Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Influenza viral glycoproteins induced cell-mediated cytotoxicity by an interferon-independent mechanism

Human natural killer (NK) cells exposed to the influenza surface antigen neuraminidase (NA) show high cytotoxic activity, as evaluated using chromium-labeled K562 target cells in a standard overnight cytotoxicity assay. The role of interferon (IFN) in the stimulation of NK cells was examined by using three separate approaches. The use of appropriate antibodies to check IFN- and NA-specific cell-mediated cytotoxicity (CMC) stimulation showed that antibodies to IFN (-alpha, -gamma) did not alter NA-induced CMC, and vice versa. The treatment of NK cells with actinomycin D, before or after stimulation with IFN and NA revealed that only IFN-induced CMC was inhibited (50 to 100%). However, NK cells that were stimulated with NA before their exposure to actinomycin D became susceptible to stimulation by IFN. The interaction kinetics between IFN and NA demonstrated the presence of two mechanisms of CMC stimulation. Taken together, the results clearly show that stimulation of CMC by a viral component is effected through an IFN-independent pathway, and that this mechanism is probably followed by IFN under certain conditions.     

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.     

Lectin-dependent cell-mediated cytotoxicity in hamsters bearing syngeneic tumors

Spleen cells from LSH hamsters inoculated with xenogeneic, allogeneic, or syngeneic (PARA-7) tumor cells were assayed for their ability to mediate direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) in a 4-hr chromium release assay. Spleen cells from animals immune to xenogeneic or allogeneic cells demonstrated specific DCMC against homologous target cells in the absence of Con A and nonspecific LDCC against both homologous and heterologous target cells in the presence of Con A. Spleen cells from animals bearing syngeneic PARA-7 tumors (TBA) failed to express DCMC against homologous or heterologous target cells; however, significant lysis of all target cells occurred in the presence of Con A. LDCC was not detectable when nonsensitized spleen cells from normal animals were employed. The LDCC reaction was dependent on the concentration of Con A and the number of effector cells present in the reaction. The development of LDCC effector cells in the TBA appeared to parallel the development of both DCMC and LDCC effector cells in immune animals.     

Inhibition of cell-mediated cytotoxicity by Corynebacterium parvum

Summary We investigated the effect of altering dose and route of Corynebacterium parvum (C. parvum) administration on the adjuvant's inhibition of cell-mediated cytotoxicity (CMC). Primary in vivo and secondary in vitro CMC of C57B1/6 mice alloimmunized to P815 were depressed if C. parvum was administered systemically (IV or IP) but not when it was given SC. Similarly, only systemic C. parvum generated cells capable of suppressing in vitro CMC. Primary and secondary CMC in spleen was equally inhibited by 700 and 70 g, whereas suppressor cell activity was marked with 700 g and minimal with 70 g. Administration of C. parvum SC admixed with alloantigen resulted in early enhancement and late depression of primary CMC. Secondary CMC was depressed but suppressor activity was absent. Dissociation of CMC depression from suppressor cell generation indicates that these phenomena can be separated under certain conditions.     

Further studies on the use of mouse epidermal cells for the in vitro induction and detection of cell-mediated cytotoxicity

Mouse epidermal cells (EC) and lymphoid cells (LC) were compared as targets of cellmediated cytotoxicity (CMC) in short-term chromium release assays where attacker cells were generated in primary mixed cultures using irradiated allogeneic EC or LC as stimulators. Three patterns of relative susceptibility to lysis of the two types of target cells were observed: (i) significantly greater lysis of LC than of EC targets; (ii) significantly greater lysis of EC than LC targets; and (iii) approximately equal susceptibility to lysis of the two targets. The first pattern was primarily associated with LC stimulators, whereas the second and third patterns were almost invariably associated with EC stimulators. Factors possibly contributing to the differences in in vitro immunogenicity and susceptibility to CMC of EC and LC were investigated, including the alteration of EC surface antigens during the trypsinization required to prepare EC suspensions, the differential expression of shared alloantigens, or the restricted expression of tissue-specific alloantigens on the two types of cells. Tests with intact and trypsinized LC on the one hand and fresh and short-term cultured EC on the other indicated that trypsinization is not responsible for the basic differences between EC and LC detected in the in vitro assays. Antibody absorption tests demonstrated that although EC and LC express approximately equal quantities of the cell surface antigens determined by the H-2D region of the H-2 complex, LC express significantly greater quantities of the antigens determined by the H-2K and I regions. In addition, the results of cold target inhibition tests suggest that tissue-specific antigens on both EC and LC also influence their relative immunogenicity and susceptibility to lysis.     

Antibody-dependent cell-mediated cytotoxicity of cryopreserved human monocytes

Human peripheral blood monocytes (PBM) modulate and participate in a variety of host defences. Cryopreservation of PBM has facilitated studies of their function. Peripheral blood samples cleared of red cells and granulocytes by centrifugation over Ficoll-Hypaque were cryopreserved at 1 degree C/min in 10% Me2SO and stored at -150 degrees C. Cryopreserved cells were thawed rapidly, diluted at a constant rate over 10 min with 9 vol of media, and washed twice prior to study. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity against anti-D-coated Rh-positive erythrocytes of both fresh and cryopreserved PBM was tested and found to be equal (52.5 vs 51%). The myeloperoxidase positive, EA-rosette-positive population in cryopreserved cells was 39% compared with 17% for fresh cells (P less than 0.0001). This difference is due to preferential recovery of cryopreserved monocytes among mononuclear cells. The proportion of cells expressing Fc receptors among the myeloperoxidase-positive mononuclear cell population increased after freezing, suggesting an alteration in membrane structure induced by cryopreservation. It is concluded that PBM can be cryopreserved in Me2SO and that ADCC function is fully retained in the cryopreserved cells. This study along with a previous study (R.S. Weiner and S.J. Norman, J. Natl. Cancer Inst. 66, 255-260, 1981) demonstrate the feasibility of using cryopreserved human PBM for functional studies.     

Ral GTPases regulate cell-mediated cytotoxicity in NK cells

NK cells are key components of the immune response to virally infected and tumor cells. Recognition of target cells initiates a series of events in NK cells that culminates in target destruction via directed secretion of lytic granules. Ral proteins are members of the Ras superfamily of small GTPases; they regulate vesicular trafficking and polarized granule secretion in several cell types. In this study, we address the role of Ral GTPases in cell-mediated cytotoxicity. Using a human NK cell line and human primary NK cells, we show that both Ral isoforms, RalA and RalB, are activated rapidly after target cell recognition. Furthermore, silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization toward the immunological synapse and the subsequent process of degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, a protein complex involved in polarized secretion. This assembly is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells. Our results thus identify a role for Ral in cell-mediated cytotoxicity, implicating these GTPases in lymphocyte function.     

Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells.

In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells.

Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab.

The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells. In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Immunogenetic studies on the cell-mediated cytotoxicity in the clawed toadXenopus laevis

In order to test for further homologies between the MHC of mammals and amphibians, experiments were conducted to assess whether lower vertebrates such as anurans were able to generate killer cells after allogeneic stimulation. The generation of cytotoxic effector cells could be obtained in outbred families and clones of isogenic frogs after in vivo priming with either irradiated allogeneic lymphocytes or with an allogeneic skin graft, provided that the immune spleen cells were restimulated in vitro with the specific irradiated cells used for priming. Effector cells generated against a definedMHC haplotype could lyse targets having one of their two haplotypes in common with the stimulators. In contrast, no lysis was observed when the target cells differed from the specific stimulators by twoMHC haplotypes.The cytotoxic activity of the MLR-restimulated lymphocytes appeared to be mediated by T cells since passage of the effector spleen cells through a nylon wool column, under conditions which removedXenopus B lymphocytes, improved killing on a per cell basis. It therefore appears that the genes responsible for the highly specialized function of T killer cells have emerged early in evolution at least at the time of the emergence of the amphibians (300 million years ago) and that they were already linked to the MHC of this species. The MHC polymorphism inXenopus seems to be lower than in mammals as evidenced by the high frequency of cross-killing observations paralleled by the high frequency of MLR identical animals found in a large outbred population.     

Inhibition of T cell-mediated cytotoxicity by anti-inflammatory steroids

We have tested the capacity of glucocorticoids to modulate the effector function of splenic cytotoxic T lymphocytes (CTL) obtained after i.p. immunization with allogeneic cells. Although acute exposure to glucocorticoids did not inhibit the activity of freshly obtained splenic CTL, preincubation of these CTL for several hours with subnanomolar concentrations of several different glucocorticoids caused marked inhibition. The relative inhibitory potency of the steroids tested correlated with their reported activity both in glucocorticoid receptor binding assays and in assays of anti-inflammatory potency in man. The inhibitory effects of low concentrations (10(-10) M to 10(-9) M) of dexamethasone were reversed by human or mouse interleukin 2 (IL 2)-containing supernatants, but were not reversed by IL 1-containing supernatants. The inhibitory effects of higher concentrations (10(-8) M to 10(-7) M) of dexamethasone could not be reversed even by very high doses of mouse IL 2. In contrast to previous reports of minimal direct glucocorticoid effects on CTL activity, the present results suggest that after preincubation, splenic CTL from in vivo-immune mice are sensitive to inhibition by glucocorticoids, and that the glucocorticoids may act both indirectly (on IL 2 production) and directly on the CTL.     

Vulnerability of human neurons to T cell-mediated cytotoxicity

Axonal and neuronal loss occurs in inflammatory diseases of the CNS such as multiple sclerosis. The cause of the loss remains unclear. We report that polyclonally activated T cells align along axons and soma of cultured human neurons leading to substantial neuronal death. This occurs in an allogeneic and syngeneic manner in the absence of added Ag, requires T cells to be activated, and is mediated through cell contact-dependent mechanisms involving FasL, LFA-1, and CD40 but not MHC class I. Activated CD4(+) and CD8(+) T cell subsets are equally neuronal cytotoxic. In contrast to neurons, other CNS cell types (oligodendrocytes and astrocytes) are not killed by T cells. These results demonstrate for the first time the high and selective vulnerability of human neurons to T cells, and suggest that when enough activated T cells accumulate in the CNS, neuronal cytotoxicity can result through Ag-independent non-MHC class I mechanisms.     

Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.     

Inhibition of NK cell-mediated cytotoxicity by oxysterols.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.