Effect of altered membrane fluidity on NK cell-mediated cytotoxicity. I. Selective inhibition of the recognition or post recognition events in the cytolytic pathway of NK cells

NK cell-mediated cytotoxicity results from membrane interactions between NK effector and target cells. The role of membrane fluidity in these events is not known. The present study was undertaken to investigate the effect of changes in membrane lipid fluidity of NK effector and NK-sensitive target cells on the lytic pathway of NK cell-mediated cytotoxicity. Fluidity was modulated by various lipids and measured by fluorescence polarization. NK effector cells treated with phosphatidylcholine complexed with polyvinylpyrrolidone (PVP) and bovine serum albumin (BSA) showed increased membrane fluidity. This fluidization of the effector cell membrane resulted in a significant inhibition of cytotoxic activity in the 51Cr-release assay. Single cell analysis revealed that the inhibition was due to a decrease in the frequency of NK target conjugates and reduced killing of conjugated targets. Rigidification of the NK effector cell membranes by treatment with cholesteryl hemisuccinate complexed with PVP and BSA also resulted in inhibition of cytotoxicity. This inhibition was post binding, because binding was increased and lysis was abrogated. Fluidization of K562 target cell membranes caused a slight but insignificant increase in their lysis by NK cells without affecting the binding step. On the other hand, rigidification of K562 membranes decreased the sensitivity of these target cells to lysis. Single cell analysis revealed that this inhibition of NK lysis is post binding, because the frequency of killers was significantly decreased. It was also shown that membrane rigidification of target cells that were programmed for lysis during the lethal hit stage and subsequently separated from effector cells, rendered the programmed cells resistant to killing during the killer cell-independent lysis step. These results demonstrate that fluidization or rigidification of the plasma membrane of either effector or target cells affect different stages of the NK cell-mediated cytolytic events.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Oncogenicity of human papillomavirus- or adenovirus-transformed cells correlates with resistance to lysis by natural killer cells.

The reasons for the dissimilar oncogenicities of human adenoviruses and human papillomaviruses (HPV) in humans are unknown but may relate to differences in the capacities of the E1A and E7 proteins to target cells for rejection by the host natural killer (NK) cell response. As one test of this hypothesis, we compared the abilities of E1A- and E7-expressing human fibroblastic or keratinocyte-derived human cells to be selectively killed by either unstimulated or interferon (IFN)-activated NK cells. Cells expressing the E1A oncoprotein were selectively killed by unstimulated NK cells, while the same parental cells but expressing the HPV type 16 (HPV-16) or HPV-18 E7 oncoprotein were resistant to NK cell lysis. The ability of IFN-activated NK cells to selectively kill virally transformed cells depends on IFN's ability to induce resistance to NK cell lysis in normal (i.e., non-viral oncogene-expressing) but not virally transformed cells. E1A blocked IFN's induction of cytolytic resistance, resulting in the selective lysis of adenovirus-transformed cells by IFN-activated NK cells. The extent of IFN-induced NK cell killing of E1A-expressing cells was proportional to the level of E1A expression and correlated with the ability of E1A to block IFN-stimulated gene expression in target cells. In contrast, E7 blocked neither IFN-stimulated gene expression nor IFN's induction of cytolytic resistance, thereby precluding the selective lysis of HPV-transformed cells by IFN-activated NK cells. In conclusion, E1A expression marks cells for destruction by the host NK cell response, whereas the E7 oncoprotein lacks this activity.     

Natural killer cells in rhesus monkeys: properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC+) fraction and decreased in the ERFC- fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16+ (Leu 11b+) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16-, CD2+ phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Participation of the CD27 antigen in the regulation of IL-2-activated human natural killer cells.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.     

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.     

Interferon and butyrate treatment leads to a decreased sensitivity of NK target cells to lysis by homologous but not by heterologous effector cells

Human K-562 and HHMS cells were pretreated with human recombinant interferon (IFN)-gamma and used as targets in NK assays against human and murine effector cells. A protective effect against NK lysis was observed only in the homologous assay, whereas no change or even a slight increase in NK sensitivity against heterologous effector cells was found. In cold target inhibition experiments IFN-treatment of K-562 cells led to a decrease in their capacity to act as competitors in the homologous NK assay, leaving their inhibitory capacity unaltered in the heterologous assay. In accordance with results observed using human NK targets, murine YAC-1 cells treated with mouse recombinant IFN-gamma did not lose their susceptibility to human NK cells. However, they were markedly less susceptible to lysis mediated by murine effectors. Butyrate, another compound causing decreased sensitivity of K-562 cells for human natural killing, also failed to reduce the susceptibility against murine NK cells. The results indicate that the NK-resistant tumor target phenotype caused by IFN or differentiation-inducing agents can only be detected by homologous but not by heterologous effector cells. This suggests that major differences exist between the inter- and intraspecies NK killing mechanisms.     

Definition of a secondary target cell trigger during natural killer cell cytotoxicity: possible role of phospholipase A2

Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined. PA-2 inhibitors, quinacrine and p-bromophenacyl bromide, inhibited NK CMC at the effector cell level, but affected neither initial target-effector cell binding nor dissociated conjugates during the length of the NK assay, suggesting that they block post-binding lytic events. A calcium pulse assay showed that PA-2 inhibitors inhibit only moderately when added after calcium and only within the first 15 min, demonstrating that these inhibitors blocked very early post-binding lytic events. Because this very early post-binding inhibitory effect was consistent with effects upon the NK trigger mechanism, the effect of exogenous PA-2 on NK lysis was tested. Pretreatment of K562 target cells but not pretreatment of peripheral blood lymphocytes (PBL) with 20 units/ml PA-2 enhanced lysis by two to eight-fold (based upon lytic units), showing its enhancing effect to be at the target cell level. Single cell assays using effector cells purified by indirect panning with monoclonal antibody NKH-1 showed that only the number of killer cells was increased. Calcium pulse assays showed that enhancement of lysis was maximum 15 min after addition of calcium and decreased rapidly thereafter, demonstrating its effect at an early post binding stage. Additionally, PA-2 was shown to overcome inhibition by the monoclonal antibody 13.3, which has been shown to affect the trigger stage of NK lysis (post-binding but prior to calcium dependent events). Thus, it appears that an NK cell-associated PA-2 could function by modulating the target cell surface, revealing a structure which acts as a "secondary" trigger, subsequent to the 13.3 "trigger", requisite for activation of the NK lytic process.     

Helper factor(s) augment in vitro induction of tumor-specific immunity

Helper factor supernatants derived from alloantigen-activated murine lymphocytes augment the generation of cytolytic effector cells to syngeneic tumor cells. The effects are dose dependent and vary with the syngeneic tumor cell system studied. The effector cells are specific for the tumor-associated antigen(s) utilized for their induction, and are sensitive to lysis with anti-T-cell serum (Thy 1.2), but are insensitive to lysis with an allogeneic anti-NK-cell serum. The helper factor supernatants also augment the production of a “tissue-culture-induced” cytolytic cell (cultured NK cell), which is resistant to treatment with both anti-Thy 1.2 and anti-NK serum.     

Inhibition of natural killer cell lysis by natural killer-inhibitory substance: mechanism of suppression

The mechanism of suppression of NK-mediated lysis by a soluble product of peritoneal cells (NK-IS, natural killer-inhibitory substance) was investigated. Pretreatment of effector cells resulted in depressed NK lysis while pretreatment of targets had no effect, indicating suppression is due to alterations in effector cell function rather than changes in target cells. NK-IS had no effect on the formation of conjugates between effectors and NK-susceptible targets. When NK-IS was added to effector-target cell mixtures after the binding step had been successfully completed, ensuing lysis was significantly depressed, confirming that NK-IS inhibited a postbinding lytic event. The degree of suppression caused by NK-IS was directly related to the duration of exposure to the inhibitory molecule. In addition, a preliminary temperature-dependent step of binding to and/or intracellular entry of NK-IS into effectors is required before suppression can occur. NK-IS prevents the activation of NK cell lysis by interferon and Corynebacterium parvum and effectively inhibits lysis mediated by already activated effectors. The potent suppression of NK lysis and prevention of interferon and C. parvum-mediated activation of NK lysis by a soluble product of peritoneal cells may explain the extremely low level of NK effector cell function within the peritoneal cavity.     

Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and by T cells. In both humans and mice, KLRG1 identifies Ag-experienced T cells that are impaired in their proliferative capacity but are capable of performing effector functions. In this study, we identified E-cadherin as a ligand for murine KLRG1 by using fluorescently labeled, soluble tetrameric complexes of the extracellular domain of the murine KLRG1 molecule as staining reagents in expression cloning. Ectopic expression of E-cadherin in B16.BL6 target cells did not affect cell-mediated lysis by lymphokine-activated NK cells and by CD8 T cells but inhibited Ag-induced proliferation and induction of cytolytic activity of CD8 T cells. E-cadherin is expressed by normal epithelial cells, Langerhans cells, and keratinocytes and is usually down-regulated on metastatic cancer cells. KLRG1 ligation by E-cadherin in healthy tissue may thus exert an inhibitory effect on primed T cells.     

Leukocyte-associated Ig-like receptor-1 functions as an inhibitory receptor on cytotoxic T cells

Leukocyte associated Ig-like receptor-1 (LAIR-1) is a surface molecule expressed on human mononuclear leukocytes that functions as an inhibitory receptor on human NK cells. In addition to NK cells, LAIR-1 is expressed on T cells, B cells, macrophages, and dendritic cells. Most cells express two biochemically distinct forms of LAIR-1, which we now show are likely alternative splice variants of the same gene. Cross-linking of LAIR-1 on human T cell clones results in inhibition of cytotoxicity only in T cell clones that lack CD28 and are able to spontaneously lyse certain targets in vitro. Moreover, the cytolytic activity of freshly isolated T cells, which is thought to be mainly due to "effector" T cells, can be inhibited by anti-LAIR-1 mAb. Thus, LAIR-1 functions as an inhibitory receptor not only on NK cells, but also on human T cells. This indicates that LAIR-1 provides a mechanism of regulation of effector T cells and may play a role in the inhibition of unwanted bystander responses mediated by Ag-specific T cells.     

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.     

Enhanced lytic susceptibility of Ha-ras transformants after oncogene induction is specific to activated NK cells

C3H 10T1/2 mouse fibroblasts were transfected with a plasmid vector composed of EJ, the mutated c-Ha-ras, and a metallothionein promotor that induced amplified ras expression when activated by culture in the presence of zinc. Experiments were conducted to compare the effect of induction on killing by activated natural killer (NK) cells, cytotoxic T lymphocytes, activated macrophages, and antibody plus complement. The only effector that recognized increased ras expression and exhibited high-inducible cytolysis was an activated NK cell. The effectors from spleen were poly I.C. boostable, Lyt-1.1 negative, NK 1.2 positive, and asialo GM1 positive. Spleen cells from T cell-deficient nude mice, but not NK-deficient beige mice, exhibited high levels of killing activity, and experiments with NK cell clones demonstrated that these lines were also highly cytolytic and killed Ha-ras transfectants in parallel to YAC. Transfection of the same fibroblast line with c-myc did not alter the level of activated NK sensitivity. Cold target competition experiments revealed that Ha-ras-transfected and non-transfected 10T1/2 fibroblasts competed equally for lysis of either YAC or Ha-ras transfectants. Rat-1 fibroblasts did not compete, but gained this capacity when transformed with the v-Ki-ras oncogene but not v-fps. These data suggest that Ha-ras acts in target cells at a post-binding step, whereas Ki-ras may affect expression of target-effector binding structures. The findings that activated NK cell lysis may be specifically influenced by ras expression support a role for NK cells in host surveillance against early neoplastic changes.     

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.     

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.     

The switch from latent to productive infection in epstein-barr virus-infected B cells is associated with sensitization to NK cell killing

Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8+ cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8+ effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.     

Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid)

Spleen cells from C.B- 17 scid mice with severe combined immunodeficiency disease exhibit natural killer cell (NK) activity against YAC lymphoma targets in a standard 4-hr 51Cr release assay. The cytolytic activity is demonstrable only at high effector to target ratios but can be augmented at least sevenfold by the interferon inducer poly I:C. The pattern of target lysis is specific, because splenocytes from poly I:C-primed C.B-17 scid mice lyse NK-sensitive YAC cells and not the insensitive P815 mastocytoma. The presence of several NK-associated antigens on C.B-17 scid splenocytes was tested by pretreating cells with the appropriate antiserum plus complement before testing for NK activity. The results indicate that a proportion of NK effectors in C.B-17 scid mice bear surface NK 2.1 and Asialo GM1 but are negative for Thy-1.