Antibody-dependent Cell-mediated Cytotoxicity Assay Research Articles

Studies on the antibody-dependent cell-mediated cytotoxicity (ADCC) of thioglycollate-stimulated and BCG-activated peritoneal macrophages

Peritoneal macrophages (mφ), activated by Bacillus Callmette-Guerin (BCG) or elicited by thioglycollate broth in vivo in C57BL/6J mice, were compared with regard to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The BCG-activated mφ had several times higher ADCC activity than did the TG-mφ against erythroid targets. When human and murine tumor cells were employed as targets in the ADCC assay, the BCG-mφ lysed each of six separate target lines considerably, while the TG-mφ had little and, occasionally no, lytic activity against the six targets. Although F c-dependent and -independent phagocytosis of the erythroid targets by the TG-mφ exceeded that by the BCG-mφ, this rapid and extensive phagocytosis did not account soley for the decreased lysis of the erythroid targets by the TG-mφ. The results indicate that BCG-activated peritoneal mφ, in comparision with TG-elicited inflammatory mφ, are potent effectors of ADCC against both erythroid and neoplastic target cells.

Role of Antibody in Cytotoxicity by Lymphocytes Armed against 253J Bladder Cancer Line

The role of antibody-dependent cell-mediated cytotoxicity (ADCC) in cytotoxicity mediated by antibody-armed effector cells was examined. Cytotoxicity mediated by unsensitized human peripheral blood mononuclear cells (MNC) directed against the human bladder cancer cell line, 253J, was significantly enhanced when MNC were preincubated with a rabbit anti-253J serum and then washed free of unbound antibody. Nonadherent, Fc receptor positive MNC were the effector cells and the arming antibody was IgG. Supernatants of 24-hour cultures and armed MNC exhibited antibody activity in ADCC assays, suggesting the ADCC was in part responsible for the arming phenomenon.

Evaluation of whole blood as effector for antibody-dependent cellular cytotoxicity against autologous targets

An assay for antibody-dependent cell-mediated cytotoxicity which uses whole blood as effector and autologous lymphocytes sensitized with rabbit anti-human lymphocyte serum as targets is assessed. Comparison with performances of purified lymphocytes shows that whole blood yields approximately the same cytotoxicity levels as the lymphocytes which can be separated from the corresponding volume of blood. This equivalence is not seen at the early stage of cytolysis, whole blood reaching its plateau stage 1 h later. A contribution of phagocytic cells to lysis of the target cells is ruled out. The presence of plasma does not modify the cytolytic capacity of the blood of healthy subjects. The test may be performed with less than 1 ml of blood and is suitable for repeated evaluation of the immunologic status of patients, for evaluation of plasma factors in diseases and of the effects of agents inducing lymphocyte redistribution.

Role of humoral presensitization in human renal transplant rejection

A prospective study of 31 cadaveric renal allograft recipients was performed to determine the significance of pretransplant presensitization undetected by the conventional microlymphocytotoxicity crossmatch. Donor-specific humoral presensitization revealed by the antibody-dependent cell-mediated cytotoxicity assay (ADCC) was associated with a high incidence of early graft rejection. Six-month graft survival was 20% in recipients with positive pretransplant ADCC and 75% in ADCC-negative recipients (P < 0.01). Among recipients highly presensitized to a random panel of HLA antigens, donor-specific humoral presensitization detected by chromium-51-release complement-dependent cytotoxicity (51Cr-CDC) was also highly correlated with accelerated rejection (P < 0.05). Pathologic study of the rejected allografts revealed antibody-mediated rejection vasculitis in all recipients. We conclude that humoral presensitization undetected by current conventional methods plays a cardinal role in early renal graft rejection and is a major factor responsible for low cadaveric renal transplant survival. This study suggests that use of the ADCC and 51Cr-CDC as routine adjunctive crossmatch procedures may contribute to improvement in renal transplant survival rates.

Evaluation of the humoral response of glioma patients to a possible common tumor‐associated antigen(S)

Cytotoxic antibodies against glioblastoma-associated antigen(s) have been sought for in glioma patient sera. Complement-dependent cytotoxicity (CDC) I and antibody-dependent cell-mediated cytotoxicity (ADCC) assays were used to test sera from 80 patients using 51 Cr labelled target cells derived from eight different glioblastoma lines. As more positive sera were detected with ADCC than with CDC, ADCC assay was used for the remainder of the study. Cytotoxic antibodies were detected in the sera from 8 of 80 glioma patients (10%) by CDC and in 20 of 143 (14%) by ADCC. Fourteen percent of 27 meningioma patients and 16% of 25 normal donors used as controls were found to react in ADCC against the same glioblastoma cell lines. The positive serum samples showed extensive cross-reactions with the different glioblastoma cells, but the pattern of reactivity was different for each serum tested. The antibodies detected did not seem to be directed against tumor-associated antigen(s), since the positive sera were found to have a similar ADCC reactivity against unrelated tumor cells and normal fibroblasts. Moreover, their antiglioma reactivity was absorbed by cells of unrelated tumors and by normal platelets. These results do not support previous reports of specific humoral responses in glioma patients against common tumor-associated antigen(s).

Antibody‐dependent cell‐mediated cytotoxicity against epstein‐barr virus membrane antigen in african Burkitt's lymphoma

Serial samples of sera from patients with African Burkitt's lymphoma were tested for antibody against Epstein-Barr virus (EBV)-specific membrane antigen (MA) by the antibody-dependent cell-mediated cytotoxicity assay (ADCC). Titers of patients in the long-term survivor group were generally higher than those found in the sera of patients in the short-term survivor group. Although ADCC titers to EBV-MA were not useful in predicting which patients would relapse there was a definite relationship between ADCC titer and prognosis. The individual differences in ADCC titers in patients in remission may explain the variability of responses that have been reported in studies on serotherapy with remission plasma.

K-cell activity in Lamina proprial lymphocytes from the human colon

Using an enzymatic method for their isolation, it has been shown that human colonic lamina proprial lymphocytes, isolated from patients with various colonic diseases and depleted of polymorphonuclear leukocytes and macrophages, demonstrated K-cell activity in two antibody-dependent cell-mediated cytotoxicity assays in vitro. No differences were found between the activities of lamina proprial lymphocytes isolated from colonic specimens involved by Crohn's disease, chronic ulcerative colitis, or colonorectal carcinoma. K-cell activity was demonstrable using lymphoid cells isolated from the colons of patients receiving steroid therapy at the time of the therapeutic resections. These findings indicate the need for further investigation of possible roles for K-cells in local immune responses in the human bowel mucosa in inflammatory and neoplastic diseases.

Evidence that Natural Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity Are Mediated in Humans by the Same Effector Cell Populations

The present study strongly suggests that, in humans, natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) are mediated by the same effector cell population. This is supported by two different experimental approaches. First, competition for NK effector cells was accompanied by simultaneous inhibition of ADCC activity. Target cells sensitive to NK activity were capable of inhibiting specifically an ADCC assay in cold target competition experiments. Second, specific removal of NK cells on monolayers formed by target cells sensitive to NK activity caused simultaneous depletion of ADCC effector cells. In association with the removal on the monolayers of effector cells for ADCC as well as NK activity, we also found a significant depletion of cells bearing Fc gamma receptors.

Antibody-Dependent Cell-Mediated Cytotoxicity against Cells Infected with Respiratory Syncytial Virus: Characterization of in Vitro and in Vivo Properties

Abstract An in vitro 51Cr release assay for human antibody-dependent cell-mediated cytotoxicity (ADCC) against HeLa cells infected with respiratory syncytial virus (RSV) has been characterized by using leukophoresed and adherent cell-depleted adult lymphocytes. Lymphocytes from RSV seronegative children were also competent as effector cells. Sera from children with: 1) primary and recurrent natural RSV infections, or 2) live attenuated RSV vaccine infection were examined to characterize the behavior of ADCC antibody in vivo. After natural RSV infection ADCC antibody rose and fell more rapidly than neutralizing antibody. In two children undergoing primary RSV infection with attenuated vaccine, neutralizing antibody was formed in the absence of detectable ADCC antibody. The nonparallel behavior of ADCC and neutralizing antibodies suggests the heterogeneity of either the antigen involved or the mechanism of antibody production in the two antibody systems.

ADVERSE PRESENSITIZATION TO RENAL TRANSPLANTS

Sera obtained before transplantation from 52 consecutive renal allograft recipients were tested for antibody-dependent cell-mediated cytotoxicity (ADCC) and for complement-dependent cytotoxic antibodies (CDC). A D locus antigen-defined lymphoblastoid cell panel (B lymphoblastoid cell panel) was used as targets for the ADCC, and a peripheral blood lymphocyte (PBL) panel from 40 donors was used as targets for the CDC. Of the 343 ADCC assays, 118 of 168 performed with pretransplant sera from 24 recipients with early graft loss were positive, whereas only 81 of 175 performed with pretransplant sera from 25 recipients with a successful graft outcome were positive (P less than 0.001). A significantly greater degree of presensitization to the B lymphoblastoid cell panel was found in the group that lost their grafts as compared to the group with successful grafts (69% versus 49%, P less than 0.001). Sera from all six recipients with hyperacute rejection were positive in the ADCC before and after absorption with pooled platelets. In contrast, pretransplant CDC results were not predictive of ultimate graft outcome. Utilizing any level of cytotoxicity against the PBL panel as an index of adverse presensitization, no significant correlation between pretransplant CDC results and graft outcome was observed. These results suggest a prognostic role for ADCC using a B lymphoblastoid cell panel as targets to screen and identify high-risk potential graft recipients.

ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY TO HERPES SIMPLEX VIRUS TYPE 2 INFECTED TARGET CELLS IN THE COURSE OF CERVICAL CARCINOMA

The antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used as a sensitive instrument to study if specific antibodies acting synergistically with lymphocytes to induce target cell destruction, could be detected in sera from patients with cervical carcinoma. Significant cytotoxicity to herpes simplex type 2 (HSV-2) infected target cells was observed in sera of 17 out of 23 (74%) surviving patients with cervical cancer who were followed with at least two serum samples. A rise in cytolytic activity was also observed in 11 out of 23 cases. Seven out of 14 tumor-bearing cervical cancer patients with progressing cervical lesions showed significant cytotoxicity, but four of the seven had lost activity in later sera. When 43 patients surviving the observation period of 24--60 months with no signs of residual tumors were compared with 23 severely ill cervical cancer patients, patients with less advanced cancer had significantly higher cytolytic activity.

Antibody-dependent cellular cytotoxicity in patients with mycosis fungoides and sézary syndrome

An antibody-dependent cell-mediated cytotoxicity (ADCC) assay was utilized to measure null cell function in 37 patients with cutaneous lymphomas including mycosis fungoides (NM and Sezary syndrome. Five patients with psoriasis vulgaris were included as controls for benign dermatologic disease. Patients with MF, non-MF cutaneous lymphomas, and psoriasis had mean ADCC levels equal to or greater than control values, while patients with Sézary syndrome had significantly depressed levels (64% of control values). Although this study did not explain the reason for the depression, it indicated that the ADCC assay may prove useful as a prognostic tool and as an additional means of identifying Sezary syndrome.

Natural Killing in Immunodeficient Patients

Abstract Natural killing (NK) capacity was evaluated in peripheral blood mononuclear cells from 14 patients with well defined primary immunodeficiency disorders and compared with the activity of those cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays against antibody-coated erythrocyte (killed primarily by monocytes) and lymphoid or tumor targets (killed exclusively by lymphoid cells). A selective inability to lyse antibody-coated lymphocyte targets was observed with cells from patients with x-linked agammaglobulinemia, suggesting the involvement of either a different lymphocyte subpopulation or membrane receptor for NK and ADCC, or that a different functional susceptibility exists for the two types of killing. The only immunodeficiency state in which lymphocyte NK activity was found to be lacking was severe combined immunodeficiency disease.

Immunologic Monitoring of Transplant Rejection: Correlation of &lt;i&gt;in vitro&lt;/i&gt; Assays with Morphologic Changes on Transplant Biopsy

The assays of lymphocyte-mediated cytotoxicity (LMC), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were correlated with histopathologic criteria of refection in 35 transplant biopsies. A positive LMC was seen with 6/8 biopsies showing moderate to severe cellular rejection and in 8/17 with mild cellular rejection. Positive ADCC and/or CDC assays were associated with 14/14 biopsies containing rejection vasculitis. These results suggest that selected in vitro assays may be useful in monitoring the immunologic events occurring in the rejecting allograft.

Reversible blocking of antibody-dependent cell-mediated cytotoxicity

The ability of human peripheral blood lymphocytes to kill antibody-coated Chang liver cells in antibody-dependent cell-mediated cytotoxicity (ADCC) can be blocked with aggregated IgG (agg-IgG) or by soluble immune complexes. Dissociation of aggregates of IgG or immune complexes from the cell surface, however, resulted in partial recovery of the ability both to bind agg-IgG and to kill in the ADCC assay. Our results indicate that “unblocking” of effector cells could occur in vivo when the concentration of circulating immune complexes is lowered.

Normal Antibody-Dependent Killer Cell Function in Patients With Chronic Lymphocytic Leukemia 2

Unfractionated mononuclear peripheral blood leukocytes (PBL) and nylon wool-nonadherent (B-cell-depleted and T- and null-cell enriched) cells from normal control individuals and untreated patients with chronic lymphocytic leukemia (CLL) were tested for killer cell function in an antibody-dependent cell-mediated cytotoxicity assay. Killer cell activity was present in unfractionated cells from normal individuals and was enriched after removal of adherent cells. Target cell killing by unfractionated mononuclear cells from CLL patients was deficient in 5 of 6 patients tested, but after removal of adherent cells it was approximately equal to that of normal nonadherent cells. Our results indicate that the apparent deficit of killer cells in unfractionated PBL from some CLL patients is due to dilution of killer cells and not to an intrinsic defect in the function of these cells, the presence of suppressor cells in the adherent population, or suppression of killer cell function by serum factors.

CORD AND MATERNAL BLOOD MONOCYTE-MACROPHAGE MEDIATED ANTIBODY-DEPENDENT CYTOTOXICITY TO HERPES SIMPLEX INFECTED CELLS

We recently reported that antibody-dependent cell-mediated cytotoxicity (ADCC) to Herpes simplex virus (HSV) infected cells could be mediated, not only by K lymphocytes, but by adherent mononuclear cells (MA) isolated from adult human peripheral blood. The MA differ from K lymphocytes in antibody requirement, killing kinetics, and selective inhibition by latex or silica in the Cr51 release assay and are predominantly monocyte-macrophages. Because of the known role of such cells in immunity to several viruses, and since HSV can cause severe neonatal disease, it was of interest to evaluate the function of cord blood MA in ADCC to HSV.Cord blood (CB) from 11 healthy term neonates and maternal blood (MB) from 7 of their postpartum mothers were analyzed for MA activity in an 18 hr. Cr51 ADCC assay against HSV infected Chang liver cells. The magnitude of ADCC produced by MA from CB and MB was not significantly different from that obtained with MA from 8 non-pregnant adult females. Together with our earlier report of cord blood lymphocytes functioning with HSV antibody in ADCC, these data suggest that the use of HSV antibodies for prevention or therapy of neonatal HSV infection requires further consideration.

The immunosuppressive mechanism of azathioprine. I. In vitro effect on lymphocyte function in the baboon.

The effect of azathioprine on in vitro baboon lymphocyte function tests was evaluated using the mitogen stimulation test, the mixed lymphocyte culture test, the migration inhibition factor test, the cell-mediated lymphocytotoxicity test and the antibody dependent cell-mediated cytotoxicity test. It was found that azathioprine inhibited phytohemagglutinin and Concanavalin A stimulation at lower concentrations than those required to inhibit pokeweed mitogen stimulation. It inhibited the MLC reaction with as little as 0.2 mug/culture in the microculture system. Azathioprine had no effect on (a) the release of migration inhibition factor, (b) the cell-mediated lymphocytotoxicity assay if presensitized cells were used, and (c) the antibody-dependent cell-mediated cytotoxicity assay. However, azathioprine inhibited CML if it was added during in vitro sensitization and induction of killer cells. These in vitro results suggest that azathioprine inhibits those reactions which require cellular division.

Antibody-dependent cell-mediated cytotoxic activity in syngeneic moouse ascites tumors.

Eight different syngeneic murine ascites tumor preparations were tested for their ability to lyse antibody-coated chicken erythrocytes in an assay for antibody-dependent cell-mediated cytotoxicity (ADCC). All of the tumor preparations demonstrated significant ADCC effector activity, even at effector-cell:target-cell ratios of less than 1:1. In contrast, none of eight in vitro murine tumor-cell lines showed any consistent ability to function as ADCC effector cells. Several cell fractionation experiments were performed to ascertain the nature of the effector cells in the ascites tumors. Adherent cells were removed from tumor preparations by passage of cells over columns of Degalan plastic beads and 19S Fc-receptor-bearing cells were removed by sedimentation of EA rosette-forming cells. Although each of these procedures depleted only a minority of the total cells, all the ADCC activity was simultaneously removed in each case. Depletion of phagocytic cells by treatment of tumor preparations with carbonyl iron and magnetism only slightly diminished the ADCC activity. These results indicate that the ADCC activity associated with ascites tumors is due to effector cells most probably of host origin, but possibly representing an atypical sub-population of tumor cells. The characteristics of the effector cell closely parallel those defined for the major non-phagocytic ADCC effector cell in normal mouse spleen and peritoneal exudate.

Antibody-dependent cell-mediated cytotoxicity for human monolayer target cells bearing blood group and transplantation antigens and for melanoma cells.

A sensitive assay for antibody-dependent cell-mediated cytotoxicity (ADCMC) was developed utilizing serum from a patient with gestational choriocarcinoma and the 3H-proline microcytotoxicity test for detection fo destruction of monolayer target cells. Conditions for optimal expression of ADCMC were investigated using serum from this patient and skin fibroblasts from her husband and daugther as target cells with semi-purified blood leukocytes from normal donors as effector cells. Factors critical for optimal expression of ADCMC in this assay are the selection of effector cell donors possessing high levels of activity in the presence of serum with known lymphocyte-dependent antibody. (LDA), the effector cell preparative technique, and incubation for up to 40 h. Tris-NH4Cl lysis of red blood cells was found significantly to reduce effector cell activity. Under optimal conditions, the LDA titer of this patient's serum was greater than 10(-4). The sensitivity of the assay was confirmed by the detection of LDA activity of an anti-blood-group antibody at dilutions not demonstrating hemagglutination, and by the induction of ADCMC for blood group A antigen-bearing target cells by normal sera of B and O blood groups. In a preliminary study of sera from 16 melanoma patients on the corresponding antologous tumor target cells, four had significant LDA. Further studies are under way to determine specificty, incidence, and relationship of LDA-positive autologous sera to course of disease.