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Natural killer cells in immune regulation
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moleculesin a novel endocyticcompartmentin B lymphocytes.Nature 369:113-120, 1994. DenzinL, RobbinsN, Carboy-NewcombC, Cresswell P: Assemblyand intracellulartransport of HLA-DM and correctionof the class It antigen-processingdefect in T2 cells. Immunity 1:595-606, 1994. PierceSK, Morris JF, GrusbyMJ et al.: Antigenpresentingfunctionof B lymphocytes.Immunol Rev 106:149-180, 1988. LanzavecchiaA: Antigen-specificinteractions betweenT and B cells. Nature314:537-539, 1985. CastenLA, Lakey EK, Jelachich ML, Margoliash E, Pierce SK: Anti-immunoglobulin augments the B-cell antigen-presentationfunction independentlyof internalizationof receptorantigencomplex. Proc Natl Acad Sci USA 82:5890-5894, 1985. Xu X, Press B, Wagle NM, Cho H, WandingerNess A, Pierce SK: B cell antigenreceptor signaling links biochemicalchanges in the class II peptide-loadingcompartmentto enhancedpro-
cessing, lnt Immunol8:1867, 1996. 37. FaassenAE, Pierce SK: Cross-linkingcell surface class II moleculesstimulatesIg-mediatedB cell antigenprocessing. J Immunol155:17371745, 1995. 38. FaassenAE, Dalke D, Berton MT, WarrenWD, Pierce SK: CD40-CD40-1igandinteractions stimulate B cell antigenprocessing.Eur J lmmunol25i3249, 1995. 39. BoackleSA, Holers VM, Karp DR: CD21 augments antigenpresentationin immuneindividuals. Eur J Immunol27:122-130, 1997. 40. CristauB, Schafer PH, Pierce SK: Heat shock enhances antigenprocessingand accelerates the formationof compact class II ab dimers. J Immunol 152:1546-1556, 1994. 41. MatzingerP: Tolerance,dangerand the extended family.Ann Rev Immunol12:991-1045, 1994. 42. MorrisJF, Hoyer JT, Pierce SK: Antigenpresentation for T cell interleukin-2secretion is a late acquisitionof neonatalB cells. Eur J Immunol
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22:2923-2928, 1992. 43. DomanicoSZ, Pierce SK: Virusinfectionblocks the processingand presentationof exogenous antigen with the majorhistocompatibilitycomplex class II molecules.Eur J lmmunol22:2055, 1992. 44. MachB, Steimle V, Martinez-SoriaE, Reith W: Regulationof MHC-class 1Igenes: Lessons from a disease. Ann Rev lmmunol 14:301-331, 1997. 45. NepomGT, Nepom B: The Major Histocompatibilty Complex. In: RheumatologyJH Klippel and PA Dieppe (Eds.). Mosby Year Book pp. 12.1-12.12, 1994. 46. Pinet V, Combe B, AvinensO: Polymorphisms of HLA-DMAand DMB genes in rheumatoid arthritis. Arthritis Rheum40:854-858, 1997. 47. ArmstrongTD, ClementsVK, MartinBK et al.: Major histocompatibilitycomplex class ll-transfected tumor cell lines present endogenousantigen and are potentstimulatorsof tumor specific immunity.Proc Natl Scad Sci USA 94:68866891, 1997.
N a t u r a l K i l l e r Cells i n I m m u n e R e g u l a t i o n Mary C. N a k a m u r a University of California, San Francisco, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 'atural Killer (NK) Cells are lymphocytes that play important roles in a variety of immune responses through their cytolytic effector function and by the production of immunoregulatory cytokines. NK cells are defined by their ability to spontaneously lyse certain tumor cells or virally-infected cells without prior sensitization. 1 In contrast to cytolytic T cells (CTL), N K cells do not require target cell expression of MHC Class I molecules for activation. NK cells are distinguished from other lymphocytes by their absence of T cell receptor or surface Ig expression and by their large, granular phenotype] -3 NK cell function has long been implicated in innate immunity against viruses, intracellular bacteria, and parasites 4'5 although the molecular basis of NK cell recognition and the receptors involved in their specificity were not defined. Recent studies have begun to define both activating and inhibitory receptors on N K cells that
N
regulate NK cell function in response to immune stimuli. 6
Missing Self Hypothesis A distinctive feature of NK cell cytotoxicity is the increased sensitivity to lysis by NK cells of target cells that do not express MHC. 7 The "missing self' hypothesis, initially proposed by K~irre, postulates that NK cells survey targets for the normal expression of self MHC Class I molecules, and lyse targets when the MHC Class I expression is aberrant or absent. 7 Thus, NK cell function would complement T cell function in situations where surface MHC Class I expression is downregulated, for example, on virally-infected cells. Without MHC Class I expression, such virally-infected cells would escape CTL surveillance, but would be more susceptible to NK cell lysis. The identification of MHC Class I-specific receptors on NK cells that mediate inhibition of NK cell function supports this model.
© ElsevierScience Inc.
Inhibitory Receptors on NK Cells That Recognize MHC Class I A major advance in understanding the molecular basis for N K cell specificity has been the recent identification of MHC Class I-specific inhibitory receptors on both human and m u r i n e NK cells. 8-1° Human NK cells express at least 2 families of KIR receptors (Killer Cell Inhibitory Receptor) specific for HLA Class I. The KIRs are type I glycoproteins of the Ig superfamily and can be divided into the KIR-2D family (2 extracellular Ig domains) and the KIR-3D family (3 extracellular Ig domains). 9 KIR 2-D receptors.(p58) recognize a polymorphic determinant of HLA-C, while certain KIR-3D (NKB 1) molecules are specific for the HLA-Bw4 allelic subset of HLA-B molecules. Studies in murine NK cells identified a distinct family of MHC Class I-specific inhibitory receptors, the Ly-49 family) Ly-49 molecules are type II integral membrane proteins that exist as homodimers,
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with an extracellular domain homologous to the C-type lectin superfamily. The ligand for the inital member of this multigene family, Ly-49A, is the Class I MHC D d or D k. 8.11 A human Ly-49 homologue has not been identified, though other lectin-like type II integral membrane proteins with MHC Class I specificity, CD94/NKG2 heterodimers, have been identified on human NK cells. 12 Despite the marked structural differences between the KIR and Ly-49 type receptors, both types of molecules bind specific MHC Class I antigens and inhibit NK cell lysis of targets expressing their respective ligands,J3 thus serving identical functions. Interestingly, both KIR and Ly-49 receptors are also expressed on a small subset of T lymphocytes suggesting that they may regulate both NK and T cell responses. TM
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nition of bone marrow grafts is highly specific and does not obey the traditional laws of transplantation. 15a6 Hybrid resistance refers to the rejection of parental bone marrow grafts by natural killer cells of F1 hybrid mice (derived from two different inbred parental strains, A x B). All NK cells express inhibitory MHC Class Ispecific receptors inhibited by self MHC. In F1 hybrid mice (A x B), some subsets of NK cells express inhibitory MHC Class 1-specific receptors that recognize one parental MHC haplotype (A) but not the other parental MHC (B). Bone marrow grafts from one parent (B) are rejected by this subset of F1 NK cells without MHC class I-specific inhibitory receptors to the parental (B) MHC-Class I. Recent studies in mice demonstrated that the Ly-49A and Ly-49C receptors are involved in hybrid resistance against bone marrow allografts. 16 Studies of NK cell receptors in human allogeneic bone marrow transplantation will be of considerable interest. Cytokine Production
MHC Class 1-specific receptors on NK cells mediate inhibition of NK cell function.
Possible Physiologic Roles of NK Cells
The exact physiological role of NK cells in the normal immune response has not yet been clearly defined, though their known functions indicate a likely role in i) regulation of immune response to pathogens by cytokine production; ii) cytolytic activity against infected cells, or infectious organisms; or iii) cytolytic activity against tumor cells. The identification of MHC Class I specific inhibitory receptors has led to considerable interest in the possible role of NK cells in allogeneic rejection of bone marrow grafts and pregnancy. Hybrid Resistance
NK cells have been demonstrated to mediate the acute and rapid rejection of allogeneic bone marrow grafts in nonimmunized, irradiated mice. NK cell mediated recog-
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NK cells are powerful secretors of interferon-y (IFN-D and granulocytemacrophage colony stimulating factors, and have also been shown to produce TNFc~, macrophage colony stimulating factor, IL-3 and chemokines (IL-8 and MIP- lo~.l NK cells can be induced to produce cytokines following stimulation with cytokines (IL- 12, TNFoc, IL- 1) or triggering by surface receptors (CD16, NKR-P1, immune complexes). 2 Through production of cytokines, NK cells participate as regulatory cells in antigen-specific immunity, particularly via production of interferon-'/. Early in the response to infection, interferon-y production by NK cells can stimulate antigen-independent activation of phagocytic cells and favor the development of T helper 1 (TH1) type antigen specific T-cells, which in turn produce IFN-y and IL-2] 8 Parasitic Infections
NK cells play a role in both limiting parasite replication and promoting the development of adaptive cell-mediated immunity. The principal affect of NK cells is via cytokine production rather than cytolytic activity. 5 However, NK cell-mediated parasite destruction has been demonstrated using extracellular tachyzoites of Toxoplasma gondii. Also, NK cells have been © ElsevierScience Inc.
shown to have cytolytic activity against macrophages infected with Plasmodium falciparum, T. gondii or Leishmania major. It remains unclear whether the direct cytolytic activity by NK cells occurs during natural infection. Indirect stimulation of NK cells by IL-12 (produced by infected macrophages or dendritic cells), leads to NK production of IFN-7 and TNF-cc, which plays a major role in activating phagocytes such as macrophages, stimulating killing of intracellular and extracellular microorganisms. In addition to mediating innate resistance in the early response to infection, NK cells may play a role in development of the TH l adaptive immune response to parasitic infection] 8 Viral Infections
The first demonstration for a role of NK cells in host defense against infection came from experimental viral infections in mice. Depletion of NK cells results in increased susceptibility of mice to murine CMV and HSV. 4 Evidence from natural infections in humans supports a role for NK cells in defense against certain human viral infections. Low NK cell cytotoxic activity correlates with increased human sensitivity to severe disseminated herpesgroup viral infections including HSV, EBV and HCMV. 4 Patients described with a complete lack of NK phenotypic cells and NK cytotoxicity, have developed severe and recurrent herpes group viral infections) 9 Viral-induced interferon oq13 activates NK cytotoxicity and blastogenesis, and these responses are associated with changes in NK cell trafficking and organ distribution. Certain viral infections can spontaneously induce IL-12 production, which stimulates IFN-y production by NK cells. 4 The NK cell response to viral infection is also affected by the ability of certain viruses to down-regulate host MHC Class I synthesis or transport. Diminished levels of MHC Class I antigens on virally-infected cells might render these cells more susceptible to NK cell-mediated cytotoxicity or augment NK cell cytokine production. Interestingly, both HCMV and MCMV have been demonstrated to encode a Class I like protein that has recently been proposed to interact with NK inhibitory-MHC specific receptors. Recent studies have suggested that the Class I like protein
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encoded by the virus is able to inhibit NK cell function, through interaction with MHC specific-inhibitory receptors on NK cells. 2°'21 This is a unique example of a viral strategy to evade immune surveillance by NK cells.
Tumor Surveillance Class I MHC down regulation is also a feature of neoplastic cells. In an experimental model, NK cells have been shown to lyse and reject a transplanted hematopoietic tumor cell line that lacks class I expression. 22 It remains to be elucidated whether or not NK cells play a role in active tumor surveillance against primary tumors.
demonstrated to recruit the SHP-1 phosphatase, and Ly-49A mediated inhibition of NK cells has been demonstrated to be deficient in motheaten mice (deficient in SHP- 1)27 Recruitment of the SHP- 1 phosphatase leads to a receptor-mediated MHC-Class I "short circuiting" of activating signals (tyrosine phosphorylation, inositol phosphate turnover, Ca 2÷ flux) required for NK cell activation. 26"28
N K
Pregnancy Maternally-derived NK cells accumulate at the sites of implantation in the decidua during pregnancy in all placental species studied. 23 There is considerable interest in the role of these cells in the unique maternal-fetal "tolerance" state of pregnancy. Recent evidence that human NK cell inhibitory receptors can interact with HLA-G, expressed by fetal trophoblast cells has demonstrated that NK cells may be "inhibited" in their ability to lyse fetal cells. 24 Still, while the exact role of decidual NK cells in normal and abnormal pregnancy remains to be elucidated, studies of NK cell receptors and decidual NK cells have provided important new insights into the complex questions of maternal-fetal immunology.
Molecular Mechanisms of Inhibition of NK Cell Function by Inhibitory Receptors Although the KIR and Ly-49 receptors have significant differences in their protein structures, they share the presence of an ITIM (immunoreceptor tyrosine based inhibitory motif) sequence in their cytoplasmic domains. 25 Previous studies in B cells had demonstrated that a phosphorylated ITIM sequence in the inhibitory Fc),RIIB receptor recruited the SHP-1 phosphatase (SH-2 domain containing protein tyrosine phosphatase), which led to inhibition of B cell activation. 26 Similarly, recruitment of SHP-1 by phosphorylated KIR receptors has been demonstrated to occur with inhibition of NK cell lysis of MHC-Class I expressing target cells. 25 The murine Ly-49A receptor has also been
cells influence the immune response via cytokines and ctyolysis.
tor B chain genes. J Exp Med 163:209, 1986. 3.
Sinkai Y, Rathbun G, et al.: RAG-2 deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855, 1992.
4.
Biron CA: Activation and function of natural killer cell responses during viral infections. Current Opinion in Immunol 9:24, 1997.
5.
Scott P, Trinchieri G: The role of natural killer cells in host-parasite interactions. Curr Opin Immunol 7:34. 1995.
6.
Lanier LL, Corliss B, Phillips JH: Arousal and inhibition of human NK cells. Immunol Rev 155:145, 1997
7.
Ljunggren H-G, K~irre K: In search of the "missing self': MHC molecules and NK cell recognition. Immunol Today 11:237, 1990.
8.
Karlhofer FM, Ribaudo RK, Yokoyama WM: MHC class I alloantigen specificity of mLy49+ IL-2 activated NK cells. Nature 358:66, 1992.
9.
Long EO et al.: Killer cell inhibitory receptors: diversity, specificity and function. Immunol Reviews 155:135, 1997.
10. Takei F, Brennan J, Maser DL: The Ly-49 family: genes, proteins and recognition of class I MHC. Immunol Rev 155:67, 1997. 11. Daniels BE Karlhofer FM, Seaman WE, Yokoyama WM: A natural killer cell receptor specific for a major histocompatibility complex class I molecule. J Exp Med 180:687, 1994.
Summary NK cells can influence the immune response through cytokine production and cytolysis of specific target cells. The identification of MHC-Class I specificreceptors on NK cells and their inhibitory effect on signal transduction has greatly enhanced our understanding of the mechanism of action of NK cells. NK cell function is dependent on the balance of inhibitory and activating signals received by the cell, from both direct interaction with target cells and from local immunoregulatory cytokines. The recent progress in understanding the molecular basis of NK cell function has renewed considerable interest in the exact biological role of these cells. The role NK cells play in cancer, infectious disease, bone marrow transplantation, pregnancy and autoimmunity are currently active areas of research as well as further studies to define the molecular basis for NK activation and specificity.
Selected References/Reviews 1.
Trichieri G: Biology of Natural Killer Cells. Adv Immunol 47:187, 1989.
2.
Lanier LL, Cwirla S, Felderspiel N, Phillips JH: Human natural killer cells isolated from peripheral blood do not rearrange T cell antigen recep-
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12. Lazetic S, Chang C, Houchins JP et al.: Human NK cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits. J Immunol 157:4741, 1996. 13. Gumperz JE, Parham P: The enigma of the natural killer cell. Nature 378:245, 1995. 14. D'AndreaA, Chang C, Phillips JH et al.: Regulation of "r cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J Exp Med 184:789, 1996. 15. Kumar V, et al.: Role of routine NK cells and their receptors in hybrid resistance. Current Opinion in Immunol 9:52, 1997. 16. Yu YYL, Kumar V, Bennett M: Murine natural killer cells and marrow graft rejection. Ann Rev lmmunol 10:189, 1992. 17. Yu YYL, George T, Dorfman JR, Roland J, et al.: The role of Ly49A and 5E6 (Ly49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts. Immunity 4:67, 1996. 18. Scharton TM, Scott P: Natural killer cells are a source of interferon y that drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice. J Exp Med 178:567, 1993. 19. Biron CA, Byron KS, Sullivan JL: Severe herpes virus infection in an adolescent without natural killer cells. New Engl J Med 320:1731, 1989. 20. Farrell HE, et al.: Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo. Nature 386:510, 1997.
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21. Reyburn, HT, et al.: The class I MHC homologue of human cytomegalovirus inhibits attack by NK cells. 1997. Nature 386:514-7 22. Glas R, Waldenstrom M, Hoglund Pet al.: Rejection of tumors in mice with severe combined immunodeficiency syndrome determined by, the major histocompatibility complex. Class I expression on the graft. Cancer Res 55:1911, 1995. 23. King A, Loke YW, Chaouat G: NK cells and reproduction. Immunol Today 18:64, 1997
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24. Rouas-Freiss N, Marchal RE et al.: The alpha 1 domain of HLA-G 1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLAG the public ligand for NK cell inhibitory receptors? Proc Natl Acad Sci USA 94:5249, 1997. 25. Burshtyn DN, et al.: Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor. Immunity 4:77, t996. 26. D'Ambrosio D, Hippen KL et al.: Recruitment and activation of PTPIC in negative regulation of antigen receptor signaling by FcTRIlB 1.
Science 268:293, 1995. 27. Nakamura MC et al.: Mouse Ly-49A interrupts early signaling events in NK Cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J Exp Med 673, 1997. 28. Valiante NM, Phillips JH, Lanier LL et al.: Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-y signaling complex in human natural killer ~NK) cells. J Exp Med 184:2243, 1996.
Rapid, Economical Testing in the Clinical Laboratory: A New Flow Cytometryobased Multiplex System Ralph L. McDade and Michael D. Spain Luminex Corporation, 12212 Technology Bh'd., Austin, Texas
Introduction he clinical immunologist is confronted with an increasingly complex and diverse array of diagnostic targets. For example, serum immunodeficiency analysis that once consisted of measuring immunoglobulins G, A, and M, has now evolved to include IgG subclasses and a long list of cytokines and chemokines. In fact, the analysis of most disease conditions has become more complicated, requiring more frequent and expensive diagnostic testing at a time when laboratory budgets are fixed or even decreasing. In response to this progressing conflict, we have developed a flow cytrometry-based multiplexed system (FlowMetrix ®) that allows for the simultaneous assay of up to 64 different analytes in a single sample at a cost that recognizes the economic restraints impacting medicine and research today. Any ligand-binding reaction, including immunoassay, enzyme assay, receptor analysis, or nucleic acid hybridization is applicable to this system. As with enzyme-linked immunosorbent assays (ELISA) performed in polystyrene microtiter plates, the multiplex system reagents are tethered to polystyrene in the form of microspheres. Up to 64 uniquely colored microsphere sets, each carrying
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the reactants of a different bioassay, can be classified in a specially-enhanced flow cytometer, allowing real time data acquisition and analysis. The basic strategy of this multiplexed assay system is to use three parameters, side scatter, orange fluorescence, and red fluorescence to identify and classify individual subsets of microspheres. A fourth parameter, green fluorescence, is reserved to quantify the ligand-receptor, chemical, or enzymatic reaction on the surface of each subset. Flow cytometric analysis of the microspheres is facilitated by hardware and software that acquires the data from the cytometer, classifies the microspheres into subsets, and collates measurement information for each assay set in real time. This blend of microsphere and flow cytometer technology with the most recent advances in digital signal processing results in a fast and precise assay platform, requiring low sample volume (20 ~1) and minimal reagent consumption.
System Design Features Microspheres Microspheres are a key component of the system. As carriers of assay reactants, they have been used in latex agglutination tests for at least twenty-five years. Over this I
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© Elsevier Science Inc.
time, the reproducibility and stability of microsphere-based assays has become established in the diagnostic industry. To ensure the uniformity of both reagent coupling and sample delivery in automated systems, microspheres of a single size are used in all our assays. With precisely the same surface features, optimization of a coupling procedure with one microsphere set suffices for all other microsphere sets. This advantage simplifies assay development. A 5.5 micron, cross-linked polystyrene microsphere with surface carboxylate groups was found to provide the best combination of thermal stability, uniformity (1% CV for diameter), and reagent density required for the wide range of possible assays. Different microsphere sets are prepared by dissolving two proprietary hydrophobic fluorophores, one emitting in the red region of the spectrum and one emitting in the orange region, into the microsphere interior. By, adjusting the relative concentrations of these two dyes, 64 unique microsphere sets, classifiable by their unique shade of red/orange, have been created (Figure 1), Flow cytometric assays Horan and Wheeless suggested the use of flow cytometers for the determination of analytes in fluid smnples in 1977.1 Fulwyler
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34. 35.
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moleculesin a novel endocyticcompartmentin B lymphocytes.Nature 369:113-120, 1994. DenzinL, RobbinsN, Carboy-NewcombC, Cresswell P: Assemblyand intracellulartransport of HLA-DM and correctionof the class It antigen-processingdefect in T2 cells. Immunity 1:595-606, 1994. PierceSK, Morris JF, GrusbyMJ et al.: Antigenpresentingfunctionof B lymphocytes.Immunol Rev 106:149-180, 1988. LanzavecchiaA: Antigen-specificinteractions betweenT and B cells. Nature314:537-539, 1985. CastenLA, Lakey EK, Jelachich ML, Margoliash E, Pierce SK: Anti-immunoglobulin augments the B-cell antigen-presentationfunction independentlyof internalizationof receptorantigencomplex. Proc Natl Acad Sci USA 82:5890-5894, 1985. Xu X, Press B, Wagle NM, Cho H, WandingerNess A, Pierce SK: B cell antigenreceptor signaling links biochemicalchanges in the class II peptide-loadingcompartmentto enhancedpro-
cessing, lnt Immunol8:1867, 1996. 37. FaassenAE, Pierce SK: Cross-linkingcell surface class II moleculesstimulatesIg-mediatedB cell antigenprocessing. J Immunol155:17371745, 1995. 38. FaassenAE, Dalke D, Berton MT, WarrenWD, Pierce SK: CD40-CD40-1igandinteractions stimulate B cell antigenprocessing.Eur J lmmunol25i3249, 1995. 39. BoackleSA, Holers VM, Karp DR: CD21 augments antigenpresentationin immuneindividuals. Eur J Immunol27:122-130, 1997. 40. CristauB, Schafer PH, Pierce SK: Heat shock enhances antigenprocessingand accelerates the formationof compact class II ab dimers. J Immunol 152:1546-1556, 1994. 41. MatzingerP: Tolerance,dangerand the extended family.Ann Rev Immunol12:991-1045, 1994. 42. MorrisJF, Hoyer JT, Pierce SK: Antigenpresentation for T cell interleukin-2secretion is a late acquisitionof neonatalB cells. Eur J Immunol
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22:2923-2928, 1992. 43. DomanicoSZ, Pierce SK: Virusinfectionblocks the processingand presentationof exogenous antigen with the majorhistocompatibilitycomplex class II molecules.Eur J lmmunol22:2055, 1992. 44. MachB, Steimle V, Martinez-SoriaE, Reith W: Regulationof MHC-class 1Igenes: Lessons from a disease. Ann Rev lmmunol 14:301-331, 1997. 45. NepomGT, Nepom B: The Major Histocompatibilty Complex. In: RheumatologyJH Klippel and PA Dieppe (Eds.). Mosby Year Book pp. 12.1-12.12, 1994. 46. Pinet V, Combe B, AvinensO: Polymorphisms of HLA-DMAand DMB genes in rheumatoid arthritis. Arthritis Rheum40:854-858, 1997. 47. ArmstrongTD, ClementsVK, MartinBK et al.: Major histocompatibilitycomplex class ll-transfected tumor cell lines present endogenousantigen and are potentstimulatorsof tumor specific immunity.Proc Natl Scad Sci USA 94:68866891, 1997.
N a t u r a l K i l l e r Cells i n I m m u n e R e g u l a t i o n Mary C. N a k a m u r a University of California, San Francisco, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 'atural Killer (NK) Cells are lymphocytes that play important roles in a variety of immune responses through their cytolytic effector function and by the production of immunoregulatory cytokines. NK cells are defined by their ability to spontaneously lyse certain tumor cells or virally-infected cells without prior sensitization. 1 In contrast to cytolytic T cells (CTL), N K cells do not require target cell expression of MHC Class I molecules for activation. NK cells are distinguished from other lymphocytes by their absence of T cell receptor or surface Ig expression and by their large, granular phenotype] -3 NK cell function has long been implicated in innate immunity against viruses, intracellular bacteria, and parasites 4'5 although the molecular basis of NK cell recognition and the receptors involved in their specificity were not defined. Recent studies have begun to define both activating and inhibitory receptors on N K cells that
N
regulate NK cell function in response to immune stimuli. 6
Missing Self Hypothesis A distinctive feature of NK cell cytotoxicity is the increased sensitivity to lysis by NK cells of target cells that do not express MHC. 7 The "missing self' hypothesis, initially proposed by K~irre, postulates that NK cells survey targets for the normal expression of self MHC Class I molecules, and lyse targets when the MHC Class I expression is aberrant or absent. 7 Thus, NK cell function would complement T cell function in situations where surface MHC Class I expression is downregulated, for example, on virally-infected cells. Without MHC Class I expression, such virally-infected cells would escape CTL surveillance, but would be more susceptible to NK cell lysis. The identification of MHC Class I-specific receptors on NK cells that mediate inhibition of NK cell function supports this model.
© ElsevierScience Inc.
Inhibitory Receptors on NK Cells That Recognize MHC Class I A major advance in understanding the molecular basis for N K cell specificity has been the recent identification of MHC Class I-specific inhibitory receptors on both human and m u r i n e NK cells. 8-1° Human NK cells express at least 2 families of KIR receptors (Killer Cell Inhibitory Receptor) specific for HLA Class I. The KIRs are type I glycoproteins of the Ig superfamily and can be divided into the KIR-2D family (2 extracellular Ig domains) and the KIR-3D family (3 extracellular Ig domains). 9 KIR 2-D receptors.(p58) recognize a polymorphic determinant of HLA-C, while certain KIR-3D (NKB 1) molecules are specific for the HLA-Bw4 allelic subset of HLA-B molecules. Studies in murine NK cells identified a distinct family of MHC Class I-specific inhibitory receptors, the Ly-49 family) Ly-49 molecules are type II integral membrane proteins that exist as homodimers,
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with an extracellular domain homologous to the C-type lectin superfamily. The ligand for the inital member of this multigene family, Ly-49A, is the Class I MHC D d or D k. 8.11 A human Ly-49 homologue has not been identified, though other lectin-like type II integral membrane proteins with MHC Class I specificity, CD94/NKG2 heterodimers, have been identified on human NK cells. 12 Despite the marked structural differences between the KIR and Ly-49 type receptors, both types of molecules bind specific MHC Class I antigens and inhibit NK cell lysis of targets expressing their respective ligands,J3 thus serving identical functions. Interestingly, both KIR and Ly-49 receptors are also expressed on a small subset of T lymphocytes suggesting that they may regulate both NK and T cell responses. TM
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nition of bone marrow grafts is highly specific and does not obey the traditional laws of transplantation. 15a6 Hybrid resistance refers to the rejection of parental bone marrow grafts by natural killer cells of F1 hybrid mice (derived from two different inbred parental strains, A x B). All NK cells express inhibitory MHC Class Ispecific receptors inhibited by self MHC. In F1 hybrid mice (A x B), some subsets of NK cells express inhibitory MHC Class 1-specific receptors that recognize one parental MHC haplotype (A) but not the other parental MHC (B). Bone marrow grafts from one parent (B) are rejected by this subset of F1 NK cells without MHC class I-specific inhibitory receptors to the parental (B) MHC-Class I. Recent studies in mice demonstrated that the Ly-49A and Ly-49C receptors are involved in hybrid resistance against bone marrow allografts. 16 Studies of NK cell receptors in human allogeneic bone marrow transplantation will be of considerable interest. Cytokine Production
MHC Class 1-specific receptors on NK cells mediate inhibition of NK cell function.
Possible Physiologic Roles of NK Cells
The exact physiological role of NK cells in the normal immune response has not yet been clearly defined, though their known functions indicate a likely role in i) regulation of immune response to pathogens by cytokine production; ii) cytolytic activity against infected cells, or infectious organisms; or iii) cytolytic activity against tumor cells. The identification of MHC Class I specific inhibitory receptors has led to considerable interest in the possible role of NK cells in allogeneic rejection of bone marrow grafts and pregnancy. Hybrid Resistance
NK cells have been demonstrated to mediate the acute and rapid rejection of allogeneic bone marrow grafts in nonimmunized, irradiated mice. NK cell mediated recog-
0197- 1859/97/$0.00 + 17.00
NK cells are powerful secretors of interferon-y (IFN-D and granulocytemacrophage colony stimulating factors, and have also been shown to produce TNFc~, macrophage colony stimulating factor, IL-3 and chemokines (IL-8 and MIP- lo~.l NK cells can be induced to produce cytokines following stimulation with cytokines (IL- 12, TNFoc, IL- 1) or triggering by surface receptors (CD16, NKR-P1, immune complexes). 2 Through production of cytokines, NK cells participate as regulatory cells in antigen-specific immunity, particularly via production of interferon-'/. Early in the response to infection, interferon-y production by NK cells can stimulate antigen-independent activation of phagocytic cells and favor the development of T helper 1 (TH1) type antigen specific T-cells, which in turn produce IFN-y and IL-2] 8 Parasitic Infections
NK cells play a role in both limiting parasite replication and promoting the development of adaptive cell-mediated immunity. The principal affect of NK cells is via cytokine production rather than cytolytic activity. 5 However, NK cell-mediated parasite destruction has been demonstrated using extracellular tachyzoites of Toxoplasma gondii. Also, NK cells have been © ElsevierScience Inc.
shown to have cytolytic activity against macrophages infected with Plasmodium falciparum, T. gondii or Leishmania major. It remains unclear whether the direct cytolytic activity by NK cells occurs during natural infection. Indirect stimulation of NK cells by IL-12 (produced by infected macrophages or dendritic cells), leads to NK production of IFN-7 and TNF-cc, which plays a major role in activating phagocytes such as macrophages, stimulating killing of intracellular and extracellular microorganisms. In addition to mediating innate resistance in the early response to infection, NK cells may play a role in development of the TH l adaptive immune response to parasitic infection] 8 Viral Infections
The first demonstration for a role of NK cells in host defense against infection came from experimental viral infections in mice. Depletion of NK cells results in increased susceptibility of mice to murine CMV and HSV. 4 Evidence from natural infections in humans supports a role for NK cells in defense against certain human viral infections. Low NK cell cytotoxic activity correlates with increased human sensitivity to severe disseminated herpesgroup viral infections including HSV, EBV and HCMV. 4 Patients described with a complete lack of NK phenotypic cells and NK cytotoxicity, have developed severe and recurrent herpes group viral infections) 9 Viral-induced interferon oq13 activates NK cytotoxicity and blastogenesis, and these responses are associated with changes in NK cell trafficking and organ distribution. Certain viral infections can spontaneously induce IL-12 production, which stimulates IFN-y production by NK cells. 4 The NK cell response to viral infection is also affected by the ability of certain viruses to down-regulate host MHC Class I synthesis or transport. Diminished levels of MHC Class I antigens on virally-infected cells might render these cells more susceptible to NK cell-mediated cytotoxicity or augment NK cell cytokine production. Interestingly, both HCMV and MCMV have been demonstrated to encode a Class I like protein that has recently been proposed to interact with NK inhibitory-MHC specific receptors. Recent studies have suggested that the Class I like protein
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encoded by the virus is able to inhibit NK cell function, through interaction with MHC specific-inhibitory receptors on NK cells. 2°'21 This is a unique example of a viral strategy to evade immune surveillance by NK cells.
Tumor Surveillance Class I MHC down regulation is also a feature of neoplastic cells. In an experimental model, NK cells have been shown to lyse and reject a transplanted hematopoietic tumor cell line that lacks class I expression. 22 It remains to be elucidated whether or not NK cells play a role in active tumor surveillance against primary tumors.
demonstrated to recruit the SHP-1 phosphatase, and Ly-49A mediated inhibition of NK cells has been demonstrated to be deficient in motheaten mice (deficient in SHP- 1)27 Recruitment of the SHP- 1 phosphatase leads to a receptor-mediated MHC-Class I "short circuiting" of activating signals (tyrosine phosphorylation, inositol phosphate turnover, Ca 2÷ flux) required for NK cell activation. 26"28
N K
Pregnancy Maternally-derived NK cells accumulate at the sites of implantation in the decidua during pregnancy in all placental species studied. 23 There is considerable interest in the role of these cells in the unique maternal-fetal "tolerance" state of pregnancy. Recent evidence that human NK cell inhibitory receptors can interact with HLA-G, expressed by fetal trophoblast cells has demonstrated that NK cells may be "inhibited" in their ability to lyse fetal cells. 24 Still, while the exact role of decidual NK cells in normal and abnormal pregnancy remains to be elucidated, studies of NK cell receptors and decidual NK cells have provided important new insights into the complex questions of maternal-fetal immunology.
Molecular Mechanisms of Inhibition of NK Cell Function by Inhibitory Receptors Although the KIR and Ly-49 receptors have significant differences in their protein structures, they share the presence of an ITIM (immunoreceptor tyrosine based inhibitory motif) sequence in their cytoplasmic domains. 25 Previous studies in B cells had demonstrated that a phosphorylated ITIM sequence in the inhibitory Fc),RIIB receptor recruited the SHP-1 phosphatase (SH-2 domain containing protein tyrosine phosphatase), which led to inhibition of B cell activation. 26 Similarly, recruitment of SHP-1 by phosphorylated KIR receptors has been demonstrated to occur with inhibition of NK cell lysis of MHC-Class I expressing target cells. 25 The murine Ly-49A receptor has also been
cells influence the immune response via cytokines and ctyolysis.
tor B chain genes. J Exp Med 163:209, 1986. 3.
Sinkai Y, Rathbun G, et al.: RAG-2 deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855, 1992.
4.
Biron CA: Activation and function of natural killer cell responses during viral infections. Current Opinion in Immunol 9:24, 1997.
5.
Scott P, Trinchieri G: The role of natural killer cells in host-parasite interactions. Curr Opin Immunol 7:34. 1995.
6.
Lanier LL, Corliss B, Phillips JH: Arousal and inhibition of human NK cells. Immunol Rev 155:145, 1997
7.
Ljunggren H-G, K~irre K: In search of the "missing self': MHC molecules and NK cell recognition. Immunol Today 11:237, 1990.
8.
Karlhofer FM, Ribaudo RK, Yokoyama WM: MHC class I alloantigen specificity of mLy49+ IL-2 activated NK cells. Nature 358:66, 1992.
9.
Long EO et al.: Killer cell inhibitory receptors: diversity, specificity and function. Immunol Reviews 155:135, 1997.
10. Takei F, Brennan J, Maser DL: The Ly-49 family: genes, proteins and recognition of class I MHC. Immunol Rev 155:67, 1997. 11. Daniels BE Karlhofer FM, Seaman WE, Yokoyama WM: A natural killer cell receptor specific for a major histocompatibility complex class I molecule. J Exp Med 180:687, 1994.
Summary NK cells can influence the immune response through cytokine production and cytolysis of specific target cells. The identification of MHC-Class I specificreceptors on NK cells and their inhibitory effect on signal transduction has greatly enhanced our understanding of the mechanism of action of NK cells. NK cell function is dependent on the balance of inhibitory and activating signals received by the cell, from both direct interaction with target cells and from local immunoregulatory cytokines. The recent progress in understanding the molecular basis of NK cell function has renewed considerable interest in the exact biological role of these cells. The role NK cells play in cancer, infectious disease, bone marrow transplantation, pregnancy and autoimmunity are currently active areas of research as well as further studies to define the molecular basis for NK activation and specificity.
Selected References/Reviews 1.
Trichieri G: Biology of Natural Killer Cells. Adv Immunol 47:187, 1989.
2.
Lanier LL, Cwirla S, Felderspiel N, Phillips JH: Human natural killer cells isolated from peripheral blood do not rearrange T cell antigen recep-
© Elsevier Science Inc.
12. Lazetic S, Chang C, Houchins JP et al.: Human NK cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits. J Immunol 157:4741, 1996. 13. Gumperz JE, Parham P: The enigma of the natural killer cell. Nature 378:245, 1995. 14. D'AndreaA, Chang C, Phillips JH et al.: Regulation of "r cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J Exp Med 184:789, 1996. 15. Kumar V, et al.: Role of routine NK cells and their receptors in hybrid resistance. Current Opinion in Immunol 9:52, 1997. 16. Yu YYL, Kumar V, Bennett M: Murine natural killer cells and marrow graft rejection. Ann Rev lmmunol 10:189, 1992. 17. Yu YYL, George T, Dorfman JR, Roland J, et al.: The role of Ly49A and 5E6 (Ly49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts. Immunity 4:67, 1996. 18. Scharton TM, Scott P: Natural killer cells are a source of interferon y that drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice. J Exp Med 178:567, 1993. 19. Biron CA, Byron KS, Sullivan JL: Severe herpes virus infection in an adolescent without natural killer cells. New Engl J Med 320:1731, 1989. 20. Farrell HE, et al.: Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo. Nature 386:510, 1997.
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21. Reyburn, HT, et al.: The class I MHC homologue of human cytomegalovirus inhibits attack by NK cells. 1997. Nature 386:514-7 22. Glas R, Waldenstrom M, Hoglund Pet al.: Rejection of tumors in mice with severe combined immunodeficiency syndrome determined by, the major histocompatibility complex. Class I expression on the graft. Cancer Res 55:1911, 1995. 23. King A, Loke YW, Chaouat G: NK cells and reproduction. Immunol Today 18:64, 1997
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24. Rouas-Freiss N, Marchal RE et al.: The alpha 1 domain of HLA-G 1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLAG the public ligand for NK cell inhibitory receptors? Proc Natl Acad Sci USA 94:5249, 1997. 25. Burshtyn DN, et al.: Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor. Immunity 4:77, t996. 26. D'Ambrosio D, Hippen KL et al.: Recruitment and activation of PTPIC in negative regulation of antigen receptor signaling by FcTRIlB 1.
Science 268:293, 1995. 27. Nakamura MC et al.: Mouse Ly-49A interrupts early signaling events in NK Cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J Exp Med 673, 1997. 28. Valiante NM, Phillips JH, Lanier LL et al.: Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-y signaling complex in human natural killer ~NK) cells. J Exp Med 184:2243, 1996.
Rapid, Economical Testing in the Clinical Laboratory: A New Flow Cytometryobased Multiplex System Ralph L. McDade and Michael D. Spain Luminex Corporation, 12212 Technology Bh'd., Austin, Texas
Introduction he clinical immunologist is confronted with an increasingly complex and diverse array of diagnostic targets. For example, serum immunodeficiency analysis that once consisted of measuring immunoglobulins G, A, and M, has now evolved to include IgG subclasses and a long list of cytokines and chemokines. In fact, the analysis of most disease conditions has become more complicated, requiring more frequent and expensive diagnostic testing at a time when laboratory budgets are fixed or even decreasing. In response to this progressing conflict, we have developed a flow cytrometry-based multiplexed system (FlowMetrix ®) that allows for the simultaneous assay of up to 64 different analytes in a single sample at a cost that recognizes the economic restraints impacting medicine and research today. Any ligand-binding reaction, including immunoassay, enzyme assay, receptor analysis, or nucleic acid hybridization is applicable to this system. As with enzyme-linked immunosorbent assays (ELISA) performed in polystyrene microtiter plates, the multiplex system reagents are tethered to polystyrene in the form of microspheres. Up to 64 uniquely colored microsphere sets, each carrying
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the reactants of a different bioassay, can be classified in a specially-enhanced flow cytometer, allowing real time data acquisition and analysis. The basic strategy of this multiplexed assay system is to use three parameters, side scatter, orange fluorescence, and red fluorescence to identify and classify individual subsets of microspheres. A fourth parameter, green fluorescence, is reserved to quantify the ligand-receptor, chemical, or enzymatic reaction on the surface of each subset. Flow cytometric analysis of the microspheres is facilitated by hardware and software that acquires the data from the cytometer, classifies the microspheres into subsets, and collates measurement information for each assay set in real time. This blend of microsphere and flow cytometer technology with the most recent advances in digital signal processing results in a fast and precise assay platform, requiring low sample volume (20 ~1) and minimal reagent consumption.
System Design Features Microspheres Microspheres are a key component of the system. As carriers of assay reactants, they have been used in latex agglutination tests for at least twenty-five years. Over this I
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time, the reproducibility and stability of microsphere-based assays has become established in the diagnostic industry. To ensure the uniformity of both reagent coupling and sample delivery in automated systems, microspheres of a single size are used in all our assays. With precisely the same surface features, optimization of a coupling procedure with one microsphere set suffices for all other microsphere sets. This advantage simplifies assay development. A 5.5 micron, cross-linked polystyrene microsphere with surface carboxylate groups was found to provide the best combination of thermal stability, uniformity (1% CV for diameter), and reagent density required for the wide range of possible assays. Different microsphere sets are prepared by dissolving two proprietary hydrophobic fluorophores, one emitting in the red region of the spectrum and one emitting in the orange region, into the microsphere interior. By, adjusting the relative concentrations of these two dyes, 64 unique microsphere sets, classifiable by their unique shade of red/orange, have been created (Figure 1), Flow cytometric assays Horan and Wheeless suggested the use of flow cytometers for the determination of analytes in fluid smnples in 1977.1 Fulwyler