Pseudorabies Virus US3 Protein Kinase Protects Infected Cells from NK Cell-Mediated Lysis via Increased Binding of the Inhibitory NK Cell Receptor CD300a.

UNLABELLED: Several reports have indicated that natural killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse mechanisms for evading NK cells, although few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The antiviral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surfaces of virus-infected cells. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties: it triggers the binding of the inhibitory NK cell receptor CD300a to the surface of the infected cell, thereby providing increased CD300a-mediated protection of infected cells against NK cell-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid ligands of CD300a and on group I p21-activated kinases. These data identify a novel alphaherpesvirus strategy for evading NK cells and demonstrate, for the first time, a role for CD300a in regulating NK cell activity upon contact with virus-infected target cells. IMPORTANCE: Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays a previously uncharacterized capacity for evasion of NK cells. Expression of US3 protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. We show that this US3-mediated increase in CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAKs). The identification of this novel NK cell evasion strategy may contribute to the design of improved herpesvirus vaccines and may also have significance for other PAK- and CD300a-modulating viruses and cancer cells.

KIR and KIR ligand polymorphism: a new area for clinical applications?

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer (NK) activity, allowing NK cells to sense and respond to human leukocyte antigen (HLA) class I downregulation, an important hallmark for viral infections and tumor transformation. KIR and HLA genes are located on different chromosomes and KIR/HLA class I interaction represents an example of genetic epistasis in which the presence of receptor/ligand pairs is necessary for the induction of functional activity, while the presence of one in the absence of the other is not sufficient to influence NK cell function. Due to the high degree of HLA class I and KIR gene variability, KIR/KIR-ligand (KIR-L) interactions are extraordinarily diverse. KIR polymorphism arises from both haplotypic and allelic variations and was shaped by natural selection. KIR variability affects NK cell education influencing the KIR repertoire, KIR expression, the strength of KIR/KIR-L interactions and the capability to deliver signals. Moreover, it may influence NK cell function during infections, autoimmune diseases, pregnancy and allogeneic transplantation. This review summarizes the genetic and functional features of KIR/KIR-L interactions and gives an overview of their potential relevance in clinical studies.

A novel 120-kD surface antigen expressed by a subset of human lymphocytes. Evidence that lymphokine-activated killer cells express this molecule and use it in their effector function.

A human cell clone (SF-16) displaying strong cytolytic activity against fresh tumor target cells was used for production of murine mAbs against surface antigens expressed by lymphokine-activated killer (LAK) cells and their peripheral blood precursors. The preliminary screening of hybridoma supernatants was performed according to the ability to bind SF-16 cells. Selected mAbs were further analyzed for their reactivity with several T and B cell lines and with peripheral blood T and non-T cell populations. A selected mAb, termed anti-LAK-1, only reacted with some T cell lines and with 15-30% of PBMC. Approximately 10-15% E-rosetting (T) cells and 40-50% E-rosette-negative cells were LAK-1+, as determined by cytofluorometric analysis. As the fluorescence distribution of LAK-1 antigen was clearly bimodal, LAK-1+ and LAK-1- cells could be separated by FACS. Positive cells were composed of large granular lymphocytes (LGL), whereas negative cells were mostly small lymphocytes and monocytes without LGL. After culture in rIL-2, purified LAK-1+ (but not LAK-1-) cells acquired the ability to lyse NK-resistant fresh melanoma target cells. In addition, only the LAK-1+ fraction of PBMC cultured for 5 d in rIL-2 lysed fresh tumor targets, thus indicating that the LAK-1 antigen is expressed also on LAK effector cells. Unlike some other LGL/NK cell markers, LAK-1 antigen is characterized by a stable expression: thus, LAK-1+ cell populations cultured for up to 20 d in rIL-2 maintained the LAK-1 antigen expression, whereas HNK-1 and, partially, CD16 were lost. Finally the cytolytic activity of LAK effector cells generated from PBMC cultured for 3 d in rIL-2 was susceptible to inhibition by the anti-LAK-1 mAb.

Involvement of T44 molecules in an antigen-independent pathway of T cell activation. Analysis of the correlations to the T cell antigen-receptor complex.

Prior studies indicate that the 9.3 monoclonal antibody (mAb) which defines a 44 kD T lineage-specific glycoprotein (T44) enhances the proliferative response of peripheral blood T lymphocytes to phytohemagglutinin (PHA) or allogeneic cells. The T44 molecule was expressed in both resting and activated T lymphocytes and in a subset of thymocytes, as assessed by indirect immunofluorescence and flow cytofluorometry. In view of the potential importance of T44 in T cell activation, we investigated the ability of the 9.3 (anti-T44) antibody to stimulate peripheral blood T lymphocytes under culture conditions giving optimal proliferative responses to anti-T3 mAb. Like UCHT1 (anti-T3) mAb, the 9.3 (anti-T44 mAb) promoted strong proliferative responses of purified T cells, provided that adherent cells were added to the culture. Maximal proliferation in response to 9.3 antibody was consistently detected at day 5 (at day 3 with anti-T3 or PHA). Moreover, triggering of T lymphocytes with 9.3 antibody (in the presence of adherent cells) resulted in strong IL-2 production that peaked at 48 h. Analysis of the physical and functional relationship between the T44 molecule and other molecules involved in T cell activation, including the clonotypically restricted Ti and the monomorphic T3 or T11 molecules, was carried out on a mutagenized jurkat T leukemia cell line. This mutant, termed JA3 (surface phenotype: T11+, T3+, 3A1+, T4-, T8-, DR-, Tac-, 4F2+, T44+) produced large amounts of IL-2 upon stimulation with PHA, anti-T3, or anticlonotypic mAb in conjunction with phorbol myristate acetate (or adherent cells). The molecules precipitated by anti-T44 mAb from 125I-labeled JA3 cells appeared as a diffuse band of Mr 40-45,000 under reducing conditions; under nonreducing conditions, a prominent band of Mr 80-85,000 was observed, while the Mr 40-45,000 band was greatly reduced. Thus, T44 molecules in both reducing and nonreducing conditions had relative molecular weights similar to that of molecules carrying clonotypic (Ti) determinants. In addition, like anti-Ti or anti-T3 mAb, anti-T44 antibody induced JA3 cells to produce large amounts of IL-2 in the presence of phorbol myristate acetate. Other similarities between T44 and molecules carrying clonotypic structures included the susceptibility to antibody-induced modulation and the late reexpression (72 h) at the cell surface after modulation. Taken together, these experiments suggest that anti-T44 mAb might recognize a monomorphic determinant of the T cell receptor molecule or be physically or functionally linked to the T3-Ti complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of hematopoiesis in vitro by alloreactive natural killer cell clones.

Natural killer (NK) cells lyse autologous and allogeneic target cells even in the absence of major histocompatibility complex (MHC) class I antigens on the target cells. Recently, however, human allospecific NK cell clones have been generated that recognize at least five distinct specificities inherited recessively and controlled by genes linked to the MHC. Because the genetic specificity of these alloreactive NK cells in vitro appears analogous to that of in vivo NK cell-mediated murine hybrid resistance, i.e., the rejection of parental bone marrow in irradiated F1 animals, we tested the ability of human alloreactive NK clones to recognize allogeneic hematopoietic progenitor cells. NK cells from two specificity 1 alloreactive NK clones, ES9 and ES10, significantly and often completely suppressed colony formation by purified peripheral blood hematopoietic progenitor cells from specificity 1-susceptible donors, but had no significant effect on the cells of specificity 1-resistant donors. Activated polyclonal NK cells were less efficient than the NK clones in inhibiting colony formation and had a similar effect on cells from both specificity 1-susceptible and -resistant donors. The alloreactive NK clones produced cytokines with a suppressive effect on in vitro hematopoiesis, such as interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), when exposed to phytohemagglutinin blasts from specificity 1-susceptible, but not -resistant donors. However, the mechanism by which alloreactive NK cells inhibit colony formation is more consistent with a direct cytotoxic effect than with the production of inhibitory cytokines because antibodies (anti-IFN-gamma, alpha-TNF-alpha, and -lymphotoxin) that completely blocked the inhibition by polyclonal NK cells had only a minimal effect on the inhibition by the alloreactive clones. Moreover, the alloreactive clones were directly cytolytic in a 51Cr release assay against enriched preparations of peripheral blood progenitor cells from specificity 1-susceptible donors. These data indicate that the alloreactive NK cells are likely the human counterpart of the cells mediating murine hybrid resistance and that these cells might play clinically important roles in rejection or in graft-versus-leukemia reactions after allogeneic bone marrow transplantation.

Diversity and structure of human T cell receptor delta chain genes in peripheral blood gamma/delta-bearing T lymphocytes.

We have investigated the diversity and repertoire of human TCR delta chain variable gene segments in the human peripheral blood CD4- CD8- (double-negative) population, using rearrangement and expression studies and sequence analyses. 20 TCR delta DNA clones were derived from the RNA of bulk-cultured double-negative T cells and their nucleotide sequences determined. These clones can be classified into six different V delta subfamilies. The distribution, however, was uneven in these cells, with 16 of 20 being derived from the V delta 1 (9) and V delta 2 (7) subfamilies. The remaining subfamilies, V delta 3, V delta 4, V delta 5, and V delta 6, were only represented by one clone each. The majority of these subfamilies seem to consist of a single member, in contrast with the closely linked V alpha subfamilies, which, in most cases, consist of multiple members. Our findings suggest that only a limited number of V delta genes are used in human peripheral blood double-negative T cells and that two major V delta subfamilies (V delta 1 and V delta 2) are used more frequently. Sequence comparison of our cDNA clones to V alpha clones indicates that there is no overlap in usage of V alpha and V delta gene segments, except for the V delta 4 (V alpha 6) subfamily. Comparison of the different V delta sequences suggests that the majority of the sequence diversity is concentrated in the junctions between V, D, and J segments and results from extensive N region diversity.

Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery.

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.

Antibody-induced modulation of the CD3/T cell receptor complex causes T cell refractoriness by inhibiting the early metabolic steps involved in T cell activation.

We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal antibody-induced surface modulation of the CD3-TCR complex. We determined whether modulation of CD3-TCR affected the early metabolic steps such as [Ca2+]i rise and InsP3 formation. A strong inhibition of the increase on [Ca2+]i mediated by either anti-TCR or anti-CD2 mAbs was detected. In contrast, surface modulation of CD2 molecules did not prevent the [Ca2+]i increase induced by anti-TCR mAb. Similarly, InsP3 increase was strongly reduced only after modulation of CD3-TCR complex (but not of CD2 molecules). Therefore, it appears that surface modulation of CD3-TCR complex causes T cell refractoriness by inhibiting the very early metabolic events that follow receptor-ligand interactions.

Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations.

In order to directly assess the distribution of cytolytic T lymphocytes (CTL) and their precursors (CTL-P) in the two major subsets of human T cells, we have used limiting dilution microculture systems to determine their frequencies. The two subsets were defined according to their reactivity (or lack thereof) with B9.4 monoclonal antibody (the specificity of which is similar, if not identical, to that of Leu 2b monoclonal antibody). Both B9+ and B9- cells obtained by sorting peripheral blood resting T cells using the fluorescence-activated cell sorter (FACS) were assayed for total CTL-P frequencies in a microculture system that allows clonal growth of every T cell. As assessed by a lectin-dependent assay, approximately 30% of peripheral blood T cells were CTP-P. In the B9+ subset (which represents 20-30% of all T cells), the CTL-P frequency was close to 100%, whereas the B9- subset had a 25-fold lower CTL-P frequency. It is thus evident that 90% and 10% of the total CTL-P in peripheral blood are confined to the B9+ or B9- T cell subsets, respectively. Analysis of the subset distribution of CTL-P directed against a given set of alloantigens confirmed these findings. CTL-P frequencies were also determined in B9+ and B9- subsets derived from T cells that had been activated in allogenic mixed leucocyte cultures (MLC). Approximately 10% of MLC T cells were CTL-P. This frequency was increased 3.5-fold in the B9+ subset, whereas the B9- subset contained only a small, although detectable number of CTL-P. Moreover, the great majority of the (operationally defined) CTL-P in MLC T cell population were found to be directed against the stimulating alloantigens, thus indicating a dramatic increase in specific CTL-P frequencies following in vitro stimulation in bulk cultures.

Clonal heterogeneity in the requirement for T3, T4, and T8 molecules in human cytolytic T lymphocyte function.

In an attempt to define the requirement of T8, T4, and T3 surface molecules in functional interactions occurring between human cytolytic T lymphocytes (CTL) and specific target cells, we have analyzed a large number of CTL clones derived from primary mixed lymphocyte culture (MLC) T cell populations for their susceptibility to inhibition by monoclonal antibodies (mAb) directed against these surface antigens. In most experiments, MLC T cells were stained with B9.4 (anti-T8) or OKT4 (anti-T4) mAb, separated into positive and negative cells using a fluorescence-activated cell sorter (FACS) and cloned under limiting conditions. While the lytic activity of the majority of T8+ CTL clones was inhibited by B9.4 mAb, approximately 15% of these clones were unaffected even in the presence of excess antibody. Flow cytofluorometric analysis of T8 antigen in individual clones did not show any correlation between the amount of T8 antigen expressed, the magnitude of cytolytic activity and the susceptibility (or lack thereof) to inhibition by B9.4 mAb. Of the 16 T4+ CTL clones analyzed, 7 were resistant to inhibition by OKT4 mAb even at doses 10-fold higher than that sufficient for complete inhibition of susceptible clones. Again, no correlation was found between the amount of T4 antigen expressed and the susceptibility to inhibition by the corresponding antibody. The same sets of T8+ and T4+ CTL clones were also analyzed for their susceptibility to inhibition by OKT3 mAb. Although all of the clones expressed the T3 surface antigen, only 15/23 T8+ clones and 9/14 T4+ clones were inhibited by anti-T3 mAb. To further document this clonal heterogeneity, we selected two T3+ T4- T8+ CTL clones that had no concomitant NK-like activity. One clone was resistant to inhibition by OKT3 mAb, whereas the other was highly susceptible. Incubation with OKT3 mAb resulted in modulation of the T3 molecules in both clones. Following modulation, however, the cytolytic activity of the resistant clones was unaffected, whereas the lytic activity of the susceptible clone was abrogated. These results thus indicate extensive clonal heterogeneity in the requirement for T3, T4, and T8 molecules in CTL function. Moreover, it appears that T3 molecules are not always physically and functionally linked to CTL receptor structures.

Anticlonotypic monoclonal antibodies induce proliferation of clonotype-positive T cells in peripheral blood human T lymphocytes. Evidence for a phenotypic (T4/T8) heterogeneity of the clonotype-positive proliferating cells.

Three previously selected monoclonal antibodies (mAb) directed against the clonotypic structure of a variant (termed JA3) of the interleukin 2 (IL-2)-producing Jurkat leukemia cell line (anti-JTi1-3 mAb) were found to induce an adherent cell-dependent proliferation of peripheral blood T cells in 20 different donors. Unlike the early cell proliferation induced by anti-T3 mAb, anti-JTi mAb-induced proliferation was detectable at day 5-6 of culture and reached peak levels at day 7-9. Less than 1% JTi+ cells were consistently detected in the starting peripheral blood lymphocytes or in control cultures in which cells were stimulated with anti-T3, phytohemagglutinin, or allogeneic cells. However, JTi+ cells were found in increasing proportions after culture with anti-JTi mAb and they were mostly represented by large blast cells expressing either the T4 or the T8 antigen, together with typical activation antigens including HLA-DR, IL-2 receptor, and 4F2. Immunoprecipitation experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that anti-JTi-reactive molecules present on antibody-stimulated lymphocytes or on JA3 cells were similar, disulphide-linked heterodimeric structures.

Surface markers of cloned human T cells with various cytolytic activities.

Human T cells stimulated in secondary allogeneic mixed lymphocyte culture (MLC) were cloned under limiting conditions in microculture systems using T cell growth factor and irradiated allogeneic cells. Clones with lytic activity against either phytohemagglutinin-induced blast cells bearing the stimulating alloantigen(s) (cytotoxic T lymphocyte [CTL] activity), L1210 mouse lymphoma cells coated with rabbit antibody (antibody-dependent cell-mediated cytotoxicity [ADCC]), or K562 human target cells were selected, expanded, and then analyzed for different surface markers, including rosette formation with sheep erythrocytes (E rosettes), receptors for the fc portion of IgG or IgM (Fc gamma R and Fc mu R), and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8,a nd OKT4. All the cytotoxic cells were E rosette+, Ia+ and 4f2+. Expression of Fc gamma R was restricted to the clones active in ADCC. CTL clones were either OKT8+ or OKT8-. Furthermore, three of the OKT8- CTL clones were OKT4+. In addition, some cytolytic clones devoid of specific CTL activity were OKT8+. It thus appears that the claim that human CTL are OKT8+, OKT4-, and Ia- is not supported by the analysis of their phenotype at the clonal level.

Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG.

Subpopulations of thymus-derived T lymphocytes bearing receptors for either IgM or IgG molecules were isolated from human peripheral blood. Those with receptors for IgM (T.M) provided help in a cell dose-dependent fashion for the pokeweed mitogen-induced differentiation of B lymphocytes in vitro, whereas cells with receptors for IgG (T.G) did not. T.G cells, on the hand, efficiently suppressed the differentiation and proliferation of B cells in the pokeweed system in the presence of helper T.M cells. This suppressive activity of T.G cells required prior interaction of the T.G cells with immune complexes. The helper activity of T.M cells was relatively radioresistant while the suppressor activity of T.G cells was radiosensitive. The results indicate that helper and suppressor functions of human T lymphocytes in this model system are mediated by different subpopulations of T cells which can be distinguished by their ability to bind IgM or IgG immune complexes, respectively.

The human leukocyte antigen (HLA)-C-specific "activatory" or "inhibitory" natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions.

Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C-specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (p58) but also as activatory (p50) receptors. Here we show that p50 and p58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas p58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions.

CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta.

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.

Human cytolytic cell clones lacking surface expression of T cell receptor alpha/beta or gamma/delta. Evidence that surface structures other than CD3 or CD2 molecules are required for signal transduction.

We have analyzed the transmembrane signaling operating in human cytolytic lymphocytes lacking surface expression of the CD3/TCR complex. Peripheral blood lymphocytes were fractionated into CD3+ and CD3- on the FACS and cloned under limiting conditions in the presence of PHA and IL-2. Approximately 90% CD3+ and 10% CD3- cells underwent clonal expansion. Clones obtained from the CD3- fraction belonged to two main phenotypic groups: CD2+ CD7+ and CD2- CD7+. Several clones were expanded and analyzed for surface phenotype and function. All of the five clones selected for detailed analysis did not express CD4, CD8, and CD28 antigens and did not release IL-2, whereas they displayed cytolytic activity against NK-sensitive, NK-resistant, and fresh tumor target cells. After stimulation with anti-CD2 mAbs or PHA a rapid increase in [Ca2+]i was detected in CD3- CD2+ CD7+ clones. This increment was caused by the release of Ca2+ from intracellular stores and by the influx from the extracellular compartment. Signaling in response to PHA did not appear to be dependent upon surface expression of CD2 molecules since antibody-induced modulation of CD2 did not prevent PHA-induced signal transduction. Similarly, in CD3- CD2- CD7+ clones [Ca2+]i increments and inositol phosphate formation occurred after stimulation with PHA. These data indicate that the functional PHA-binding structures, expressed in both groups of CD3- clones, are distinct from CD3/TCR complex and CD2 molecules.

P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities.

Human CD3-16+56+ natural killer (NK) cells have been shown to display a clonally distributed ability to recognize major histocompatibility complex (MHC) class I alleles. Opposite to T lymphocytes, in NK cells, specific recognition of MHC class I molecules appears to induce inhibition of cytolytic activity and, thus, to protect target cells. Since a precise correlation has been established between the expression of the NK-specific GL183 and EB6 surface molecules (belonging to the novel p58 molecular family) and the specificity of NK clones, we analyzed whether p58 molecules could function as receptors for MHC in human NK cells. NK clones displaying the previously defined "specificity 2" and characterized by the GL183+EB6+ phenotype, specifically recognize the Cw3 allele and thus fail to lyse the Fc gamma R+ P815 target cells transfected with Cw3. On the other hand, NK clones displaying "specificity 1" and expressing the GL183-EB6+ phenotype failed to lyse Cw4+ target cells. Addition of the F(ab')2 fragments of either GL183 or EB6 mAb as well as the XA141 mAb of IgM isotype (specific for the EB6 molecules) completely restored the lysis of Cw3-transfected P815 cells by the Cw3-specific NK clones EX2 and EX4. Similarly, both the entire EB6 mAb, its F(ab')2 fragment and the XA141 mAb reconstituted the lysis of C1R, a Fc gamma R- target cell expressing Cw4 as the only serologically detected class I antigen. Thus, it appears that masking of different members of p58 molecules prevents recognition of "protective" MHC class I alleles and thus the delivering of inhibitory signals. Further support to the concept that p58 molecules represent a NK receptor delivering a negative signal was provided by experiments in which the entire anti-p58 mAbs (of IgG isotype) could inhibit the lysis of unprotected Fc gamma R+ P815 target cells, thus mimicking the inhibitory effect of MHC class I molecules.

Amino acid substitutions can influence the natural killer (NK)-mediated recognition of HLA-C molecules. Role of serine-77 and lysine-80 in the target cell protection from lysis mediated by "group 2" or "group 1" NK clones.

Natural killer (NK) cells have been shown to express a clonally distributed ability to recognize HLA class I alleles. The previously defined NK clones belonging to "group 1" recognize HLA-C*0401 (Cw4) and other HLA-C alleles sharing Asn at position 77 and Lys at position 80. Conversely, the "group 2" NK clones recognize HLA-Cw*0302 (Cw3) and other HLA-C alleles characterized by Ser at position 77 and Asn at position 80. We assessed directly the involvement of these two residues in the capacity of NK cell clones to discriminate between the two groups of HLA-C alleles. To this end, Cw3 and Cw4 alleles were subjected to site-directed mutagenesis. Substitution of the amino acids typical of the Cw3 allele (Ser-77 and Asn-80) with those present in Cw4 (Asn-77 and Lys-80) resulted in a Cw3 mutant that was no longer recognized by group 2 NK cell clones, but that was recognized by group 1 clones. Analysis of Cw3 or Cw4 molecules containing single amino acid substitutions indicates roles for Lys-80 in recognition mediated by group 1 clones and for Ser-77 in recognition mediated by group 2 clones. These results demonstrate that NK-mediated specific recognition of HLA-C allotypes is affected by single natural amino acid substitutions at positions 77 and 80 of the heavy chain.