction with ITAM-containing adaptor molecules including Killer cell-Activating Receptor Activating Protein (KARAP, DAP12) in the case of activating KIR, or the Fc-γ receptor chain in the case of ILT. Phosphorylated ITAM recruit the protein tyrosine kinases Syk and ZAP70 and, thereby, induce cellular activation. ITIM and ITAM determine whether NK cells will be functionally inactive or active towards their target cells.
HLA specific inhibitory receptors found on NK and memory CD8+ T-cells can be grouped into the killer cell Ig-like receptors (KIR) and the killer cell lectin-like receptors (KLR). In human NK cells, the immunoglobulin superfamily (Ig-SF) receptors dominate the C-type lectin receptors. So far, only CD94, the NKG2 family and CD69 Citation33,Citation34 could be identified as C-type lectin receptors in human NK cells, whereas in rodents most receptors belong into the C-type lectin group. Recently, three novel NK cell specific Ig-like receptors (NKp46, NKp30, NKp44) and the C-type lectin receptors (NKp80) have been identified as Natural Cytotoxicity Receptors (NCR) that seem to be important for the attack of tumours Citation35. However, it is not clear yet whether NKp80 that contains tyrosine-based motifs in the cytoplas-mic tail, function as receptors or co-receptors. Most NCRs are connected to signal adaptor molecules like CD3ζ, FCεRγ and KARAP/DAP12. Although NCRs are known to possess activating properties, the recognition structure that is responsible for the mechanisms by which NK cells become activated to respond are still not completely understood.
Human 2B4 is rather an Ig-SF co-receptor for the triggering receptor NKp46 than a receptor on its own. The natural ligand for 2B4 on NK cells is GPI-anchored CD48 Citation35. In addition to NK cells, 2B4 is also found on some CD8 or CD4 positive T-cells, monocytes, basophils and γ/δ T-cells. Human 2B4 contains a long cyto-plasmic tail with four tyrosine-based motifs and associates with the linker for activation of T-cells (LAT). NK cell triggering results in tyrosine phosphorylation of both 2B4 and linker of activation of T-cells Citation35. As a co-receptor, 2B4 is discussed to be important to enhance the cytotoxic activity of other NCRs.
C-type lectin receptor family
CD94 is a 25 000 ± 30 000 Da protein that is covalently associated with a partner molecule of the NKG2 family (43 000 Da). Dependent on the partner receptor of CD94, inhibitory (CD94/NKG2A) or activating (CD94/NKG2C) functions can be fulfilled by the effector cell Citation34,Citation36. CD69 and CD161 (NKR-P1A, Mw 40 000 Da, reducing conditions) are other members of the C-type lectin family that mediate activating signals Citation34,Citation37.So far, three rodent NKR-P1 genes have been identified; in humans only one gene has been reported. Although several Human Leucocyte Antigens (HLA) or allele groups have been identified as inhibitory signals for NK cells, less is known about activating signals. According to the missing self theory, NK cells preferentially kill tumour target cells that either lack expression of MHC class I molecules or exhibit a modified MHC class I expression Citation28. As mentioned before, the question 'What turns on NK cells’ appears to be more complex. The CD94/NKG2C heterodimers recognize non-classical MHC class I molecules including HLA-E that present leader sequences of classical HLA-A, -B, and -C molecules Citation38 in a TAP-dependent manner. An association of the adaptor molecule DAP12 (14 000 Da phosphoprotein, also termed Killer cell Activating Receptor Associated Protein, KARAP) with the cytoplasmic tail of the activating receptor complex results in a triggering signal for NK cells Citation36,Citation39,Citation40. In contrast to NKG2A, NKG2B (a splice variant of NKG2A), NKG2C or NKG2E that are found as heterodimers together with CD94, the C-type lectin receptor NKG2D forms homodimers with activating properties that are associated with the DAP10/KAP10 molecules that recruit phos-phatidylinositol 3-kinase. DAP 10 maps centromeric to the Sialic acid-binding Ig-like lectin (Siglec) multigene family on human chromosome 19. NKG2D is involved in the stress response, it interacts with the stress-inducible MHC class I chain related MICA and MICB gene products that are encoded in the human MHC complex on chromosome 6 Citation41. Moreover, an interaction with gylcosyl-phosphatidyl-inosito l (GPI)-linked UL16-binding proteins (ULBP 1, 2, 3), the Retinoic acid early 1 (Rael) protein and H60 (a minor histocompatibility antigen) have been defined as murine NK cell triggering ligands for NKG2D Citation42–44. The expression of NKG2D is not restricted to NK cells, also NK like T-cells, monocytes and macrophages, and T-cell subsets do express them. However, in contrast to T-cells, most NK cells are able to express more than one receptor on one cell. The precise mechanism regulating the expression levels of inhibitory and activating signals remains to be determined.
Immunoglobulin (Ig)-like superfamily (Ig-SF) receptor family
According to the number of extracellular localized immunoglobulin (Ig) domains (2–4), and the size of the intracellular part, the KIR and ILT can be grouped into functionally distinct families Citation45,Citation46. Killer cell receptors with long cytoplasmic ITIM motifs mediate inhibitory signals following binding of the extracellular receptor part to MHC class alleles. In contrast, contact between the same ligands to receptors with short cytoplasmic ITAM motifs mediate activating signals. Although functionally inhibitory and activating receptors clearly differ from each other, the extracellular expressed part of both receptors are highly homologous. Since antibodies directed against KIR are unable to distinguish between the inhibitory and activating form of the receptor, flow cytometry analysis of viable NK cells with intact plasma membrane cannot describe the functional status of the cells.
summarizes NK cell receptors and co-receptors with known ligand specificity that are involved in NK-mediated function. First column: name and cluster of differentiation (CD) nomenclature Citation47, second: inhibitory or activating function, third: Ig or C-type lectin receptor family, fourth: known interacting ligand, fifth: name of the gene, sixth: chromosomal localization and references in brackets.
Table I. Interacting partners of immunoglobulin superfamily (Ig-SF) and C-type lectin (C-type) receptors of the chromosome 19q leukocyte receptor complex and the chromosome 12p NK gene complex. References in brackets.
Hsp70-NK cell interaction
Hsp70 expressing tumour cells are more sensitive to lysis mediated by IL-2 stimulated, transiently plastic adherent NK cells, as compared to Hsp70 membrane negative tumour cells Citation22. This finding has been assessed with allogeneic tumour cell lines and within an autologous human colon carcinoma cell system CX+ and CX−, that differs only with respect to the capacity to express Hsp70 on the cell surface Citation22,Citation23. All other tested cell surface markers including MHC and adhesion molecules have been found to be identically expressed on CX+ and CX− cells. Selectively transiently plastic adherent NK cells, but not T-cells, exhibit an increased cytolytic activity against Hsp70 membrane positive tumour target cells. This might be due to the fact that membrane-bound Hsp70 is not associated with immunogenic peptides. Antibody and Hsp70 protein blocking studies revealed that, indeed, plasma membrane localized Hsp70 provides a target recognition structure for NK cells Citation20,Citation23,Citation24. The Hsp70 specific monoclonal Antibody (mAb) clone C92 F3B1 with inhibitory properties recognizes an epitope of the highly stress-inducible Hsp70 in the C-terminal substrate binding domain and does not cross-react with the homologous constitutive form Hsc70.
In an H-3 thymidine uptake assay Citation49 it was demonstrated that co-incubation of isolated NK cells with recombinant Hsp70 protein results in a moderate increase in proliferation of NK cells between 10–25%. Furthermore, the cytolytic activity against Hsp70 membrane positive tumour cells was significantly enhanced following a co-incubation of NK cells with Hsp70 protein for at least 3 days Citation50. In contrast, other homologous members of the HSP70 group, including the constitutive form Hsc70 (homology to Hsp70: 84%) or the E.coli derived Hsp70 homologue DnaK (homology to Hsp70: 50%) did not stimulate an immunological response in transiently plastic adherent of NK cells Citation50. These findings provided a first hint that NK cells interact specifically with Hsp70, the major stress inducible member of the Hsp70 group. It appeared that only a distinct concentration range of Hsp70 protein efficiently stimulates an immune response.
In an effort to determine which part of Hsp70 is responsible for the interaction with NK cells, deletion mutants that either lack the C- or the N-terminal domain of Hsp70 have been tested Citation51. In addition to full length Hsp70 protein, only the C-terminal substrate binding domain of Hsp70 exhibits a stimulatory activity on NK cells Citation50. In order to further characterize the relevant Hsp70 epitope that interacts with NK cells, peptide studies were performed.
Hsp70 peptide-NK cell interaction
As mentioned before, plasma membrane localization of Hsp70 on tumour cells was detectable with an Hsp70 specific mAb clone, C92 F3B1, that specifically reacts with an epitope of the substrate binding domain of Hsp7O Citation20,Citation22,Citation51. Since this antibody inhibits the cytolytic activity of NK cells against Hsp70 expressing tumour cells Citation22, it was assumed that the epitope might be important for the interaction of NK cells with Hsp70. By peptide scanning Citation52 of the complete substrate binding domain (aa 384–618), an 8-mer sequence NLLGRFEL (aa 454–461) could be identified as the relevant recognition structure for the Hsp70 mAb. Based on this 8-mer antibody epitope, several peptides with different length have been produced in order to test their immunostimulatory capacity on NK cells. As summarized in , only a 14-mer Hsp70 peptide derived from the C-terminal substrate binding domain termed TKD (aa 450–463, TKDNNLLGRFELG) exhibits immunostimulatory capacity on NK cells Citation53. For full length Hsp70 protein, a concentration of 1 µg/ml has been defined as optimal to stimulate NK cells Citation50. Regarding the molecular weight of the Hsp70 peptide TKD (1563 Da), the concentration which is equivalent to full length Hsp70 protein (72 000 Da) was calculated as 0.2 µg/ml. Indeed, this Hsp70 peptide concentration could be determined as an effective dose to stimulate NK cell activity Citation49. These data show that, similar to full length Hsp70 protein, Hsp70 peptide also stimulates NK cell activity at equivalent concentrations. Next, the question arises whether NK cells, similar to APC, do express Hsp70 specific receptors.
Table II. Summary of amino acid (aa) sequences of stimulatory and non-stimulatory peptide sequences in the C-terminal substrate binding domain Citation49. Comparison of the amino acid (aa) sequences of Hsp70 derived peptides with NK-stimulatory and non-stimulatory capacity. Amino acid residues which appear to be key for the interaction with the NK cell receptor are indicated in bold.
Hsp70 specific receptors on NK cells?
The α2-macroglobulin receptor CD91 and TLR-2/TLR-4 have been denned as potential receptors for gp96 and for HSP70 on antigen presenting cells Citation12,Citation13,Citation54. It is also stated that the LPS receptor CD14 acts as a co-receptor for Hsp70 mediated signalling in human monocytes Citation14. Since neither CD91, TLR-2/TLR-4 nor CD14 are expressed on Hsp70 specific NK cells Citation55, these molecules could be excluded as Hsp70 receptors on NK cells. The hypothesis that NK cells use different HSP-receptors as compared to APC was supported by the finding that NK cells selectively interact with the inducible form of Hsp70 but not with other members of the HSP70 group. Furthermore, the concentration range for a specific interaction of Hsp70 protein with NK cells and with APC has been found to be significantly different; the optimal concentration for NK cell stimulation has been determined as 1 µg/ml, whereas APC exhibit specific interaction up to a concentration of 200 µg/ml Citation54. Also, functionally, Hsp70 receptors on NK cells differ from those on APC. As stated earlier, HSP70 and HSP90 peptide complexes become internalized by APC and HSP-cha-peroned, immunogenic peptides are re-presented on MHC class I molecules via endosomal or ER-golgi pathways. In NK cells, interaction with Hsp70 or the Hsp70 peptide TKD results in an activation of the cytolytic and proliferative function. Concomitant with an increased cytotoxic activity, long-term incubation of NK cells with Hsp70 protein or Hsp70 peptide, results in an upregulation in the mean fluorescence of the killer cell activating receptor CD94 Citation50,Citation53. Regarding these results, one might speculate about CD94 as a potential interaction partner for Hsp70. Hsp70 protein and peptide binding studies using an NK cell line with differential CD94 expression support this hypothesis Citation55. The known ligands for CD94/NKG2A (inhibitory) or CD94/NKG2C (activating) heterodimers are HLA-E molecules presenting leader peptides of classical MHC class I molecules Citation38. Since Hsp70 membrane positive CX+ and Hsp70 negative CX− tumour cells did not differ with respect to the expression of non-classical MHC class I molecules, as determined by flow cytometry with the anti-body supernatants 87G (IgG2a, D. Geraghty) or TP25.99 (IgGl, S. Ferrone), it is unlikely that HLA-G or -E molecules play a role in the interaction of CD94 positive NK cells with Hsp70. A reduced MHC expression has been found to provide a signal for the attack of NK cells Citation28. Since Hsp70 membrane positive and negative tumour target cells did not differ with respect to their MHC class I expression Citation23, an involvement of MHC in the interaction of NK cells with Hsp70 could be excluded. Unlike conventional MHC class I, non-classical MICA and MICB are minimally expressed under physiological conditions. However, following stress their expression is upregulated Citation41. One could imagine that concomitant with MICA and MICB, the amount of membrane-bound Hsp70 might also be upregulated. This hypothesis is unlikely, since differences in the cytolytic response of NK cells against Hsp70 membrane positive and negative tumour cell types are detectable already under physiological conditions and did not alter following non-lethal heat stress. Furthermore, by flow cytometry, no differences in Hsp70 cell membrane expression were detectable in heat-stressed CX+ and CX− cells Citation23.
In summary, the data provide evidence that, membrane-bound Hsp70 or the extracellular exposed Hsp70 peptide TKD might provide a tumour-selective target recognition structure for CD94 positive NK cells. The elucidation of potential co-receptors or partner molecules of CD94 that mediate triggering signals following contact with Hsp70 protein or Hsp70 peptides are currently under investigation.
Acknowledgement
This work was supported by the BMBF grant 0312338 BioChance.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Lindquist S, Craig EA. The heat shock proteins. Amu Rev Genet 1988; 22: 631–677
- DeNagel DC, Pierce SK. A case for chaperones in antigen processing. Immunol Today 1992; 3: 86–89
- Hartl FU. Molecular chaperones in protein folding. Nature 1996; 381: 571–580
- Srivastava PK, Deleo AB, Old LJ. Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc Natl Acad Sci USA 1986; 83: 3407–3411
- Srivastava PK. Heat shock proteins in immune response to cancer: The fourth paradigm. Experientia 1994; 50: 1094–1160
- Tamura Y, Peng P, Liu K, Daou M, Srivastava PK. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997; 278: 117–120
- Schild H-J, Arnold-Schild A, Lammert E, Rammensee H-G. Stress proteins and immunity mediated by cytotoxic T lymphocytes. Curr Opin Immunol 1999; 11: 109–114
- Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 1995; 269: 1585–1587
- Srivastava PK, Menoret A, Basu S, Binder RJ, McQuade KL. Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Immunity 1998; 8: 657–662
- Udono H, Srivastava PK. Heat shock protein 70-associated peptides elicit cancer immu nity. J Exp Med 1993; 178: 1391–1396
- Arnold-Schild D, Hanau D, Spehner D, Schmid C, Rammensee H-J, Salle EH, Schild H. Receptor-mediated endocytosis of hsp by professional antigen-presenting cells. J Immunol 1999; 162: 3757–3760
- Binder RJ, Han DK, Srivastava PK. CD91: A receptor for heat shock protein gp96. Nat Immunol 2000; 1: 151–155
- Basu S, Binder RJ, Ramalingham T, Srivstava PK. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 2001; 14: 303–313
- Asea A, Kraeft S-K, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK. Hsp70 stimulates cytokine production through a CD14-dependent pathway, demonstrating ist dual role as a chaperone and cytokine. Nat Med 2000; 6: 435–442
- Castellino F, Boucher PE, Eichelberg K, Mayhew M, Rothman JE, Houghton AN, Germain RN. Receptor mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways. J Exp Med 2000; 191: 1957–1964
- Udono H, Levey DL, Srivastava PK. Cellular requirements for tumor-specific immunity elicited by heat shock proteins: tumor rejection antigen gp96 primes CD8 + T cells in vivo. Proc Natl Acad Sci USA 1994; 91: 3077–3081
- Ferrarini M, Heltai S, Zocchi MR, Rugarli C. Unusual expression and localization of heat-shock proteins in human tumor cells. Int J Cancer 1992; 51: 613–619
- Piselli P, Vendetti S, Poccia F, Cicconi R, Mattei M, Bolognesi A, Stripe F, Colizzi V. In vitro and in vivo efficacy of heat shock protein specific immunotoxins on human tumor cells. J Biol Regul Homeost Agents 1995; 9: 55–62
- Tamura Y, Tsuboi N, Sato N, Kikuchi K. 70 kDa heat shock cognate protein is a transformation-associated antigen and a possible target for the host's anti-tumor immu nity. J Immunol 1993; 151: 5516–5524
- Multhoff G, Botzler C, Wiesnet M, Müller E, Meier T, Wilmanns W, Issels RD. A stress- inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer 1995; 61: 272–279
- Altmeyer A, Maki RG, Feldweg AM, Heike M, Protopopov VP, Masur SK, Srivastava PK. Tumor-specific cell surface expression of the -KDEL containing, endoplasmic reticular heat shock protein gp96. Int J Cancer 1996; 69: 340–349
- Multhoff G, Botzler C, Wiesnet M, Eissner G, Issels RD. CD3− large granular lympho cytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcoma cells. Blood 1995; 86: 1374–1382
- Multhoff G, Botzler C, Jennen L, Schmidt J, Ellwart J, Issels RD. Heat shock protein 72 on tumor cells. A recognition structure for Natural Killer cells. J Immunol 1997; 158: 4341–4350
- Botzler C, Issels R, Multhoff G. Heat-shock protein 72 cell-surface expression on human lung carcinoma cells in associated with an increased sensitivity to lysis mediated by adherent natural killer cells. Cancer Immunol Immunother 1996; 43: 226–230
- Botzler C, Kolb H-J, Issels RD, Multhoff G. Noncytotoxic alkyl-lysophospholipid treat ment increases sensitivity of leukemic K562 cells to lysis by natural killer cells (NK). Int J Cancer 1996; 65: 633–638
- Multhoff G. Hsp72, a hyperthermia-inducibb immunogenic determinant on leukemic K562 and Ewing's sarcoma cells. Int J Hyperthermia 1997; 13: 39–48
- Trinchieri G. Biology of natural killer cells. Adv Immunol 1989; 47: 187
- Ljunggren HG, Kärre K. In search of the missing self: MHC molecules and NK recog nition. Immunol Today 1990; 11: 237–244
- Siliciano RF, Pratt JC, Schmidt RE, Ritz J. Reinherz EL. Activation of cytolytic Tlymphocytes and NK cell function through the Til sheep erythrocyte binding protein. Nature 1985; 317: 428–430
- Lanier LL, Ruitenberg JJ, Phillips JH. Functional and biochemical analysis of CD16 antigen on NK cells and granulocytes. J Immunol 1988; 141: 3478–3485
- Long EO. Regulation of immune responses through inhibitory receptors. Annu Rev Immunol 1999; 17: 875–904
- Vales-Gomez M, Reyburn H, Strominger J. Molecular analyses of the interactions between human NK receptors and their HLA ligands. Human Immunol 2000; 61: 28–38
- Zingoni A, Palmieri G, Morrone S, Carretero M, Lopez-Botet M, Piccoli M, Frati L, Santoni A. CD69-triggering ERK activation and functions are negatively regulated by CD94/NKG2A inhibitory receptor. Eur J Immunol 2000; 30: 644–651
- Lopez-Botet M, Perez-Villar JJ, Carretero M, Rodriguez A, Melero I, Bellon T, Llano M, Navarro F. Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules. Immunol Rev 1997; 155: 165–172
- Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R. Activating receptors and coreceptors involved in human NK cell mediated cytolysis. Annu Rev Immunol 2001; 19: 197–223
- Lanier LL, Corliss B, Wu J, Phillips JH. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 1998; 8: 693–701
- Lanier LL, Chang C, Phillips JH. Human NKR-Pla. A disulphide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes. J Immunol 1994; 153: 2417–2423
- Braud VM, Allan DSJ, O'Callaghan CA, Soderstrom K, D'Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH, Lanier LL, McMichael JA. HLA-E binds to natural killer cell receptors CD94/NKG2A, B, and C. Nature 1998; 391: 795–798
- Lanier LL. Turning on natural killer cells. J Exp Med 2000; 191: 1259–1262
- Lanier LL. On guard—activating NK cell receptors. Nat Immunol 2001; 2: 23–27
- Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T. Activation of NK cells and T cells by NKG2D, a receptor for stress inducible MICA. Science 1999; 285: 727–730
- Cosman D, Mullberg J, Fanslow W, Armitage R, Chin W, Cassiano I. The human cytomegalivirus (HCMV) glycoprotein, UL16, binds to the MHC class I-related protein, MICB/PERB11, and to two novel, MHC class I related molecules ULBP1 and ULBP2. Faseb J 2000; 14: 1018–1023
- Diefenbach A, Jamieson AM, Liu SD, Shastri N, Raulet DH. Ligands for the murine NKG2D receptor: Expression by tumor cells and activation of NK cells and macrophages. Nat Immunol 2000; 1: 119–126
- Malarkannan S, Skih PP, Eden PA, Horng T, Zuberi AM, Christianson G, Roopenian D, Shastri N. The molecular and functional characterization of dominant minor H antigen H60. J Immunol 1998; 161: 3501–3509
- Wagtmann N, Biassoni R, Cantoni C, Verdiabnoi S, Malnati MS, Vitale M, Bottino C, Moretta L, Moretta A, Long EO. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both extra and intracellular domains. Immunity 1995; 2: 439–449
- Moretta A, Sivori S, Vitale M, Pende D, Morelli L, Augugliaro R, Bottino C, Moretta L. Existence of both inhibitory (p58) and activating (p50) receptors for HLA-C molecules in human natural killer cells. J Exp Med 1995; 182: 875–879
- Andre P, Biassoni R, Colonna M, Cosman D, Lanier LL, Long EO, Lopez-Botet M, Moretta A, Moretta L, Parham P, et al. New nomenclature for MHC receptors. Nature Immunol 2001; 2: 661
- Welch WJ, Suhan JP. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol 1986; 103: 2035–2052
- Strong DM, Ahmed AA, Thurman GB, Sell KW. In vitro stimulation of murine spleen cells using a microculturesystem and a multiple automated sample harvester. J Immunol Methods 1973; 2: 279–284
- Multhoff G, Mizzen L, Winchester CC, Milner CM, Wenk S, Kampinga HH, Laumbacher B, Johnson J. Heat shock protein 70 (Hsp70) stimulates proliferation and cytolytic activity of NK cells. Exp Hematol 1999; 27: 1627–1636
- Botzler C, Li G, Issels R, Multhoff G. Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response. Cell Stress Chaperones 1998; 3: 6–11
- Reineke U, Ehrhard B, Sabat R, Volk H-D, Schneider-Mergener J. Mapping discontin uous epitopes using-cellulose-bound peptide libraries. Immunobiology 1996; 196: 96–104
- Multhoff G, Pfister K, Gehrmann M, Hantschel M, Gross C, Hafner M, Hiddemann WA. 14-mer Hsp70 peptide stimulates NK cell activity. Cell Stress Chaperones 2001; 6: 337–344
- Binder RJ, Harris ML, Menoret A, Srivastava PK. Saturation, competition, and specifi city of heat shock proteins (hsp) gp96, hsp90, and hsp70 with CDllb+ cells. J Immunol 2000; 165: 2582–2586
- Gross C, Hansch D, Gastpar R, Multhoff G. Binding of Hsp70, Hsp70C, and the Hsp70 peptide TKD to NK cells involves receptor CD94. Biol Chem 2003; 384: 267–279
- Milani V, Noessner E, Ghose S, Kuppner M, Ahrens B, Scharner A, Gastpar R, Issels RD. Int J Hyperthermia 2002; 18: 563–575