Low affinity E-rosette formation by the human K cell.

Human T lymphocytes were separated into two subsets on the basis of relative affinity for sheep red blood cells (E), and these T cell fractions were examined for cytotoxic reactivity against antibody-sensitized Change liver cells (ADCC). High affinity E-rosette-forming cells (E-RFC) (55 +/- 6% of peripheral blood mononuclear cells), capable of rosette formation despite elevated temperatures of incubation (29 degrees C) and a limited concentration of E, contained few antibody-dependent cytotoxic cells (K cells). In contrast, low affinity E-RFC (23+/-7% of mononuclear cell suspensions) requiring cool temperatures of incubation (4 degrees C) and an excess of E to form rosettes, were highly enriched for ADCC activity. The majority of K cells exhibited low affinity interactions with E. T cells in thymus, tonsil, and lymph node formed high affinity E-rosettes and exhibited little reactivity in ADCC. Only peripheral blood and spleen contained easily identified low affinity E-RFC and anti-body-dependent cytotoxic cells. The proportion of low affinity E-RFC in the peripheral blood of normal subjects correlated closely with reactivity in ADCC, making it possible to predict cytotoxic potential for the E-rosette pattern. These data indicate that the human K cell may belong to a previously unappreciated but functionally important subset of thymic dependent mononuclear cells.     

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO₄ for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the ⁵¹Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.     

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil mediated antibody-dependent cellular cytotoxicity

The human lymphokine, leukocyte inhibitory factor (LIF), was investigated for its effect on neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) for K562 targets. Highly purified LIF (0.5 to 2 U/ml) induced a significant dose-dependent potentiation of neutrophil ADCC by up to 54.9% (p less than 0.001). Higher concentrations of LIF inhibited cytotoxicity. The degree of cytotoxicity was found to correlate (r = 0.99) with the increased secretion of superoxide after neutrophil-target cell interaction. Anaerobic conditions inhibited cytotoxicity mediated by both control and LIF-treated neutrophils. The latter observation lends support to the concept that enhanced ADCC was mediated through increased superoxide production and not through the induction of a separate pathway. Increased superoxide production may have resulted from an upregulation of the transduction mechanism leading to neutrophil stimulation through the Fc receptor. In addition, we demonstrated an increased capacity of the neutrophil to adhere to its target (average 3.3:1 effector:target ratio in untreated cells to 4.8:1 after treatment with LIF), and this may also have been responsible for the increase in the respiratory burst and subsequent enhanced ADCC. These observations provide potential support for an in vivo role for LIF in tumor immunity.     

Isolation of functional subsets of human peripheral blood monocytes.

Monocytes were isolated by counterflow centrifugation of Ficoll-Hypaque separated peripheral blood mononuclear cells. The monocytes formed a bimodal volume distribution of "large" and "small" phagocytic esterase-positive, peroxidase-positive cells with peaks at 470 and 410 mu3, respectively. The large monocytes were predominately Fc receptor positive, and were able to lyse both sensitized human and chicken erythrocyte targets in ADCC assays, whereas the small monocytes were largely FcR negative and were inactive against sensitized human erythrocyte targets. However, ADCC against chicken erythrocyte targets was seen in some fractions containing small monocytes and was probably due to FcR+ lymphocytes (K cells) in those fractions. These experiments establish that monocytes are effectors of ADCC against both human and chicken erythrocyte targets and that the peripheral blood monocyte is heterogeneous in size, function, and surface receptor distribution.     

Divergence in activation by poly I:C of human natural killer and killer cells

Summary Interferons consistently enhance spontaneous cellular cytotoxicity (SCC) mediated by natural killer (NK) cells. More controversial is the ability of interferons to enhance antibody-dependent cellular cytotoxicity (ADCC) mediated by killer (K) cells. Since NK and K cells appear to represent overlapping subpopulations of lymphocytes, the present study was undertaken to examine in greater detail the relationship between NK and K cell functional modulation by the potent interferon inducer, poly I:C. Utilizing peripheral mononuclear cells from a panel of 21 healthy individuals, treatment in vitro with poly I:C resulted in modulation of both SCC and ADCC. SCC was significantly enhanced in 52 of a series of 55 trials (95%), whereas ADCC was significantly enhanced in parallel in only 18 of the trials (33%). Cells which mediated enhanced ADCC were plastic-nonadherent, which is characteristic of K cells. SCC was consistently enhanced in all but two of the 14 individuals who were tested two or more times. By contrast, the ability of poly I:C to enhance ADCC varied between trials in 11 of these individuals. In the other three, ADCC enhancement never occurred. No correlation existed between SCC and ADCC augmentation despite use of the same target cell to assess the two lytic activities in parallel. Poly I:C exclusively enhanced SCC in 36 trials (65%) and exclusively enhanced ADCC in two trials (4%). Discordance between SCC and ADCC enhancement also occurred in three of eight trials (38%) in which lymphocytes were treated directly with interferon a. Results in long-term (18-h) ⁵¹Cr-release assays indicated that poly I:C accelerated the kinetics of ADCC without affecting the proportion of target cells lysed by K cells. By contrast, an increased proportion of target cells was killed by poly I:C-stimulated NK cells. These results suggest that the controversy concerning relative interferon effects upon NK and K cells derives from differences both quantitative and qualitative in nature. K cell activity is enhanced but at a relatively low frequency. Enhancement of NK cell activity is selective in the sense that it occurs independently of and with greater frequency than enhancement of K cell activity. Distinct biological mechanisms may, therefore, be involved in regulation and expression of NK and K cell activation by interferons.     

Enhancement of the antibody-dependent cellular cytotoxicity of human peripheral blood lymphocytes with interleukin-2 and interferon α

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3^–, CD56⁺, CD16⁺ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon (rIFN) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFN induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15–30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20–50 U/ml. We now show that activation of the NK cells with a combination of rIL-2and rIFN induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFN was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of FcRIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this FcR was essential for ADCC of the human effector cells.Supported by a grant from the Dutch Cancer Society (grant NKI-84-14)     

Apparent sensitivity of human K lymphocytes to the spatial orientation and organization of target cell-bound antibodies as measured by the efficiency of antibody-dependent cellular cytotoxicity (ADCC)

Although monoclonal antibodies (mAb) can elicit potent ADCC by human K lymphocytes, different mAb, even of the same antibody subclass or even of the same target antigen specificity, vary considerably as to their efficiency in eliciting ADCC. The extensive variability in ADCC efficiencies of murine IgG2a mAb is analyzed here. In cold-target inhibition experiments it was found that only cells coated with "ADCC-efficient" IgG2a mAb, and not "ADCC-inefficient" IgG2a mAb, inhibit K effector cell lysis of radiolabeled target cells by ADCC. This result indicates that the spatial orientation of the antibodies on the target cell membrane influences the net efficiency of ADCC reactions by affecting the efficiency of interaction between antibody and the Fc receptors (FcR) of K cells. It is proposed that a "favorable" orientation of antibodies on the target cell membrane is required for efficient ADCC reactions. This proposal is directly supported by the observation that one IgG2a mAb (20.8.4), which cross-reacts with several different H-2 alloantigens, was found to elicit efficient ADCC only when bound to certain of its possible target cell antigens. It was also observed in these studies that the organization of antibodies on a target cell membrane influences the net efficiency of ADCC reactions. It is proposed that a "favorable" antibody organization on the target cell membrane is also required for efficient ADCC reactions. This proposal is supported by the observation that certain antihuman beta 2m (anti-Hu beta 2m) IgG2a mAb, which elicit efficient ADCC lysis of human target cells, fail to elicit the lysis of murine cells having Hu beta 2m molecules coupled randomly to their external membrane surfaces. The differences in the way the Hu beta 2m was organized on the surfaces of the human cells and the murine-Hu beta 2m cell conjugates presumably caused differences in the way the bound antibodies were organized on the cell surfaces, which in turn resulted in the ADCC efficiency differences observed for the same mAb with the different target cell types. Because ADCC reactions appear to be sensitive to both the orientation and the organization of cell surface-bound antibodies, certain types of structural alterations or variations in the membrane molecules (relative to other neighboring structures on the target cell membrane) are potentially detectable by quantitative differences or variations in ADCC reactions.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

Antibody-dependent murine macrophage-mediated damage to Schistosoma mansoni schistosomula in vitro

Antibody-dependent cell-mediated cytotoxicity (ADCC) against schistosomula of the human parasite Schistosoma mansoni was demonstrated using antisera from mice plus peritoneal exudate cells (PEC). PEC were divided into plastic-adherent (96% macrophages, 4% lymphocytes) and nonadherent (92% lymphocytes, 8% macrophages) cell populations. Four criteria of ADCC were used, including minimal and maximal cell attachment, and death of and uptake of trypan blue by schistosomula. Using cells from normal mice and antisera from schistosome-infected mice, macrophages adhered to, damaged the tegument and underlying structures of, and killed schistosomula when observed following 18 hr incubation. In homologous systems, the results were similar when outbred CD-1 and inbred BALB/c mice were compared, except that potency of antisera from the latter mice decreased after 6–7 weeks postinfection, whereas the opposite was true for the former strain of mice. Nonadherent cells also exhibited ADCC against schistosomula, but the potency was considerably lower than that of adherent cells. These complement-independent ADCC reactions were stage-specific for the schistosomulum in that no reactions occurred with adult worms.     

Expression of human T lymphocyte antigens by killer cells.

K cells, the effectors of antibody-dependent cell-mediated cytotoxicity, were found to express human T but not B lymphocyte antigens detected by rabbit anti-HTLA and anti-HBLA. Pretreatment of effector cells with anti-HTLA+C inhibited ADCC by specifically lysing K cells: no inhibition of ADCC by anti-HTLA occurred when deltaC was substituted for C. By contrast, pretreatment of effector cells with anti-HBLA nonspecifically inhibited ADCC, probably for forming antigen-antibody complexes with HBLA+ cells in effector suspensions: a) treatment with anti-HBLA deltaC was more inhibitory of ADCC than treatment with anti-HBLA+C, and b) the inhibitory effect of anti-HBLA on ADCC was either eliminated or markedly reduced if effector suspensions were first passed through a nylon fiber column, a procedure that removed most HBLA+ cells without affecting K cell activity. HTLA antigens expressed by K cells and NK cells are the same as HTLA antigens expressed by thymocytes since thymocytes completely absorb the anti-K cell and NK cell reactivity of anti-HTLA.     

Identification of target antigen for antibody-dependent cellular cytotoxicity on cells carrying Epstein-Barr virus genome

The target antigen for antibody-dependent-cellular cytotoxicity (ADCC) on Epstein-Barr virus-(EBV) carrying lymphoblastoid cells expressing EBV-specific membrane antigen (MA) were examined with human serum antibody and adult human peripheral lymphocytes as effector cells. These studies confirmed that anti-MA-positive but not MA-negative sera were reactive in the ADCC. The ADCC reaction was positive with cells in which the MA consisted of late (LMA) and early (EMA) components. These included 1) MA-positive cells prepared by EBV antigen-adsorption, 2) cells carrying de novo-synthesized MA without adsorbed MA, and 3) EBV-producer cells expressing MA spontaneously. In all these preparations, the target cells were lysed roughly in parallel with the frequency of MA-positive cells. Inhibition of LMA synthesis in EBV-superinfected cells by phosphonoacetate (PA) reduced ADCC sensitivity significantly and to a far greater extent than MA synthesis as measured by immunofluorescence. This suggests that a target for ADCC is the PA-sensitive LMA. No ADCC reaction occurred with the cell preparation comprised of a high percentage of MA-positive cells induced by 5-iodo-2'-deoxyuridine, which is believed to be EMA only. These results strongly suggest that the target antigen for ADCC in EBV-positive cells is a late MA but not early MA.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.     

Virus-induced enhancement of lymphocyte-mediated antibody-dependent cytotoxicity (ADCC) in vitro

The effect of Parotis virus on antibody-dependent cellular cytotoxicity in vitro (ADCC) of human lymphocytes was investigated in a 51Cr-release assay and, at the effector cell level, in an ADCC plaque assay. Target cells were bovine or chicken erythrocytes, which are not susceptible to natural cytotoxicity (NK) of human lymphocytes. They were not killed when incubated with virus-treated lymphocytes in the absence of antibodies. Treatment of the lymphocytes or the target cells with small amounts of virus, however, resulted in a very significant enhancement of ADCC. The same results were obtained with live or UV-inactivated virus, suggesting that enhancement was a passive phenomenon not requiring infection. Enhancement was already significant after 3 hr of incubation, indicating that it was independent of endogenously released interferon. Enhancement of ADCC by virus was due to effector cell recruitment rather than due to the increase of the cytotoxic potential of the individual K cell. The highest frequency of effector cells was present in Percoll fractions enriched in large granular lymphocytes (LGL). Virus treatment resulted in recruitment of effector cells carrying T cell markers such as the T3 antigen (OKT3+), receptors for sheep erythrocytes, or Fc receptors for IgM. In contrast, the absolute number of K cells carrying the HNK-1 marker (Leu-7) or receptors for C3 fragments was not changed by the virus. It is concluded that Parotis virus enhances ADCC by improving effector cell-target cell contacts, resulting in recruitment of effector cells with T cell characteristics. Recruitment is accompanied by a significant reduction of the antibody concentration needed for ADCC induction. This virus-mediated enhancement of ADCC may be of importance for protection of the host in the early phases of a virus infection in which the amounts of anti-viral IgG antibodies capable of inducing cellular cytotoxicity may yet be very small.     

Natural killing and antibody-dependent cellular cytotoxicity are mediated by different mechanisms and by different cells.

Natural killing (NK) in humans, as well as in other species, has been shown to be specific for antigenic determinants present on the surfaces of a variety of tumor cells. Physical separation of NK cells from K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC), has not been successful; however, there is indirect evidence suggesting that these activities are distinct. To further study the relationship between NK and K cells, competitive inhibition techniques were employed. NK cells can be blocked via two mechanisms: 1) by direct inhibition with NK-sensitive tumor cells binding to NK receptor sites present on the effector cells and 2) by steric inhibition resulting from the binding of antibody-coated cells to the FcR on the effector cells. K cells, however, lack the NK receptor site(s) but are FcR+, and can therefore be blocked only by antibody-coated cells. We therefore postulate that NK and K cells are two separate lymphoid populations. NK cells bear receptor site(s) for NK determinants and FcR, whereas K cells bear only FcR.     

抗单纯疱疹病毒单克隆抗体的研究——Ⅱ.单克隆抗体介导细胞毒效应的研究

建立了单克隆抗体(McAb)介导细胞毒作用(ADCC)~(51)Cr释放试验的测定力法。确定了最适工作条件。ADCC测定结果表明,5株抗HSV McAb介导ADCC的活性不同:McAb 1A12、2A8和1G8无ADCC活性;而1D10和2C5两株McAb作1:10稀释时,~(51)Cr释放率分别为27.09％和25.07％,稀释至1:100或1:1000时仍有ADCC活性。结果提示,不同的McAb抗原决定族诱导产生的抗体,在介导ADCC免疫保护作用上有差异,并为McAh治疗临床单纯疱疹病毒感染的可能性提供了实验资料。     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells

Summary The effects of human recombinant interleukin-6 (hrIL-6) on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h⁵¹Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.1A and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100–400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity. hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy.     

Dual mode of action of a human anti-epidermal growth factor receptor monoclonal antibody for cancer therapy

Epidermal growth factor receptor (EGF-R) overexpression is common in a large number of solid tumors and represents a negative prognostic indicator. Overexpression of EGF-R is strongly tumor associated, and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1kappa) directed against EGF-R, developed using human Ig transgenic mice. mAb 2F8 effectively blocked binding of EGF and TGF-alpha to the EGF-R. At saturating concentrations, 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concentrations, associated with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo analysis of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor saturation and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we observed potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.