Enhancement Of Antibody-dependent Cellular Cytotoxicity Research Articles

Enhanced Natural Killer Cell Binding and Activation by Low-Fucose IgG1 Antibody Results in Potent Antibody-Dependent Cellular Cytotoxicity Induction at Lower Antigen Density

Recent studies have revealed that fucose removal from the oligosaccharides of human IgG1 antibodies results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to FcgammaRIIIa. In this report, we investigated the relationship between enhanced ADCC and antigen density on target cells using IgG1 antibodies with reduced fucose. Using EL4 cell-derived transfectants with differential expression levels of exogenous human CC chemokine receptor 4 or human CD20 as target cells, ADCC of fucose variants of chimeric IgG1 antibodies specific for these antigens were measured. We further investigated IgG1 binding to natural killer (NK) cells and NK cell activation during ADCC induction to elucidate the mechanism by which low-fucose IgG1 induces ADCC upon target cells with low antigen expression. Low-fucose IgG1s showed potent ADCC at low antigen densities at which their corresponding high-fucose counterparts could not induce measurable ADCC. The quantitative analysis revealed that fucose depletion could reduce the antigen amount on target cells required for constant degrees of ADCC induction by 10-fold for CC chemokine receptor 4 and 3-fold for CD20. IgG1 binding to NK cells was increased by ligating IgG1 with clustered antigen, especially for low-fucose IgG1. Up-regulation of an activation marker, CD69, on NK cells, particularly the CD56(dim) subset, in the presence of both the antibody and target cells was much greater for the low-fucose antibodies. Our data showed that fucose removal from IgG1 could reduce the antigen amount required for ADCC induction via efficient recruitment and activation of NK cells.

The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.

Prestimulation of monocytes by the cytokines GM-CSF or IL-2 enhances the antibody dependent cellular cytotoxicity of monoclonal antibody 17-1A.

Previously, we have shown that the cytokines IFN-alpha, IFN-gamma and IL-2 significantly enhance the antibody dependent cellular cytotoxicity (ADCC) exerted by the monoclonal antibody (mAb) 17-1A which recognizes the tumor associated antigen EpCAM. ADCC was assessed by a new flow cytometric cytotoxicity assay using the PKH2 labeled colorectal tumor cell line HT29 as target cells and peripheral blood mononuclear cells as effectors. Monocytes are assumed to be one of the major effectors for ADCC. However, isolated monocytes have a rather low ADCC capacity while addition of CD4+ lymphocytes optimizes ADCC. Since such an interaction between immune cells may act through cytokines we investigated whether a seven-day-prestimulation of monocytes by the cytokines M-CSF, GM-CSF, IFN-gamma, IFN-alpha and IL-2 enhances ADCC. Thereafter, we added for three days IL-2 and IFN-alpha with or without the mAb 17-1A for terminal activation of monocytes. Interestingly, GM-CSF which was ineffective in terminal activation, significantly enhanced ADCC of monocytes when it was used for prestimulation. Similar results were obtained with IL-2. IFN-gamma and M-CSF were also active but less than GM-CSF. Astonishingly, IFN-gamma and IFN-alpha prestimulation of monocytes suppressed the enhancement of ADCC exerted by GM-CSF and IL-2, respectively. Our experiments suggest that the timing of cytokine application is critical for the induction of optimal ADCC. Subcutaneous pretreatment with GM-CSF or IL-2 followed by the combination of IL-2/IFN-alpha/17-1A should be evaluated in a phase I clinical trial in patients with colorectal cancer.

Enhancement of antibody-dependent cellular cytotoxicity of neonatal cells by interleukin-2 (IL-2) and IL-12.

Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.

Immunostimulatory Oligodeoxynucleotides Containing CpG Motifs Enhance the Efficacy of Monoclonal Antibody Therapy of Lymphoma

AbstractBacterial DNA and synthetic oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets, including natural killer cells and macrophages. We evaluated whether the combination of CpG ODN and antitumor monoclonal antibody is effective at preventing tumor growth in an immunocompetent murine lymphoma model. CpG ODN–activated murine splenocytes induced lysis of tumor targets more effectively than unactivated splenocytes. These effector cells were also superior to unactivated splenocytes or cells activated with a control methylated ODN at inducing antibody-mediated lysis of 38C13 murine lymphoma cells. In vivo, CpG ODN alone had no effect on survival of mice inoculated with 38C13 cells. However, a single injection of CpG ODN enhanced the antitumor response to antitumor monoclonal antibody therapy. Ninety percent of mice treated with monoclonal antibody alone developed tumor compared with 20% of mice treated with antibody and CpG ODN. These antitumor effects were less pronounced when treatment consisted of an identical ODN containing methylated CpG dinucleotides. A single dose of CpG ODN appeared to be as effective as multiple doses of interleukin-2 at inhibiting tumor growth when combined with antitumor monoclonal antibody. We conclude that immunostimulatory CpG ODN can enhance antibody dependent cellular cytotoxicity and warrant further evaluation as potential immunotherapeutic reagents in cancer.

Granulocyte-macrophage colony-stimulating factor and the immune system.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine currently used for the reversal of neutropenia associated with cytotoxic chemotherapy, bone marrow and haemopoietic stem cell transplantation. GM-CSF also modulates the function of differentiated white blood cells. In the context of local inflammatory responses, GM-CSF stimulates macrophages for antimicrobial and antitumor effects. GM-CSF further enhances healing and repair by its actions on fibroblasts and epidermal cells. GM-CSF is the pivotal mediator of the maturation and function of dendritic cells, the most important cell type for the induction of primary T cell immune responses. GM-CSF may enhance antibody dependent cellular cytotoxicity (ADCC) in several cell types, and the generation and cytotoxicity of natural killer (NK) cells. On this basis, GM-CSF may be useful for inducing or augmenting antibody responses to antimicrobial vaccines, to enhance killing of intracellular microorganisms, to accelerate epidermal and mucosal wound healing, and to stimulate protective immunity against tumors.

Macrophage colony-stimulating factor enhancement of antibody-dependent cellular cytotoxicity against human colon carcinoma cells

Antibody-dependent cellular cytotoxicity (ADCC) has been suggested to be an important defense mechanism against tumors. The effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) on ADCC activity of human monocytes were investigated. Human peripheral monocytes were pre-incubated for 72 h with rhM-CSF at various concentrations (50, 100, 200, 400 U/ml) and then used as effector cells in a 24-h 111-Indium release assay. Human carcinoma cell lines LS-174T, CBS, and KLE were used as targets to react with anti-carcinoma monoclonal antibodies (mAbs: murine D612, murine CC49, and chimeric CC49). A significant increase in ADCC activity was observed after monocytes were incubated in 100–400 U/ml of human rhM-CSF. Variation in ADCC activity of monocytes among donors was observed. The enhancement of ADCC activity was blocked by the addition of a neutralizing antibody to rhM-CSF. Less D612 mAb was required for the rhM-CSF-treated monocytes to mediate an equivalent level of ADCC activity as compared to the untreated monocytes. Because of the low levels of rhM-CSF required in these studies to enhance ADCC, treatment of monocytes alone with comparable levels of rhM-CSF did not enhance antibody-independent cytotoxicity. Moreover, it is demonstrated here that recombinant human interleuken-4 (rhIL-4) and rhM-CSF can have a synergistic effect of monocyte-mediated ADCC on human tumor cells. These results thus indicate that rhM-CSF augments ADCC of human peripheral blood monocytes using mAbs to human carcinomas, suggesting a potential role for rhM-CSF in cancer immunotherapy.

Synergetic effect of interleukin-2 and cellular cytotoxicity against a novel tumor-associated carbohydrate antigen Le(a)/Le(a) (dimeric Le(a)) mediated by monoclonal antibody NCC-ST-421 in adoptive immunization using SCID mice.

The murine IgG3 monoclonal antibody NCC-ST-421, raised against a human gastric cancer, shows strong reactivity with dimeric Le(a) (Le(a)/Le(a); V4FucIII4FucLc6Cer) expressed on gastrointestinal cancer cells. ST-421 reacted minimally with non-dimeric or simple Le(a) expressed on normal tissues. ST-421 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) with human peripheral blood lymphocytes, and complement-dependent cytotoxicity with human complement. Interleukin-2 (IL-2) modulates the function of immunocytes, in particular inducing lymphokine-activated killer (LAK) cell activity and enhancing ADCC. We therefore employed combination immunotherapy with IL-2, LAK, and ST-421-induced ADCC in vitro and in mice with severe combined immunodeficiency (SCID), using target tumor cells expressing Le(a)/Le(a) antigen. ADCC against human colon cancer cell lines in vitro was enhanced three to four times after preincubation with IL-2. Addition of IL-2 reduced the amount of ST-421 required for efficient ADCC 10- to 100-fold. ADCC was activated by IL-2 earlier (1 day) than the generation of LAK cells (3-4 days), and at lower concentration of IL-2. These effects were specific for ST-421, as demonstrated by experiments with irrelevant antibody or irrelevant target cells. An anti-(Fc receptor) antibody blocked the ADCC but not the LAK activity in vitro. The enhancement of ADCC by IL-2 may be caused by activation of effector cells expressing Fc receptors. In vivo experiments using SCID mice inoculated with human colon cancer showed a significant tumor-growth-suppressive effect after combined therapy using human peripheral blood lymphocytes, LAK, IL-2, and ST-421. In summary, adoptive immunization with human lymphocytes activated by IL-2 and ST-421 effectively suppressed growth of gastrointestinal cancer cells expressing Le(a)/Le(a).

Melatonin‐induced enhancement of antibody‐dependent cellular cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) is a lytic mechanism in which a specific antibody acts cooperatively with leukocytic effector cells to induce target cell lysis. In this paper, the effect of exogenous melatonin on ADCC was examined. It was found that two evening intravenous injections of melatonin (1 mg/kg b.w.) was sufficient to enhance the capacity of splenocytes to mediate ADCC. This augmented activity returned to normal levels by day 6. Moreover, the opioid antagonist, naloxone, was unable to inhibit the ADCC enhancement, suggesting that melatonin did not operate through a naloxone-sensitive opiatergic mechanism. These results further support the modulatory action of melatonin on immune responses.

Peripheral blood monocytes derived from HIV + individuals mediate antibody-dependent cellular cytotoxicity (ADCC)

Monocytes/macrophages serve a number of immunologic functions and play a major role in the host defense against infection. Abnormal functions of monocytes have been reported in AIDS and HIV + individuals. A recent report from our laboratory demonstrated that peripheral blood monocytes (PBM) derived from AIDS patients were de novo “activated” as assessed by direct cell-mediated cytotoxicity (CMC) and secretion of cytotoxic factors and tumor necrosis factor-α (TNFα). Thus, both the direct cytotoxicity as well as the antibody-dependent cellular cytotoxicity (ADCC) exerted by the monocytes may contribute to the destruction of HIV-infected/coated cells and the immunopathogenesis of AIDS. The present study investigated the ability of HIV + PBM to mediate ADCC against antibody-coated target cells in an 18-hr 51Cr release assay. Initial studies examined ADCC using a macrophage resistant target Raji and rabbit anti-Raji serum. The results show that the majority of PBM from HIV + individuals mediate ADCC activity while the majority of PBM from normal healthy controls was not cytotoxic. While activation of PBM with recombinant interferon-γ (rIFN-γ) enhances the ADCC activity of normal PBM, treatment of HIV + PBM with IFN-γ resulted in significant enhancement of ADCC. Both untreated and treated PBM from HIV + individuals had significantly higher ADCC than PBM from normal individuals. Of interest, a significant ADCC activity was found by PBM derived from two HIV − high risk individuals whether untreated or treated with rIFN-γ. The ADCC results with RAJI target cells prompted us to investigate whether ADCC can also be obtained using HIV-infected T4 + cells. We selected a macrophage and TNF resistant T4 + CEM cell line as target for ADCC. The target was coated with inactivated HIV and pooled human anti-HIV serum was used. Studies with a few HIV + individuals demonstrate that significant ADCC is obtained with PBM from HIV + individuals but little or no ADCC by normal PBM and the ADCC was specific for HIV. The ADCC was also significantly enhanced by treatment of PBM with rIFN-γ. The results of this study clearly indicate that PBM from HIV + individuals are endowed with the capacity to mediate ADCC against HIV-infected/coated cells and thus, we postulate that PBM may play a direct role in vivo in lysis or suppression of HIV-coated/infected cells and in the pathogenesis of AIDS.

C1q enhancement of antibody-dependent granulocyte-mediated killing of nonphagocytosable targets in vitro.

A possible role for C1q in antibody-dependent granulocyte-mediated killing of nonphagocytosable targets was investigated utilizing IgG-dependent granulocyte cytotoxicity directed against microfilariae of Dirofilaria immitis. Granulocyte-mediated killing of microfilariae is enhanced by addition of fresh serum. Lack of C4 did not significantly reduce the observed increase in cytotoxicity. The addition of highly purified monomeric human Clq (0.2 microgram/ml) in the presence of immune IgG resulted in a two- to fivefold enhancement of killing (P less than 0.025). C1q enhancement of killing occurred in the absence of fluid-phase IgG, but killing was significantly less than when both fluid-phase IgG and C1q were present. The effect of C1q was inhibited by the addition of solubilized type I collagen (44-92% inhibition of killing, P less than 0.05). Significant 125I-Clq binding to microfilariae occurred only in the presence of immune IgG. In addition, C1q in concentrations ranging from 0.5 to 2.0 micrograms/ml resulted in a dose-dependent increase in binding of 125I-immune IgG to microfilariae. Finally, when purified C1q was added to preopsonized, washed microfilariae, granulocyte production of superoxide was increased from 0.25 +/- 0.07 to 0.68 +/- 0.07 nm/10(6) cells.10 min (P less than 0.01). These results describe a novel functional role for C1q in enhancement of antibody-dependent cellular cytotoxicity towards nonphagocytosable targets.

Effects of in vitro asbestos exposure on natural killer and antibody-dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) were exposed in vitro to asbestos fibers. Antibody-dependent cellular cytotoxicity (ADCC) activity and natural killer (NK) activity were examined by a chromium-51 release assay. There was a statistically significant enhancement of ADCC and NK activity by chrysotile and crocidolite fibers when cultured together with PBL for a period of 42 hr in medium containing a concentration of at least 2.5% fetal calf serum (P less than 0.05). Isolation of large granular lymphocytes to measure NK activity, however, showed the opposite effect when exposed to asbestos fibers (P less than 0.05). Our results indicate that asbestos fibers can directly affect lymphoid cytotoxic responses in vitro and may provide clues to immunopathogenic mechanisms for the occurrence of neoplasms in vivo.

Cyclophosphamide inhibits antibody-dependent cellular cytotoxicity (ADCC) suppression exerted by lymph node cells

The effect of Cyclophosphamide (Cy) on suppression of antibody-dependent cellular Cytotoxicity (ADCC) by lymph node cells (LNC) was evaluated. The results show that the suppression of ADCC exerted by LNC was abrogated when mice had been treated with Cy. Moreover, it was shown that ADCC inhibition induced by LNC was mediated by soluble factor(s) and that treatment with a single dose of 200 mg/kg ip of Cy, significantly decreased its release. In addition, suppressor activity of normal LNC was enriched by depletion of adherent cells and was not affected by treatment with monoclonal anti-Thy 1.2 plus complement. These observations indicate that modulatory cells are nonadherent and lack characteristic T-cell markers. Thus, we conclude that this suppressor system, which normally controls ADCC activity, can be inhibited by treatment of mice with Cy and that this effect may explain the enhancement of ADCC observed in splenocytes of Cy-treated animals.

Mapping the functional topography of Fcγ with monoclonal antibodies: localization of epitopes interacting with the binding sites of Fc receptor on human K cells

A panel of monoclonal antibodies (mAb) specific for the C gamma 2, C gamma 3 or inter C gamma 2/C gamma 3 domain epitopes was tested for inhibition of antibody-dependent cellular cytotoxicity (ADCC) specific for anti-D IgG-coated erythrocytes. Significant inhibition of ADCC was demonstrable for some antibodies having specificity for C gamma 2 or C gamma 3 domain epitopes, while others gave no inhibition. Fab fragments of a representative C gamma 2-specific antibody (A55) and C gamma 3-specific antibody (x3a8) retained their inhibitory capacity in lymphocyte-mediated ADCC, but only A55 Fab inhibited monocyte-mediated lysis. Furthermore, the Fab portion of A55 completely abolished the complement-dependent enhancement of ADCC mediated by concanavalin A-stimulated cells, while x3a8 Fab had no effect in this system. On the other hand, x3a8 Fab inhibited the binding of anti-D IgG-sensitized erythrocytes to lymphocytes while A55 Fab did not influence this latter interaction. The results suggest that C gamma 2 domain-FcR interaction is essential for the triggering of lytic process both in lymphocyte and in monocyte-mediated ADCC, while C gamma 3 domain has no role in the latter but is responsible for the appropriate contact between effector lymphocytes and target cells. A site in the region of Lys274 appears to be critical for triggering of both lymphocyte and monocyte-mediated ADCC.

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

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.

Differential interference by azathioprine and 6-mercaptopurine with antibody-mediated immunoregulation: synergism of azathioprine and antibody in the control of an immune response.

Comparative studies on the effects of immunosuppressive agents azathioprine (AZ) and 6-mercaptopurine (6-MP) in antibody-mediated immunoregulation are reported. To determine the degree of interference of these agents with the generation of antibody molecules controlling an anti-SRBC response, the donors of mouse anti-SRBC sera were given daily doses of the drugs. Primary antisera from AZ-treated donors were not significantly inhibited by daily doses of 180, 270, and 360 mumol/kg in their ability to suppress the direct PFC response; on the other hand 6-MP was inhibitory at the two higher equimolar dose levels. When the antisera were used in the antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (AD phi) assays, similar differential effects of the two agents were found. Multiple doses of AZ and 6-MP given to the donors of ADCC effector cells enhanced the capacity of these cells to lyse antibody-sensitized SRBC target cells. The enhancement of ADCC with 6-MP was less manifest than that with AZ. When injected before the antigen, AZ acted synergistically with the antibody in the control of the direct PFC response. Possible mechanisms by which AZ can interact with antibody-mediated immune regulation are discussed.

Potentiation of Antibody-Dependent Cell-Mediated Cytotoxicity by Target Cell-Bound C3b

Abstract Antibody-dependent cytolytic effector lymphocytes are known to possess, in part, receptors for activated C3. Employing a model system consisting of 51Cr-labeled chicken erythrocytes and purified human peripheral lymphocytes, we investigated the effect of target cell bound C3b on antibody-dependent cellular cytotoxicity (ADCC). At concentrations of anti-target cell antibody too low to cause effective ADCC, target cell bound C3b cooperated with antibody to produce marked target cell lysis. In the presence of a 1/6.25 × 106 dilution of antichicken erythrocyte rabbit IgG, cell lysis increased from 20% to 65% by the attachment of 18,000 C3b molecules per cell. C3b-dependent enhancement of ADCC was dose dependent. It was augmented by attachment of activated properdin (P̄) to the C3b-bearing target cells.

Turner's syndrome in monozygotic twins.

Comparative studies on the effects of immunosuppressive agents azathioprine (AZ) and 6-mercaptopurine (6-MP) in antibody-mediated immunoregulation are reported. To determine the degree of interference of these agents with the generation of antibody molecules controlling an anti-SRBC response, the donors of mouse anti-SRBC sera were given daily doses of the drugs. Primary antisera from AZ-treated donors were not significantly inhibited by daily doses of 180, 270, and 360 mumol/kg in their ability to suppress the direct PFC response; on the other hand 6-MP was inhibitory at the two higher equimolar dose levels. When the antisera were used in the antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (AD phi) assays, similar differential effects of the two agents were found. Multiple doses of AZ and 6-MP given to the donors of ADCC effector cells enhanced the capacity of these cells to lyse antibody-sensitized SRBC target cells. The enhancement of ADCC with 6-MP was less manifest than that with AZ. When injected before the antigen, AZ acted synergistically with the antibody in the control of the direct PFC response. Possible mechanisms by which AZ can interact with antibody-mediated immune regulation are discussed.