comparison of bovine mononuclear cells with other Species for cytolytic activity against virally-infected cells

Veterinary Immunology and Immunopathology, 20 (1989) 239-261 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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Comparison of Bovine Mononuclear Cells with Other Species for Cytolytic Activity Against Virally-Infected Cells CAROL G. COOK and GARY A. SPLITTER

Department of Veterinary Science, University of Wisconsin-Madison, 1655 Linden Drive, Madison, W153706 (U.S.A.) Supported in part by the College of Agriculture and Life Sciences and USDA grants 86-CRSR2-2848, 87-CRSR-2-3128 and BARD US- 1009-85. TABLE OF CONTENTS 1.0 INTRODUCTION 2.0 COMPARISON OF UNPRIMED, NON-MHC-RESTRICTED EFFECTOR CELLS LYTIC FOR VIRALLY-INFECTED TARGET CELLS 2.1 Effector cell populations 2.1.1 Natural killer cells 2.1.2 Natural cytotoxic cells 2.1.3 Monocytes/Macrophages 2.2 Morphological and phenotypic characteristics 2.3 Cell lineage 2.4 Recognition of target cell 2.5 Role of gamma interferon 3.0 COMPARISON OF VIRALLY-STIMULATED EFFECTOR CELLS LYTIC FOR VIRALLY-INFECTED TARGET CELLS 3.1 Viral and MHC restriction 3.2 Recognition of target cell 3.3 MHC unrestricted lysis 4.0 CONCLUSIONS 5.0 REFERENCES

1.0 INTRODUCTION

Cell-mediated immunity appears to play a major role in defense against many viral infections. Both natural cellular defenses and virus-primed specific defenses mediate resistance to viral replication and dissemination. Natural cellular defenses include natural killer (NK) cell, natural cytotoxic (NC) cell and macrophage-mediated lytic activity against virally-infected cells. These effector cells do not need prior sensitization, but appear capable of lysing cells infected with any one of a variety of viruses when first encountered. 0165-2427/89/$03.50

© 1989 Elsevier Science Publishers B.V.

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In addition, these effector cells are not major histocompatibility (MHC) restricted, with lytic activity against virally-infected autologous, allogeneic or xenogeneic target cells (Mogensen, 1979; Herberman and Ortaldo, 1981). In contrast to natural cellular defenses, lytic activity by cytotoxic T lymphocytes (CTL) is evident only if the animal has first been sensitized to the virus. CTL are antigen specific, possessing a T cell receptor that recognizes specific antigenic determinants in the context of self MHC molecules. Therefore, CTL are MHC restricted, lysing only target cells which share common MHC molecules. In some viral systems, cells taken directly from an animal at the optimal time after priming will lyse virally-infected target cells. However, in other systems, no lysis is detected after in vivo priming. If in vivo primed cells are cultured in vitro without virus in some cases, or with inactivated virus in other cases, cytotoxic T lymphocytes are then detected. A variety of bovine effector cells mediating killing of virally-infected target cells have been described. The goal of this review is to compare and contrast phenotypic and functional characteristics of cytolytic bovine mononuclear cells with those of other species. 2.0 COMPARISON OF UNPRIMED, NON-MHC-RESTRICTED EFFECTOR CELLS LYTIC FOR VIRALLY-INFECTED TARGET CELLS

2.1 Effector cell populations 2.1.1 Natural killer cells Evidence from human and mouse systems indicates NK cells are important in mediating resistance to viral replication and dissemination. In general, NK cells are nonadherent, nonphagocytosing lymphocytes without B or T lymphocyte markers. NK cells lyse certain tumor cells and virally-infected cells without prior sensitization and in an MHC-nonrestricted manner. Functional activity of NK cells can be augmented by interferon (IFN) (Colmenares and Lopez, 1986) and interleukin 2 (IL-2) (Shaw et al., 1985). Natural killer cells, which are present as large granular lymphocytes, are identified by their lytic activity against virally-infected cells as part of the first line of defense against viral infections. Another role for NK cells includes surveillance for tumor cells, with lysis of these abnormal cell populations. In addition, NK cells induce graft-versus-host reactions in bone marrow transplant recipients and mediate hemopoetic homeostasis and bone marrow graft rejection (Lopez, 1985). NK cells have been shown to limit viral infections both in vitro and in vivo. NK effector cells added to herpes simplex virus 1 (HSV-1)-infected human fibroblasts, limited replication of the virus when compared to infected fibroblasts with no effector cells present (Fitzgerald et al., 1985; Leibson et al., 1986). This inhibition of viral replication was shown to be due to cytotoxic NK

241 cells lysing HSV-l-infected fibroblasts before production of new viral progeny and independent of interferon production (Bishop et al., 1983a; Fitzgerald et al., 1985). Another important observation was that fresh peripheral blood mononuclear cells lysed HSV-l-infected autologous lymphoblastoid cell line target cells (Yasukawa and Zarling, 1983). These in vitro results indicate a role for NK cells in inhibiting viral infections in vivo, since NK cells limited viral replication and lysed virally-infected autologous cells. Bovine NK cell activity against virally-infected target cells has been described by several authors using different virally infected cells. In the first system, nonadherent, nonphagocytic peripheral blood mononuclear (PBM) cells ( < 0.1% phagocytic cells ) from parainfluenza virus 3 (PI-3) serologically negative cattle lysed PI-3-infected target cells (Campos et al., 1982). As with lysis of HSV-l-infected human fibroblasts, lysis of PI-3-infected bovine embryo cells, Georgia bovine kidney (GBK) cells or bovine testicular cells was detected in a long-term ( 18 h) assay with effector to target cells ratios of 100:1 and 25:1 (Campos et al., 1982). In the second system, P B M cells from normal sheep and cattle lysed fetal lamb kidney (FLK) cells persistently infected with bovine leukosis virus (BLV) (Yamamoto et al., 1985). Seventy to 80% of the FLK cells expressed BLV antigens on their cell surface. Sheep P B M cells lysed FLK target cells, and cattle PBM cells also lysed FLK target cells but at a reduced level (Yamamoto et al., 1985). There was no lysis of YAC-1 (a murine NK cell sensitive target), normal sheep testis or normal bovine kidney cells with either sheep or cattle effector cells (Yamamoto et ah, 1985). In contrast, others (Bielefeldt Ohmann et al., 1985) demonstrated YAC-1 cell lysis by bovine PBM cells. Populations of both adherent and nonadherent cells from cattle and sheep were cytotoxic against FLK target cells (Yamamoto et al., 1985), suggesting the participation of multiple effector cells or that bovine NK cells were a heterogeneous population. In the third system, nonadherent PBM cells from bovine herpesvirus 1 (BHV-1) nonimmune animals lysed BHV-l-infected xenogeneic A549 tumor target cells (Cook and Splitter, 1989). Monoclonal antibody (mAb) and complement (C') depletions and percoll density gradient enrichments were used to identify effector cells as nonadherent, large, low density, non T, non B mononuclear cells (Cook and Splitter, 1989). These effector cells resembled natural killer cells, lysing virally-infected target cells without prior sensitization as described in other species. NK cells have been shown to be important in the resistance to mouse cytomegalovirus (MCMV) infections in vivo. Depletion of NK cells in mice by injection of antibody to asialo GM1 resulted in increased synthesis of MCMV (Bukowski and Welsh, 1985 ). NK cells express high levels of glycosphingolipid asialo GM1 on their cell surface compared to T cells which express only low levels of asialo GM1 (Dennert et al., 1981 ). Therefore, in vivo treatment with

242 antibody to asialo GM1 allows for a fairly specific depletion of NK cells. In addition, mice with low NK cell activity associated with youth, strain, immunosuppressive drug treatment, or beige mutation are more susceptible to MCMV (Bukowski and Welsh, 1985). However, with other viruses, such as lymphocytic choriomeningitis virus (LCMV), no in vivo role for NK cells against LCMV appears to be present. Mice injected with antibody to asialo GM1 to deplete NK cells showed no change in LCMV synthesis (Bukowski and Welsh, 1985 ). Therefore, NK cells appear to be important effector cells in vivo against some viruses, but not others. The reason for these differences is under investigation, specifically addressing the role of gamma interferon (7-IFN), lytic capabilities and pathogenesis of the virus. Organ distribution of NK cell activity within an animal varies from species to species. Human (Nieminen and Saksela, 1985), mouse (Tagliabue et al., 1982), rat (Reynolds et al., 1982), dog (Loughgram et al., 1985) and cattle (Cook and Splitter, 1989) are similar, with high natural cytolytic activity found in lymphocytes obtained from peripheral blood or spleen and little or no NK cell activity in bone marrow lymphocytes. In contrast, little or no NK activity has been detected in cells from cat peripheral blood (Kooistra and Splitter, 1985; Tompkins and Tompkins, 1985), while high NK cell activity has been detected in cat spleen cells (Tompkins and Tompkins, 1985 ). In addition, little or no NK cell activity was found in mouse (Tagliabue et al., 1982), or rat (Reynolds et al., 1982) peripheral lymph nodes or thymus. NK cell activity in specific-pathogen-free miniature swine was high in cells from peripheral blood but no NK cell activity was detected in cells from thymus, mesenteric lymph nodes, bone marrow, tonsil or spleen (Kim et al., 1980). In contrast, other workers report that lymphocytes from both spleen and blood of adult pigs could lyse PI-3-infected Vero cells (Yang et al., 1987). 2.1.2 Natural cytotoxic cells Originally NK and NC cell differences were based on target cell selection and kinetics of lysis. NK cells lysed nonadherent lymphoid target cells in 4-6 h while both NK and NC cells lysed adherent target cells in 16-24 h (Stutman et al., 1980). Exceptions to this generalization have been observed, with nonlymphoid tumor targets being susceptible to NK and NC cell lysis (Tompkins et al., 1983). On the basis of cold target inhibition studies, NK and NC cells share common target cell recognition structures (Lattime et al., 1982b). Like NK cells, NC cells are nonadherent, nonphagocytosing, non B, non T lymphocytes. NK and NC cells differ phenotypically and functionally. Unlike NK cells, NC cells have no lytic activity in a 4-h assay (Lattime et al., 1982a). In addition, the NK cell deficient beige mouse has normal NC activity (Lattime et al., 1982a). While NK activity may be augmented with interferon, NC cells are not responsive to interferon (Colmenares and Lopez, 1986). NC cell activity was augmented by IL-3 while NK cells were not responsive to IL-3 (Lattime

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et al., 1983). NC cell activity can be distinguished from NK cell activity by treating the effector population with mAb to NK cell determinants (e.g., Qa 5 or asialo GM1) and C' to eliminate NK cells (Colmenares and Lopez, 1986). Therefore, numerous differences have been identified to distinguish NK and NC populations. Just as NK cells are found preferentially in specific tissues, NC cells are found in specific tissues depending on animal species. High NC activity was found in human and cat nonadherant PBM cells, cat and mouse spleen, and mouse bone marrow (Tompkins and Tompkins, 1985). Little NC activity was detected in mouse peripheral blood lymphocytes, lymph node cells, or thymus (Tompkins and Tompkins, 1985). Discrimination between lytic activity mediated by NK and NC cells in domestic species has been difficult for a number of reasons. Most significant is the lack of adequate mAb to cell surface determinants, which would allow for the identification of NK and NC cells. Other difficulties include the limited number of characterized target cells available and the lack of NK or NC deftcient animal models. There is a population of bovine effector cells that do not fit into typical NK or NC populations. These effector cells are a nonadherent, nonspecific esterase-negative population whose lytic activity can only be detected after 3 days in culture (Brigham and Rossi, 1986; Chung and Rossi, 1987). PBM cells obtained from BHV-1 nonimmune animals and cultured for 3 days in media alone lysed BHV-l-infected GBK cells in a 22-h assay (Brigham and Rossi, 1986). Freshly isolated PBM cells from the same animal did not lyse the virally-infected target cells (Brigham and Rossi, 1986). Results from further work showed that this same cultured effector population lysed BHV-l-infected bovine embryo kidney (BEK) cells in a 4-6-h assay (Chung and Rossi, 1987). It was concluded that the effector cell was enriched or generated by culturing or that a population, possibly T suppressor cells, was depleted by culturing (Chung and Rossi, 1987). The effector cell was not a cytotoxic T cell since there was no prior sensitization or MHC restriction of killing. It was also not a K cell, which mediates antibody-dependent cellular cytotoxicity (ADCC), for two reasons. First, no ADCC activity was detected with the addition of antibody to BHV-1 (Chung and Rossi, 1987), and, second, previous work has failed to detect a K cell in the bovine PBM cell population (Campos and Rossi, 1985a). This effector population was not considered a lymphokine-activated killer (LAK) cell, since there was no killing of uninfected target cells. The same workers have shown that bovine PBM cells from BHV-1 nonimmune calves cultured 6 days with lymphokine-rich supernatant lysed both BEK cells and BHV-l-infected BEK cells (Campos and Rossi, 1986b). One interpretation of these results would suggest that a natural killer-like effector cell from nonimmune animals was mediating the killing. The quiescent effector cells are activated by culturing on plastic or by factors produced by other activated cells.

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2.1.3. Monocytes / Macrophages In addition to NK and NC cells, macrophages appear to be important mediators of natural resistance to virus infections. Macrophages may play a role in resistance to HSV-1 infections. One reason newborn mice were more susceptible to lethal HSV-1 infections than adult mice was the inability of newborn peritoneal macrophages to restrict viral replication (Lopez, 1985). In vitro, 7-IFN-activated peritoneal macrophages from adult mice mediated lysis of HSV-l-infected target cells (Koff and Dunegan, 1986). Human monocytes activated with ?,-IFN lysed HSV-l-infected whole human embryo and xenogenic BALB/c 10EZ cells but not uninfected cells (Koff et al., 1984). In addition to mature human macrophages and monocyte-mediated lysis of HSV-infected cells, an immature monocyte population can also mediate lysis. Human peripheral blood monocytes, isolated after 1 h of adherence on plastic, lysed NK cell tumor target cells (Fisher et al., 1982). The authors concluded these effector cells were the immature monocyte population since effectortarget cell binding assays determine only 15% of monocytes lysed target cells and the monocytes lost their cytolytic activity over time. When bovine PBM cells were evaluated for cytolytic activity, a heterogeneous population of blood monocytes was evident that mediated lytic activity against both tumor and virally-infected target cells (Bielefeldt Ohmann et al., 1985). As with human monocyte-mediated lysis, the heterogeneity of bovine monocytes could be due to cells being at different stages of maturity. PBM cells expressed lytic activity against the mouse NK cell target YAC-1 at an effector to target cell (E:T) ratio of 50:1 (Bielefeldt Ohmann et al., 1985). Unlike the 4-h assay for NK cell activity against YAC-1 in the mouse (E:T, 60:1 ), bovine P B M cells required 10-16 h of incubation to obtain appreciable lysis (Bielefeldt Ohmann et al., 1985). Adherent cells (86% nonspecific esterase-positive) treated with anti-monocyte antibody and complement decreased the number of nonspecific esterase-positive cells with a corresponding decrease in lytic activity. In addition, bovine P B M cells expressed low, but consistent lytic activity against BHV-l-infected fibroblasts (Bielefeldt Ohmann et al., 1985). The lysis of BHV-l-infected low passage fibroblasts was not dependent on age or BHV-1 serological status of the animal (Bielefeldt Ohmann et al., 1985). Similarly, adherent P B M cells from BHV-1 nonimmune animals lysed BHV-l-infected xenogeneic tumor target cells but not uninfected cells (Cook and Splitter, 1989). Using BHV- 1-infected autologous or allogeneic bovine testicular cells as targets, others were unable to detect lysis by freshly isolated PBM cells from BHV-1-nonimmune animals (Campos and Rossi, 1985b ). However, earlier results by the same workers detected lysis of parainfluenza virus-3 (PI-3)-infected bovine embryo cells, GBK cells, and bovine testicle cells by freshly isolated PBM cells from PI-3-nonimmune animals (Campos et al., 1982 ). Removal of adherent cells on Sephadex G-10 columns reduced lytic activity of the PBM

245 cells, indicating that macrophages were involved as effector cells, activators of other cytolytic effectors or both (Campos et al., 1982). Alveolar macrophages from PI-3 immune or nonimmune calves lysed PI-3-infected primary calf kidney cells (Stott et al., 1975 ). However, lymph node cells and PBM cells isolated from the same animals did not lyse PI-3-infected target cells (Stott et al., 1975 ). In conclusion, it appears that naturally cytotoxic bovine effector cells including NK cells, NC cells and macrophages, are a very heterogeneous population. Macrophages definitely play a role in vitro by killing BHV-l-infected target cells. In addition, a nonadherent cell population has lytic activity against vitally-infected target cells. Reportedly, fresh cells serve as effector cells in certain systems, while only cultured cells are effector cells in other systems. The development of mAbs against molecules expressed on macrophages, T cell subsets and NK cells will aid in defining natural cytolytic activity in cattle.

2.2 Morphological and phenotypic characteristics Phenotypically the best characterized NK cells are from the human. Human NK cells are a subpopulation of large granular lymphocytes (LGL) (Ferrarini and Grossi, 1984) with up to 70% of LGL mediating NK activity (Timonen et al., 1982). LGL are nonadherent, nonphagocytic, non T and non B lymphocytes. LGL express very avid Fc receptors for IgG and contain intracytoplasmic azurophilic granules (Ferrarini and Grossi, 1984). The human NK cell population is very heterogeneous with regard to cell surface markers. For example, NK cells mediating killing against HSV-l-infected targets differ from NK cells mediating killing against K562 cells (Fitzgerald et al., 1982). These workers found that NK cells mediating lysis of HSV-1-infected fibroblasts were Leu 1-, Leu 2-, Leu 3-, Leu 4-, Ia +, and OKM1 +, while NK cells mediating lysis of K562 cells were Leu 1°/+, Leu 2 -, Leu 3 -, Leu 40/+, Ia-, and OKM1 + + (Fitzgerald et al., 1982 ). However, competition studies using cold target inhibition showed an overlap in the effector populations mediating lysis against HSV-l-infected cells and K562 cells (Fitzgerald et al., 1982; Yasukawa and Zarling, 1983 ). Phenotypically, human NK cells have been shown to express the Fc receptor for IgG (detected by mAb B73.1), C3b receptor and the determinants identified by mAbs OKM1, O K T l l A , and HNK-1 (Yasukawa and Zarling, 1983). NK cells do not express T3 or the T cell receptor (Burns et al., 1985). Like T cells, NK cells express IL-2 receptors and proliferate in response to IL-2 alone (Burns et al., 1985). Interferon alpha, beta, and gamma all augment NK cell function (Burns et al., 1985). Human NK cells also synthesize and secrete many cytokines including IL-1, IL-2, CSF, and a - I F N and fl-IFN. Although NK cells are frequently considered heterogeneous in phenotype, one characteristic of NK cells that appeared quite homogeneous was density, with enrich-

246 ment for NK cell activity occurring in low density percoll fractions (Bishop et al., 1983b ). Also mouse NK cells can be characterized as low density lymphocytes (Bukowski and Welsh, 1985) and are within the LGL population (Minato et al., 1985 ). Adult NK cells are nylon wool nonadherent and phenotypically express asialo GM1, NKI.1 (or NK1.2), Ly 5, and Qa 5. They do not express Thy 1 or MHC class II molecules (Bukowski and Welsh, 1985; Colmenares and Lopez, 1986). Rat NK cells also fall within the LGL population and are low density lymphocytes (Reynolds et al., 1982). Canine NK cells have many characteristics in common with mouse, rat and human NK cells (Loughgram et al., 1985). Morphologically, canine NK cells are LGL and can be characterized as low density lymphocytes (Loughgram et al., 1985). NK activity was found in the nylon wool nonadherent population and effector cells were phenotypically heterogeneous, with some cells expressing MHC class II molecules and some cells expressing the CD8 homologue (Loughgram et al., 1985). Similar to other species, porcine NK cells were nonadherent, esterase-negative lymphocytes (Yang et al., 1987). However, unlike other species, porcine NK cells were not LGL but small and medium sized lymphocytes with few cytoplasmic granules (Yang et al., 1987). Bovine nonadherent cells mediating natural killer activity were large, low density mononuclear cells (Cook and Splitter, 1989). These effector cells did not express MHC class II molecules. Unlike human LGL, with prominent intracytoplasmic azurophilic granules, identification of granules in bovine effector cells has been difficult, with no conclusive results (Cook and Splitter, unpubl., 1988). NC cells are also within the LGL population. In the mouse, these nonadherent cells differ phenotypically from NK cells, with mouse NC cells not expressing asialo GM1 or Qa 5 (Colmenares and Lopez, 1986). In other species, NC cells have been poorly characterized. Many mAbs have been produced to identify cell surface molecules on macrophages. It is not within the scope of this review to address the many determinants that have been identified. However, it can be noted that human NK cells and macrophages share the cell surface determinant OKM1 (Ferrarini and Grossi, 1984). Monoclonal antibody WIG4, that identifies the canine monocyte/macrophage population, did not react with canine NK cells (Loughgram et al., 1985 ). Bovine nonadherent effector cells were not identified by the mAb C5B6 (Eskra et al., 1989), that recognizes the C D l l c equivalent on bovine monocytes/macrophages (Cook and Splitter, 1989). In human and mouse, NK cells express an FcR receptor for IgG (with the human receptor possessing higher avidity). Similarly, all macrophages express Fc receptors for IgG. In addition, both macrophages and NK cells express the C3b receptor (London et al., 1985). A characteristic that distinguishes macrophages from NK cells is

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esterase staining. Macrophages are positive for nonspecific esterase, in contrast to NK cells which are negative for esterase.

2.3 Cell lineage There are no concrete conclusions as to the lineage of NK cells, but several arguments have been put forth concerning the origin of NK cells. In light of the differences in activity of cells from different species and cells from different organs within a species, a discussion of cell lineage appears in order. There is general agreement that NK cells are a very heterogeneous population. Therefore, determining cell lineage on the basis of phenotypic markers could be very misleading. As already discussed, NK cells and macrophages share some cell surface molecules (Ferrarini and Grossi, 1982). In addition, like macrophages, NK cells can synthesize and secrete IL-1 (Burns et al., 1985). However, NK cells also share characteristics with T lymphocytes: as mentioned previously both have IL-2 receptors and can proliferate in response to IL-2 (Burns et al., 1985). Also, NK and T cells are both nonadherent, nonphagocytic lymphocytes. Functional evidence has accumulated that argues for a macrophage or independent lineage for NK cells. NK cell activity against HSV-l-infected fibroblasts was normal in cells from patients with severe combined immunodeficiency (individuals with essentially no B or T cells) (Lopez, 1985). In addition, cells from some patients with a monocyte/macrophage deficiency had normal NK activity against HSV-l-infected fibroblasts while cells from others had diminished activity (Lopez, 1985). This information would suggest NK cells were not derived from differentiated monocytes but may have originated from a common precursor cell. Others (Burns et al., 1985) conclude that the majority of circulating NK cells may be pre-thymic T cells or may represent different stages of development of an unique lineage of lymphocytes. Cloned T l l +, T 3 - NK cells expressed truncated T cell receptor beta chain RNA transcripts but no alpha chain RNA transcript and no T cell receptor cell surface protein (Ritz et al., 1985). These workers conclude NK cells appear to be derived from T-lineage precursors. However, these results must be interpreted with some caution as to the derivation of freshly isolated NK cells versus cloned NK cells due to changes that could take place with culture conditions necessary for cloning. Evidence for an independent lineage comes from the following observations. Firstly, NK cells contain mature electron-dense granules indicating cell maturity (Ferrarini and Grossi, 1982). Secondly, NK cells are derived from bone marrow stem cell precursors in a thymic-independent manner (Lopez, 1985). Thirdly, analysis of the constant beta chain genes of the T cell receptor showed germline arrangement in freshly isolated NK cells, in contrast to the rearranged constant beta chain genes found in T cells in early thymic ontogeny

248 (Lanier et al., 1986). Fourthly, LGL cells are present in the earthworm, with the only other mononuclear cells in the earthworm being phagocytic cells (Ferrarini and Gross, 1982). Lastly, NK cells appear to interact with normal bone marrow cells in vivo and possess the ability to destroy these cells. NK cells fail to rearrange T cell antigen receptor genes, and, by this criterion are considered distinct from the T cell lineage (Tutt et ah, 1986, 1987; Young et ah, 1986). Additional evidence separating NK cells from the T lineage is provided by studies in mice with the severe combined immunodeficiency disease genetic defect. These genetically defective mice fail to develop T and B progenitor cells. The presence of normal NK progenitor cells in the bone marrow of these animals demonstrates that NK cells are distinct from both the B and T lineage (Hackett et al., 1986). However, the existence of a common progenitor cell for T and NK cells might be possible prior to the commitment of pre-T cells to rearrange the T cell antigen receptor genes during fetal development. If this developmental pathway exists, it would be very early in cell differentiation. Can the organ distribution of NK cell activity give insight into cell lineage? In the mouse, no NK activity is found in the thymus (Tagliabue et al., 1982) which fits with a thymic-independent cell. Little or no NK activity was found in peripheral lymph nodes while high NK cell activity was found in the spleen and peripheral blood (Tagliabue et al., 1982). Since macrophages and T lymphocytes are found in all three compartments, does the absence of NK cells in peripheral lymph nodes point to an independent lineage or just differential function? Finally, what role might an immature macrophage population play in natural cytotoxicity? A low density, nonadherent, nonphagocytosing macrophage in an early stage of differentiation was obtained from the spleen of cyclophosphamide-treated mice (Baccarini et al., 1986). These effector cells lysed Candida and YAC-1 tumor target cells but not the NK-insensitive lines EL4 and P815 (Baccarini et ah, 1986). These results should suggest caution in interpretation of results indicating NK activity in species without well defined mononuclear cell markers. Certainly a low density, nonadherent nonphagocytosing mononuclear cell could be considered an NK cell or an immature monocyte if no antibodies to cell surface antigens have been well defined. Even with defined mAbs, the fact that NK cells and monocytes share cell surface determinants would make results difficult to interpret. 2.4 Recognition of target cell Human NK cells need expression of cell surface viral glycoproteins on HSV1-infected target cells to mediate lysis (Bishop et ah, 1983b). For example, virally-infected target cells treated with emetine, to inhibit protein synthesis, were not lysed by NK cells (Bishop et al., 1983b). Alternatively, to determine

249 the importance of individual viral glycoproteins, target cells were infected with glycoprotein C (gC) m u t a n t virus or treated with 2-deoxy-D-glucose to inhibit N-glycosylation and prevent expression of glycoprotein B (gB) by the mutant HSV-1 virus. With these targets, effector cells could still lyse all targets but at a lower level than control target cells infected with wild type of HSV-1. The results indicated HSV-1 glycoproteins B, C, and D were each important in NK cell recognition of virally-infected target cells (Bishop et al., 1983b). Further evidence that cell surface glycoproteins are important in recognition comes from blocking studies with mAbs to HSV-1 glycoproteins (Bishop et al., 1986). Monoclonal antibody to gB or gC blocked lysis of HSV- 1-infected target cells (Bishop et al., 1986). In addition, a minor alteration in gB affected the ability of NK cells to recognize and kill virally-infected target cells with the altered glycoprotein (Bishop et al., 1986). Natural killer activity of bovine P B M cells against BHV-l-infected target cells requires BHV-1 major cell surface glycoprotein expression (Cook et al., 1989). Target cells treated with tunicamycin, to inhibit N-glycosylation, did not express BHV-1 glycoproteins gI, gIII and gIV on the cell surface. These drug-treated target cells were not lysed by bovine effector cells, indicating the importance of viral glycoprotein expression (Cook et al., 1989). However, analysis of NK cell recognition of vesicular stomatitis virus (VSV)infected target cells suggested NK cells do not recognize VSV proteins but some alteration in the cell membrane caused by the virus (Welsh, 1986 ). This hypothesis was supported by the observation that cells transfected with VSV major protein G gene were not lysed by NK cells (Welsh, 1986). Therefore, NK cells appear specific for antigens of certain viruses or altered host antigens induced by other viruses. Further, a role for the antiviral action of NK cells cannot be established with some viruses. For example, NK cells did not mediate antiviral action in mice infected with LCMV (Bukowski and Welsh, 1985). Evidence suggests the LCMV-infected target cells were protected from NK cell mediated lysis by interferon (Welsh, 1986). This may be one way for the virus to evade the immune system. Target cell structures for NK cell binding versus killing may be different (Welsh, 1986). When target cells were treated with IFN and protected from lysis, the NK cell still bound to the target cell but cytotoxic factors were not released (Welsh, 1986). Interferon appeared to induce a change in the target cell (Welsh, 1986). Current speculation is that recognition and conjugate formation between NK cells and the target cell probably involve a lectin-type receptor on the NK cell. The NK cell binds to specific, differentiation-associated carbohydrate moieties on the target cell (Burns et al., 1985). To actually lyse the target cell there must be receptor sites for the cytolytic factor (Burns et al., 1985 ). NK cell mediated lysis of target cells may be inhibited by an mAb against structures associated with the T200 molecule (Burns et al., 1985). In

250 addition, NK cell mediated lysis may be blocked by mAb to the leukocyte function associated antigen-1 (LFA-1) adhesion molecule (Burns et al., 1985). In general, target cells with high levels of MHC class I molecule expression are less susceptible to NK cell mediated lysis (Piontek et al., 1985). This resistance to killing appeared to be the result of high levels of sialoglycoprotein (on MHC class I molecules) (Welsh, 1986) and not the result of target cell protection by interferon (Piontek et al., 1985). Therefore, it was hypothesized that NK cells may function in vivo to eliminate host cells that fail to express MHC molecules after IFN stimulation during an immune response (Piontek et al., 1985 ). These host cells could be a great threat since they could escape T cell recognition despite viral or tumor antigen expression (Piontek et al., 1985 ). The recognition mechanism used by macrophages to identify virally-infected target cells has not been well established. In one example, reovirus type 1 determinants necessary for macrophage recognition of the virally-infected target cell were characterized (Letvin et al., 1982 ). Macrophages isolated from unprimed mouse spleen cells lysed reovirus type 1-infected L cells (fibroblasts) but not reovirus type 3-infected target cells (Letvin et al., 1982 ). Using intertypic recombinant viruses, it was shown that macrophage lytic specificity was due to a property of the viral hemagglutinin (Letvin et al., 1982). In other systems macrophages do not show such viral antigen specificity. In hamsters immunized with vaccinia virus, macrophages lysed vaccinia virusinfected target cells as well as HSV-infected target cells, but not uninfected target cells (Chapes and Tompkins, 1979).

2.5 Role of gamma interferon Gamma interferon has been shown to have a major role in the augmentation of NK cell activity (Burns et al., 1985). Treatment with 7-IFN increases lytic efficiency of mature NK cells and recruits immature cells to become mature functional cytolytic cells (Lopez, 1985 ). NK cell mediated lysis in conjunction with IFN activity appears to be important in early antiviral activity against both VSV (Welsh, 1986) and influenza virus (Lewis et al., 1986). The role of IFN appears to depend on the viral infection and the species being infected. In mice infected with HSV-1, IFN appears to mediate natural resistance to HSV-1 in an NK cell-independent manner (Welsh, 1986). HSV replication was not altered in mice depleted of NK cells in vivo with antibody to asialo GM1 (Bukowski and Welsh, 1986). These results do not rule out the importance of NK cells later in infection or in a latent reaction but do suggest IFN is of great importance in natural resistance to HSV-1 in mice (Bukowski and Welsh, 1986). In contrast, lysis of HSV-l-infected target cells by human NK cells was independent of IFN production (Bishop et al., 1983a; Fitzgerald et al., 1985). When NK cells were treated with actinomycin D to inhibit protein production,

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lytic activity remained constant, but IFN synthesis was greatly inhibited (Bishop et al., 1983a). Also, if antibody to y-IFN was added to the assay, there was no change in lytic activity when compared with controls (Bishop et al., 1983a). However, pretreatment of effectors with y-IFN augmented cytolytic activity (Fitzgerald and Lopez, 1982; Bishop et al., 1983a; Yasukawa and Zarling, 1983 ). A similar phenomenon of nonspecific cytotoxic lymphocyte generation was reported by Casali et al. (1981). The novel aspect of this study was that the use of purified soluble glycoproteins of measles and lymphocytic choriomeningitis virus could enhance human peripheral blood lymphocyte killing of a variety of target cells including autologous and heterologous human fibroblasts, and tumor cells. No interferon could be detected in the culture fluids, and the inability of supernates to recruit cytotoxic activity from newly added lymphocytes distinguished this type of nonspecific human cytotoxicity from the interferon-associated enhancement of NK cell activity. Because these cells could be activated within 2-4 h after the exposure of lymphocytes to glycoproteins and interferon release was not required, these cells could function early in defense against virus infection. The combined action of killer cells induced by virus glycoproteins, virions, and interferon may function to limit both the spread of virus and the numbers of infected cells in primary infections until specific CTL and antiviral antibodies are generated later. Failure of these early nonspecific defense mechanisms might favor the virulence of the virus over the host's immune system later in infection. Finally, other roles for interferon in natural resistance to viral infections have been described. It has been known for several decades that interferon can inhibit viral replication and protect cells from viral infection (Joklik, 1985). Gamma IFN also plays a role in macrophage activation, whose lytic activity was discussed previously. The mechanisms involved in interferon inhibition of viral replication (Hamilton and Adams, 1987) or activation of macrophages (Bielefeldt Ohmann et al., 1987 ) are tangential to aims of the review and have been reviewed by others. 3.0 COMPARISON OF VIRALLY-STIMULATED EFFECTOR CELLS LYTIC FOR VIRALLY-INFECTED TARGET CELLS

3.1 Viral and M H C restriction

Classically, the functional activity of cytolytic T lymphocytes (CTL) has been shown to be restricted by recognition of virus in the context of autologous MHC class I molecules (Doherty and Zinkernagel, 1974). This lytic activity is specific, with no killing of noninfected target cells, virally-infected allogeneic cells or autologous cells infected with another virus. Also, effector cells express the CD8 homologue.

252

The role of cytolytic T lymphocytes in herpesvirus infections has recently been reviewed by Rouse and Horohov ( 1984 ). The following discussion will be confined primarily to the bovine system and to recent developments in recognition of herpesvirus-infected target cells. MHC and antigen restriction of bovine cytolytic cells has been difficult to demonstrate. Until recently only two systems have demonstrated MHC and antigen restriction in cattle. First, primary cytotoxic bovine PBM cells generated in a primary mixed lymphocyte reaction with stimulators of the BoLA w8/wl 1 phenotype lysed all targets bearing w8, w l l or both w8 and w l l (Teale et al., 1985 ). MHC class I molecules have been the principal recognition structures for alloreactive CTL in other species (Alter et al., 1973; Eijsvoogel et al., 1973; Thistlethwaite et al., 1984). Results suggest these bovine alloreactive cytotoxic cells recognize determinants identical with or closely related to determinants characterized by BoLA w8 and w l l alloantisera (Teale et al., 1985). In the second system, MHC and antigen-restricted cytotoxic T cells were generated against Theileria parva-infected cells (Eugui and Emery, 1981 ). Effector cells from cattle immunized and challenged with T. parva lysed autologous infected target cells and not infected allogeneic cells or uninfected xenogeneic cells (Eugui and Emery, 1981). Later work confirmed the presence of antigen specific and MHC-restricted cytotoxic T lymphocytes from Theileria parva-immune cattle and also analyzed cytotoxic cells generated in vitro by autologous theilerial-lymphocyte cultures (Emery and Kar, 1983). Cells with classic CTL activity and cells with natural killer-like activity were detected at E:T cell ratios of 40:1 (Emery and Kar, 1983). Similarly, in calves undergoing immunization with Theileria annulata, cytotoxic cells detected early in the response were MHC-restricted, while MHC-nonrestricted cytotoxic cells appeared later in the response (Preston et al., 1983). Results suggested that MHC-restricted cytolytic cells were generated from memory cytotoxic T cells, while MHC-nonrestricted cells were natural killer-like cells (Preston et al., 1983) or, alternatively, could represent lymphokine-activated killer cells. Recent advances in the ability to clone bovine lymphocytes have allowed the detection of MHC and virally restricted bovine CTL (Splitter et al., 1988). PBM cells from BHV-l-immunized cattle were stimulated in vitro with ultra violet-irradiated BHV-1 and cloned cells were established. Cloned cells were evaluated for the ability to lyse virally-infected day 3 concanavalin A-stimulated lymphocytes in a 6-h assay. Cloned cells were specific for BHV- 1-infected autologous cells at an E : T cell ratio of 1:1, with no lysis of uninfected cells (Splitter et al., 1988). There was no lysis of bovine herpesvirus 2 or pseudorabies virus-infected autologous cells. Also, allogeneic BHV-l-infected or uninfected target cells were not lysed. These results support an antigen-specific role for bovine cytotoxic T lymphocytes in the immune response to BHV-1 (Splitter et al., 1988).

253

Additional evidence for MHC-restricted cytolysis by bovine peripheral blood lymphocytes from BHV-1 hyperimmune animals has been reported (Campos and Rossi, 1986a). Effector cells were evaluated for the ability to lyse virallyinfected day 5 pokeweed-stimulated lymphocytes in a 7-h assay. Cytotoxic lymphocytes, generated by in vitro stimulation with ultra violet-inactivated BHV-1 for 7 days, lysed BHV-l-infected autologous target cells but not BHV1-uninfected allogeneic target cells or xenogeneic tumor target cells (K562) (Campos and Rossi, 1986a). However, after four in vitro BHV-1 stimulations at weekly intervals, cytotoxic lymphocytes lysed autologous and to a lesser degree, heterologous BHV-l-infected target cells. After in vitro stimulation with BHV-1, the phenotype of the effector cell population was determined. Most cells were recognized by mAb 15, shown to react with bovine T lymphocytes (Campos and Rossi, 1986a). One reason cytotoxic cells may lose specificity is long-term culture in IL-2-containing medium (Brooks et al., 1985). It is interesting to note that the four examples cited above all use lymphoblasts as target cells. Many studies were unsuccessful in demonstrating MHC and antigen restriction using adherent cell lines such as fibroblasts (Rouse and Babiuk, 1977) and testicular cells (Campos and Rossi, 1985b). Analysis of MHC molecule expression on lymphoblast target cells versus fibroblasts could yield valuable information concerning antigen recognition by bovine cytolytic cells. MHC-restricted killing of HSV-l-infected cells in mice was dependent on MHC class I molecule expression (Pfizenmaier et al., 1977) while MHC-restricted killing of HSV- 1-infected cells by human CTL clones was class II molecule dependent (Yasukawa and Zarling, 1984; Zarling et al., 1986b). Defining the role of MHC molecule expression on target cells will depend on the population of CTL effector cells analyzed. CTL may differ if generated by ultra violet-inactivated virus or fixed vitally-infected monolayers, or if cloned cells are analyzed instead of bulk cultured cells.

3.2. Recognition of target cell Identifying antigenic determinants important in cytolytic T cell recognition will greatly aid in the understanding of the immune response to viruses and vaccine development. The role of major glycoproteins of HSV has been analyzed in mouse and human cytolytic T cell response. Murine CTL generated by foot pad immunization with HSV-1 were obtained only after culturing lymphocytes from draining lymph nodes for 3 days (Rosenthal et al., 1987 ). Mouse L cells transfected with genes coding for gB-1, gC-1 or gE-1 served as target cells (Rosenthal et al., 1987). CTL were MHC class I molecule restricted and recognized only gC-1 with no lysis ofgB-1 or gE-1 expressing target cells (Rosenthal et al., 1987). Similarly, limiting dilution analysis was performed on HSV-l-specific CTL from draining lymph nodes of foot pad immunized mice (Glorioso et aL, 1985 ). Using target cells infected with a HSV-1 mutant lack-

254

ing gC-1 expression, the majority of cloned cells did not recognize target cells lacking gC-1 expression, although most clones recognized wild type virus-infected target cells (Glorioso et al., 1985). Interestingly, target cells infected with gC-1 mutant viruses, defined by the lack of mAb recognition (mar mutants), but still expressing an altered gC product, can be recognized by CTL (Glorioso et al., 1985). These results identify HSV-1 gC as the immunodominant antigen for HSV-l-specific memory cytotoxic T lymphocytes of mice (Glorioso et al., 1985). In humans, CTL recognition of HSV-1 has been studied with peripheral blood lymphocytes from HSV-1 seropositive individuals stimulated with cloned gB-1 or gD-1 genes expressed in vaccinia virus or with ultra violet-inactivated HSV-1 (Zarling et al., 1986a,b). Of 33 CTL clones from one donor stimulated with gD-1, all clones lysed gD-1 recombinant targets (Zarling et al., 1986a). CTL clones stimulated with either gB-1 or gD-1 lysed HSV-l-infected target cells at an E : T cell ratio of 20:1 (Zarling et al., 1986b). Therefore, a difference may exist between species with regard to immunodominant determinants recognized by similar lytic effector cells. 3.3 M H C unrestricted lysis An earlier report showed antiviral cytotoxicity by freshly isolated bovine lymphocytes was not MHC restricted (Rouse and Babiuk, 1977). This study demonstrated PBM cells from vaccinia or BHV-1 immunized animals were not MHC restricted, lysing infected autologous fibroblasts and infected allogeneic fibroblasts equally well. Results suggested that cells mediating the lysis were T cells since removal of phagocytic cells and Fc receptor-bearing cells showed enhanced lysis as compared with the total cell population. Effector cells from nonimmune animals had only low levels of cytotoxicity against autologous and allogeneic noninfected and virus-infected target cells (Rouse and Babiuk, 1977). In contrast, more recent results described Sephadex G-10 adherent effector cells from B H V - l - i m m u n e or hyperimmunized cattle that lysed BHV-I-infected autologous or allogeneic bovine testicular target cells (Campos and Rossi, 1985b). PBM cells from nonimmune animals did not lyse BHV-l-infected bovine testicular target cells. This adherent effector population was characterized by the requirement for a long assay time (18 h) and a variance in lytic ability with time after inoculation (peak response occurring on day 6). In contrast, natural cytotoxicity against PI-3-infected cells did not vary with time after inoculation (Campos and Rossi, 1985b). Natural cytotoxic activity has been described against PI-3-infected targets mediated by Sephadex G-10 nonadherent effector cells (Campos et al., 1982). Lysis by the adherent effector cells was not enhanced by addition of anti-BHV-1 antibody and was not MHC restricted (Campos and Rossi, 1985b). Considering the above evidence, it ap-

255 peared t h a t the cytotoxic m e c ha ni s m was not ADCC, natural cytotoxicity or C T L (Campos and Rossi, 1985b ). One i n t e r p r e t a t i o n of the data would suggest t h a t the effector cell from immunized animals was an activated monocyte with specificity for B H V - l - i n f e c t e d cells (Campos and Rossi, 1985b). 4.0CONCLUSIONS W h a t in vitro role against pathogens can be assigned to cytolytic bovine m o n o n u c l e a r cells? As in other species, antigen and M H C - r e s t r i c t e d bovine C T L operate in certain systems. In addition, both a d h e r e n t m a c r o p h a g e / m o n ocyte cells and n o n a d h e r e n t , non B, non T cells from n o n i m m u n e animals lyse virally-infected target cells. One example, t h a t was in contrast to results in o t h e r species, described a n o n a d h e r e n t cytolytic cell from n o n i m m u n e animals which lysed virally-infected cells only after in vitro culture. Also, an adherent cell population from i m m u n e animals lysed virally-infected targets in an antigen-restricted manner. T h e in vivo role of these cytolytic cells deserves att e n t i o n in future research. A comparison of the known phenotypic antigens present on bovine N K cells, TABLE1 Comparison of antigenic phenotype of human and bovine NK cells, macrophages and CTL Antigen

NK c e l l

Macrophage

CTL

Human

TCR CD2 CD3 CD4 CD5 CD7 CD8 CDlla(LFA-1) CD16 (FcR) Leu 19 (NKH-1) MHC class II Esterase positive

No Subset No No No Yes Subset ND Yes Yes ND No

No No No No No No No Yes Yes ND Yes Yes

Yes Yes Yes Subset Yes Yes Subset Yes Subset Yes No No

Bovine

CD2 CD4 CD5 CD8 CDll~(LFA-1 ) CD1 lc (p150,95) MHC class II Esterase positive

No No No ND ND No No No

No No No No Yes Yes Yes Yes

Yes ND Yes Yes ND ND No No

ND, not determined.

256 TABLE 2 Functional characteristics of NK cell, macrophages, and cytotoxic T lymphocytes killing of virally-infectedtarget cells Characteristic

NK cell

NC c e l l

Macrophage Tc-1

Antigen priming Target cell antigen

No Unknown

No No Unknown Unknown

MHC restriction Effector c e l l molecule Lyric molecule Cytoskeletal reorganization Lytic assay

Tc-2 Yes Viralantigen only

No No No U n k n o w n Unknown Unknown

Yes Viral antigen and MHC I or MHC II Yes TCR

Perforin Yes

Unknown Many Unknown Yes

Perforin Yes

Perforin Yes

4-18 h

16-24 h

4-6 h

4-6 h

10-20 h

No TCR

macrophages and C T L with h u m a n effector cells is summarized in Table 1. A more extensive review of monoclonal antibodies specific for molecules on bovine cell surfaces will be pr e s ent ed at a forthcoming workshop on differentiation antigens of cattle. I n f o r m a t i o n regarding the functional characteristics of N K cells, macrophages and C T L regardless of species is present ed in Table 2. M H C - u n r e s t r i c t e d C T L (To-2) can be f ur t her subdivided into cells expressing c~/fl-TCR or ~/~-TCR. Evidence indicates cytotoxic cellular defenses play a role in resistance to viral infections. F u t ur e studies to identify viral d e t e r m i n a n t s recognized by bovine cytolytic cells should aid in developing effective vaccines. P h e n o t y p i c characterization of different cytolytic effector cells with mAbs will aid in the elucidation of the different types of effector cells, their ontogeny, prevalence in body tissues and in vivo importance in protection against infection. Finally, through rigorous characterization of the different effector cells, the regulating mechanisms t h a t control these cells can be identified and understood. Use of targeted exogenous cytokines or synthetic immunomodulatory compounds may augment the numbers and function of these cells in vivo to protect the host when challenged with a pathogen.

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