Characterization with monoclonal antibodies of human lymphocytes active in natural killing and antibody-dependent cell-mediated cytotoxicity of dengue virus-infected cells.

Non-immune human peripheral blood lymphocytes (PBL) lyse dengue virus-infected cells to a greater degree than uninfected cells. In the present study, the PBL active in lysing dengue virus-infected Raji cells are characterized using monoclonal antibodies and are compared to lymphocytes that lyse K562 cells. Leu11+ cells lyse dengue virus-infected cells and K562 cells. Leu11- cells lyse dengue virus-infected cells, but not K562 cells. In the Leu11+ fraction, Leu11+ Leu7- cells are more active than Leu11+ Leu7+ cells in lysing dengue virus-infected cells. T3+ cells also lyse dengue virus-infected cells, but they do not lyse K562 cells. T3- cells lyse both target cells. These results, along with the observation that Leu11+ cells and T3+ cells are different subsets of PBL, indicate that the PBL that are active in lysing dengue virus-infected cells are heterogeneous and are contained in Leu11+ and T3+ subsets. Leu11+ cells are more active than T3+ cells. Leu11+ cells are active in lysing dengue virus-infected cells by antibody-dependent cell-mediated cytotoxicity, whereas T3+ cells are not active.     

Inhibition of murine natural killer cell-mediated cytotoxicity by pretreatment with ammonium chloride.

In the present study the effect of ammonium chloride on murine natural killer (NK) cell-mediated cytotoxicity to T cell lymphoma, YAC-1 was studied. It was found that ammonium chloride treatment significantly reduced the cytotoxicity of splenic NK cells without any detectable change in cell viability. It is, therefore, suggested to avoid ammonium chloride treatment in order to obtain the realistic reflection of murine NK cell activities.     

B cell antigens on effectors for natural cell-mediated cytotoxicity.

Cell suspensions enriched for N cells, the effector cell for human natural cell-mediated cytotoxicity, were observed to react strongly with a rabbit antiserum directed against an antigen associated with B cells. Treatment of the effector suspension with the antiserum resulted in inhibition of natural cell-mediated cytotoxicity. The blocking occurred, however, only with the intact antibody indicating that inhibition was caused through the formation of antigen-antibody complexes. The antiserum was then employed to arm natural effector cells to react specifically with B cell lines. Specific arming was achieved after the formation of antigen-antibody complexes was prevented by initially reacting the effector suspension with F(ab')2 of the anti-B cell antibody. This study also supports the identity of the subset of cells responsible for antibody-dependent and natural cell-mediated cytotoxicity and the similarity in their mechanisms.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

Monoclonal antibodies in cell-mediated cytotoxicity against human melanoma and colorectal carcinoma.

Hybridoma-derived monoclonal anti-melanoma antibodies and anti-colorectal carcinoma antibodies were found to mediate in vitro antibody dependent cell-mediated cytotoxicity (ADCC) reactions against melanoma and colorectal carcinoma cells, respectively. The antigen(s) detected in ADCC on melanoma cells maintained for more than one hundred passages in tissue culture were also found on two recently established melanoma cell lines. These antigens were not detected on skin fibroblasts of the same patients from whom the melanomas were obtained. The ADCC reactivities of anti-melanoma and anti-colorectal carcinoma antibodies were found to be specific for melanoma cells and colorectal carcinoma cells, respectively.     

Studies of human natural killer cells. III. Neutropenia associated with unusual characteristics of antibody-dependent and natural killer cell-mediated cytotoxicity

A 52-year-old Caucasian man with chronic neutropenia and recurrent infections was found to have an increased proportion of peripheral T lymphocytes having Fc receptors for IgG (T(). Although levels of antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killing (NK) by unfractionated lymphocytes were similar to those of a control donor, the frequency of NK cells was markedly increased. Removal of E rosette-forming cells eliminated both NK and ADCC by the patient's peripheral blood, in marked contrast to theenhanced cytotoxicity seen with control lymphocytes. Both normal and patient ADCC and NK functions were removed by depletion of Fc receptor-bearing cells. These depletion experiments proved that all of the patient's killer cells were E rosetteforming T cells, in contrast to the heterogeneous pattern of Null and T killer cells seen in the blood of normal donors. The homogeneity of the T proliferation suggested that ADCC and NK were mediated by the same cell type, albeit acting by different mechanisms. The addition of the patient's serum and lymphocytes to chromiumlabeled normal granulocytes caused a low but significant level of cytotoxicity, indicating that the patient's neutropenia may have been caused by a similar mechanismin vivo. There was no evidence of complement-dependent serum antibody-mediated neutrophil lysis, but one serum sample taken over the course of the patient's disease agglutinated granulocytes from four of five donors tested.     

In vitro activity of anti-DNA antibodies from SLE patients in antibody-dependent cell-mediated cytotoxicity.

Peripheral blood lymphoid cells from normal individuals were cytotoxic to target cells coated with DNA when incubated with serum from patients with systemic lupus erythematosus (SLE) but not when incubated with serum from normal subjects. Sera from SLE patients with clinically acitve disease were more active than sera obtained from those patients in remission. In the absence of normal lymphocytes, SLE sera were not cytotoxic to the DNA-coated cells. The active fraction in the serum appeared to be IgG anti-DNA antibodies. These studies indicate that anti-DNA can operate through the antibody-dependent cell-mediated cytotoxicity mechanisms in vitro.     

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, during an attempt to identify novel NK receptors or co-receptors, we found that CD59 functions as a co-receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in markedly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3zeta chains associated with these NCR, but not those associated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physical interaction of this GPI-linked protein with primary triggering NK receptors.     

Role of specific cell-mediated cytotoxicity in protecting fish from viral infections

We report the in vivo role of specific cytotoxic cells in protecting fish from acute viral infections. Specifically, we found that (1) there is an inverse relationship between cytotoxic activities and viral load and (2) adoptive transfer of immune leukocytes prevented viral infection. Crucian carp hematopoietic necrosis virus (CHNV), which has a virulence to ginbuna crucian carp, was recently found and identified in Japan. Specific cell-mediated cytotoxicity of ginbuna leukocytes against CHNV-infected syngeneic cells was induced as a result of intraperitoneal inoculation with CHNV. This cytotoxicity was not induced against either virus-infected allogeneic cells or eel rhabidovirus- (EVA) infected syngeneic cells. In these respects, the cytotoxic activity was similar to that of mammalian cytotoxic T-lymphocyte (CTL) activity. Viral titers of tissues from infected fish were remarkably reduced 8 days after infections, when specific cytotoxic activity reached a peak. This result suggested that specific cytotoxic cells were responsible for the early control of CHNV replication. On the other hand, CHNV-specific antibody was greatly increased when the virus was eliminated by cytotoxic activities. The effectiveness of the virus-specific cytotoxicity was evaluated using adoptive cell transfer. Recipients that received leukocytes from immune syngeneic donors escaped CHNV infection. These findings suggest that virus-specific cytotoxic cells have a role in controlling viral infections in a fish.     

Inhibition of natural killer cell-mediated cytotoxicity by lipids extracted from Mycobacterium bovis BCG.

Several studies have demonstrated an augmentation of natural killer (NK) cell-mediated cytotoxicity by various adjuvants including BCG. Inhibitory effects of BCG have also been reported, particularly for relatively high doses. Because the cell wall of Mycobacterium bovis BCG contains a high proportion of lipids, the possibility was considered that these lipids may modulate NK activity. A total lipid fraction was extracted from Mycobacterium bovis BCG and used for the lipid modulation of NK effector and target cells. Treatment of effector or target cells resulted in decreased membrane fluidity and decreased NK cell-mediated cytotoxicity in both cases. Pretreatment of target cells did not affect the binding between target and effector cells, as shown in the single cell assay, whereas pretreatment of effectors resulted in inhibition of conjugation. It was further demonstrated that treatment of target cells which were first programmed for lysis protected these cells from subsequent lysis during the killer cell independent lysis stage. The results of this study suggest that adverse effects of BCG treatment on immune functions may be mediated by BCG derived lipids.     

Development of antibody-dependent cell-mediated cytotoxicity in the respiratory tract after natural infection with respiratory syncytial virus.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured in nasopharyngeal secretions collected from 42 infants and young children at various intervals after primary or secondary infection with respiratory syncytial virus. ADCC was determined by specific immune release of 51Cr from respiratory syncytial virus-infected HEp-2 cell culture monolayers, with lymphocytes from adult volunteers as effector cells. Specific ADCC responses in nasopharyngeal secretions after primary infection were observed as early as 3 days after the onset of clinical symptoms, and peak activity was observed 14 to 29 days after the onset of illness. ADCC responses after reinfection were significantly greater in both the acute and convalescent phases (P less than 0.05) than were ADCC responses after primary infection. ADCC in secretions was mediated primarily by the immunoglobulin G isotype of respiratory syncytial virus antibody.     

Staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity

T cells equipped with sophisticated TCR and MHC recognition structures, an efficient cytokine communication network and lethal cytotoxic effector functions constitute one of the bulwarks of the mammalian immune system. However, infective agents have developed strategies to undermine T-cell immunity; for example, certain bacterial toxins serve as 'superantigens' by binding to preserved determinants on MHC class-II-encoded proteins and activating T cells expressing particular sequences of TCR V beta gene products. In this paper, Mikael Dohlsten and colleagues present evidence suggesting that these bacterial superantigens direct T cells to eradicate MHC class-II-expressing antigen-presenting cells, thus counteracting specific T-cell functions.     

Specificities in natural cell-mediated cytotoxicity against lymphoblastoid cell lines. I. Selective inhibition by cross-competition.

The apparent nonselective effects of natural cell-mediated cytotoxicity tested directly on different lymphoblastoid cell targets were found to be quite specific in the cross-competition assay. The specificity was detected through inhibition of cytotoxicity by competitor cells sharing common specificities with the target cells. Cross-competition tests were performed employing eight lymphoblastoid lines including four T and four B cells. Selective inhibition observed between lymphoblastoid cell lines indicated that T and B cell lines were more antigenically similar within each group than between them. The target antigens that distinguish T cell from B cell lines have been tentatively called TA-T and TA-B.     

Antibody-dependent cell-mediated cytotoxicity to Varicella zoster.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against Varicella zoster (VZ) infected fibroblasts is described. ADCC requires antibody to VZ and is greater with heavily infected targets. It is not dependent on the immune status of the effector cells. The effector cells responsible for ADCC are present in sheep red cell (E) positive and E-fractions of peripheral blood, and in the G10 non-adherent population. The ADCC activity is present in microexudate non-adherent cells and in the adherent population to a lesser extent. The technique provides a means to study host defence against VZ infection in immune compromised individuals.     

Specificity of cell-mediated transplantation reactions: II. Studies with a sensitive assay of cell-mediated cytotoxicity

To study the specificity of transplantation immunity in vitro the capacity of two aggressor cell populations from allografted mice to destroy target cells obtained from a panel of strains having different H-2 types was assessed. ⁵¹Chromium-labelled macrophages were found to be suitable target cells to allow for quantitative comparisons of cytotoxicity. Spleen cells from skin-grafted mice lysed only target cells of strains which shared at least one H-2 region (K or D) with the donor. By contrast, non-adherent peritoneal exudate cells, produced as a consequence of ascites tumour allografts, exhibited a cytotoxic potential approximately 16- to 32-fold greater than the above spleen cells and were capable of lysing third-party target cells of strains having disparity with the graft donor at both H-2 regions. The difference between the two systems appears to be quantitative. These findings appear to reconcile previously conflicting observations, and suggest minimal or no participation of public H-2 specificities in cell-mediated reactions.     

Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines

The lack of classical HLA-class I molecules on trophoblast is necessary to prevent allorecognition by maternal CTL, but may induce activation of NK cells. A protective role against NK cells equipped of suitable inhibitory receptors has been proposed for nonclassical HLA-class I molecules including HLA-E and HLA-G. In the present study we show that the NK-mediated killing of two choriocarcinoma cell lines, JAR and JEG3, is induced upon engagement of natural cytotoxicity receptors (NCR) with their specific ligands. In particular, we show that NKp44, a triggering receptor expressed at the NK cell surface only after in vitro culture in the presence of IL-2, plays a central role in triggering NK cytotoxicity against trophoblast cells. Also NKp46 appear to contribute to this function by cooperating with NKp44. On the other hand, other triggering receptors such as NKp30, 2B4, and NKG2D are not involved in killing of choriocarcinoma. Our findings suggest that resistance of trophoblast to NK-mediated cytotoxicity is the result of insufficient activating interactions between the various triggering NK receptors and their target cell ligands. On the other hand, the interaction of nonclassical HLA class I molecules with inhibitory NK receptors appears to play only a marginal role in regulating the susceptibility of choriocarcinoma to NK mediated cytotoxicity.     

Monoclonal antibodies against leucoagglutinin-reactive human T lymphocyte surface components. Two antibodies which inhibit cell-mediated cytotoxicity at a post-binding stage

Two out of 20 monoclonal antibodies (IgM, kappa), mAb 3192 and mAb K3G, raised against leucoagglutinin-reactive components on human T cells, effectively blocked lymphocyte-mediated cytotoxicity in vitro. No antigenic polypeptide reactive with these antibodies has been identified thus far. However, they have previously been shown to react specifically with certain neutral glycolipids obtained from spleen. Both mAb inhibited the cytotoxicity of natural killer (NK) cells against K562 cells, antibody-dependent cellular cytotoxicity (ADCC) towards antibody-coated bovine erythrocytes and cytotoxic T lymphocyte activity against allogeneic target cells. In both NK and ADCC, preincubation of the lymphocytes with different antibody concentrations resulted in a dose-dependent reduction of cytotoxicity. In contrast, preincubation of the target cells had no effect indicating that the mAb inhibited cytotoxicity at the effector cell level. When studied at the single-cell level, the mAb did not alter the number of lymphocytes forming conjugates with K562 but significantly reduced the frequency of conjugates containing dead target cells. Addition of the mAb to preformed conjugates resulted in a dose-dependent reduction in the proportion of conjugates containing dead target cells. Furthermore, mAb 3192 did not reduce the number of lymphocytes forming rosettes with bovine erythrocytes, indicating that inhibition of ADCC was not due to blocking of the effector cell-target cell interaction mediated by the Fc receptor of the effector cells. Taken together, these results suggest that the mAb inhibited cytotoxicity by interfering with a post-binding step common for the different cytotoxicity systems.     

Inhibition of natural killer cell-mediated cytotoxicity by Kaposi's sarcoma-associated herpesvirus K5 protein

Kaposi's sarcoma-associated herpesvirus (KSHV) K3 and K5 proteins dramatically downregulate MHC class I molecules. However, although MHC class I downregulation may protect KSHV-infected cells from cytotoxic T lymphocyte recognition, these cells become potential targets for natural killer (NK) cell-mediated lysis. We now show that K5 also downregulates ICAM-1 and B7-2, which are ligands for NK cell-mediated cytotoxicity receptors. As a consequence, K5 expression drastically inhibits NK cell-mediated cytotoxicity. Conversely, de novo expression of B7-2 and ICAM-1 resensitizes the K5-expressing cells to NK cell-mediated cytotoxicity. This is a novel viral immune evasion strategy where KSHV K5 achieves immune avoidance by downregulation of cellular ligands for NK cell-mediated cytotoxicity receptors.