Modulation of Antibody-Dependent Cellular Cytotoxicity Against Chicken Erythrocyte Targets by Adriamycin and Daunorubicin

Abstract

Peritoneal exudate cells isolated from Adriamycin (AM) or daunorubicin (DM)-treated mice demonstrated an increased ability to mediate antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken red blood cell targets. Following initial suppression of this cell-mediated function 3 days after a single injection, increased effector cell efficiency occurred to an equal degree in both groups of drug treated mice by day 10 compared to controls. This increase in ADCC activity occurred in parallel with a decrease in the total number of peritoneal cells recovered. It is hypothesized that the drugs acted to modulate ADCC in two ways: 1) suppression by induction of suppressor cell activity, and 2) enhancement by elimination of suppressor cells which resulted in increased effector activity of the remaining cells.     

Antibody-Dependent Cell-Mediated Cytotoxicity in Cattle: Activity Against 51Cr-Labeled Chicken Erythrocytes Coated with Protozoal Antigens

Bovine mononuclear cells in the presence of bovine anti-chicken erythrocyte sera at high dilutions induce release of chromium-51 from labeled chicken erythrocytes. Bovine effector cells are capable of recognizing both bovine immunoglobulin G(1) and bovine immunoglobulin G(2); in contrast, human effector cells only recognize immunoglobulin G(1). Effector cell activity of bovine mononuclear cells is equally distributed between peripheral blood and spleen. As in other species, thymus and lymph node cells exert no antibody-dependent effect, although some direct cytotoxicity by lymph node cells may be observed. Antibody-dependent cell-mediated cytotoxicity against a bovine cell line can also be detected. By using a tannic acid technique, it was found that chicken erythrocytes coated with Theileria parva piroplasm antigen or with Trypanosoma rhodesiense variant-specific coat antigen form suitable targets for bovine antibody-dependent cell-mediated cytotoxicity assays. By using such targets, a moderate degree of direct cytotoxicity by bovine mononuclear cells, in the absence of antibody, is always observed; this may be reduced by choosing optimal conditions of tannic acid treatment and antigen sensitization and by the use of short incubation periods for the cytotoxicity assay. Observations have been made on the variant specificity, time course of appearance, and association with immunoglobulin G(1) of the antibody activity responsible for cell-dependent cytotoxicity against chicken erythrocytes coated with T. rhodesiense antigens. The potential usefulness of this technique in the analysis of protective immune responses against protozoal infections is discussed.     

Selective Inhibition of Human Natural Killing and Antibody-Dependent Cellular Cytotoxicity by a Polyanion

A high molecular polyanion, Liquoid, was found to inhibit at nontoxic concentrations (12-50 micrograms/ml) the natural killing (NK) and the antibody-dependent cellular cytotoxic (ADCC) activity of human peripheral blood mononuclear cells selectively. Whereas NK of the K 562 target cell was slightly or not at all affected, the spontaneous lysis of PDe-B-1, an EBV-transformed B-cell line, was strongly inhibited or even completely abolished. ADCC activity could only be inhibited by Liquoid if the target cells were mycoplasma-free, while the polyanion had no effect when mycoplasma-contaminated target cells were used. Liquoid did not alter the target binding capacity of the NK effector cells and did not activate monocytes or induce other suppressive cells. Alpha interferon, but neither beta nor gamma interferon, was able to neutralize the NK reduction. These results suggest that Liquoid inhibits a target cell-related, selective process in the post-binding stage of NK cell lysis.     

Characterization of Effector Cells Mediating IgG and IgM Antibody-Dependent Cellular Cytotoxicity

Spleen effector cells for IgG- and IgM-induced antibody-dependent cellular cytotoxicity (ADCC) were characterized with respect to density and cell surface markers by using sheep erythrocytes (SRBC) coated with hybridoma-derived monoclonal anti-SRBC IgG or anti-SRBC IgM antibodies as targets. While basically the same effector cells are cytolytic for IgG and IgM antibody-coated SRBC, they differ with respect to their relative killing capacity for IgG- versus IgM-coated target cells. On the basis of physical and biochemical properties three populations with cytolytic capacity could be separated: (I) A light fraction of large cells had high cytolytic potential for both IgG- and IgM-coated SRBC. The cells were negative for the Fc receptor for IgG (Fc gamma-R-) and the C3-receptor (C3-R-), they carried the receptor for Helix pomatia A agglutinin (HP-A+), and reactivity was strongly reduced after treatment with anti-Thy-1 and complement (C). (II) High activity was also observed with a medium-dense fraction, preferably lysing IgG antibody-coated cells. The cells were Fc gamma-R+, partly C3-R+, mostly HP-A-, and only a minor portion of the cells were Thy-1+. (III) A dense fraction, displaying on a per cell basis low cytolytic potential, was more active in IgM than IgG ADCC. The cells were Fc gamma-R+, HP-A+ and Thy-1+. All three effector cell populations were non-adherent, non-phagocytic, and surface immunoglobulin-negative (s-Ig-).     

Interleukin-10 Down-Regulates Oxidative Metabolism and Antibody-Dependent Cellular Cytotoxicity of Human Neutrophils

The authors investigated the ability of interleukin-10 (IL-10) to modulate some constitutive or interferon-γ (IFN-γ)-enhanced activities of human neutrophils. An 18 h culture of neutrophils with IL-10 dose-dependently down-regulated their capacity to produce O₂⁻ and lucigenin-amplified chemiluminescence in response to n-formyl-methionyl-leucylphenyl-alanine (FMLP). Furthermore, treatment of neutrophils with IL-10 decreased in a dose-dependent fashion, their capacity to lyse antibody-coated sheep erythrocytes. Membrane expression of FcγRI, FcγRII, FcγRIII, CR1, CR3 and FcγR- and CR-mediated phagocytosis were not modified by the cytokine. Culture of neutrophils with IFN-γ (100 U/ml) did not modify their FcγR- and CR-mediated phagocytosis, but significantly up-regulated FcγRI and CR3 membrane expression as well as their oxidative metabolism and antibody-dependent cellular cytotoxicity (ADCC). When IL-10 and IFN-γ were added simultaneously to neutrophil culture, IL-10 dose-dependently reduced IFN-γ-induced increase of CR3 expression, O₂⁻ production (in response to both FMLP and phorbol 12-myristate 13-acetate, or PMA) and ADCC, but did not change FcγRI expression on phagocytes. These results demonstrate that IL-10 is a significant neutrophil deactivator and provide new information on the role of IL-10 in the regulation of neutrophil-mediated inflammatory processes.     

Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

The Effects of Cyclophosphamide on Mitogen-Induced and Antibody-Dependent Cellular Cytotoxicity in Guinea Pigs

Abstract

Lymph node cells from normal guinea pigs, when stimulated with the non-specific mitogen PHA were transformed to activated killer cells, capable of lysing ⁵¹Cr labeled mouse fibroblasts. Similarly, these lymphoid cells effected lysis of antibody coated chicken red cells in an antibody-dependent cellular cytotoxicity assay. Following cyclophosphamide, 150 mg/kg IP, the reactivity of an aliquot of lymph node cells to effect either cytotoxic reaction was not diminished. These results indicate that this immunosuppressant does not promote a selective decrease in either lymphoid effector. Rather, they are diminished in parallel with the generalized lympholysis resulting from drug administration. During the recovery phase, lymph node cells showed increased ability to lyse target cells, suggesting a rebound phase of heightened activity. Thymic cells from normal and cyclophosphamide treated animals were poor effectors in either cytotoxic assay. The addition of an equal number of thymocytes to lymph node cells resulted in decreased mitogen-induced cytotoxicity. Thymic inhibitory activity was mediated only by viable cells and the phenomenon did not represent an altered shift in PHA sensitivity. This suppressive activity persisted when thymic cells from cyclophosphamide treated-animals were employed, indicating that inhibitory cells were also not selectively depleted by this drug. In antibody-dependent cellular cytotoxicity assays, thymocytes from normal or cyclophosphamide-treated animals did not alter the cytotoxicity capacity of lymph node cells.     

The Effects of Cyclophosphamide on Mitogen-Induced and Antibody-Dependent Cellular Cytotoxicity in Guinea Pigs

Lymph node cells from normal guinea pigs, when stimulated with the non-specific mitogen PHA were transformed to activated killer cells, capable of lysing ⁵¹Cr labeled mouse fibroblasts. Similarly, these lymphoid cells effected lysis of antibody coated chicken red cells in an antibody-dependent cellular cytotoxicity assay. Following cyclophosphamide, 150 mg/kg IP, the reactivity of an aliquot of lymph node cells to effect either cytotoxic reaction was not diminished. These results indicate that this immunosuppressant does not promote a selective decrease in either lymphoid effector. Rather, they are diminished in parallel with the generalized lympholysis resulting from drug administration. During the recovery phase, lymph node cells showed increased ability to lyse target cells, suggesting a rebound phase of heightened activity. Thymic cells from normal and cyclophosphamide treated animals were poor effectors in either cytotoxic assay. The addition of an equal number of thymocytes to lymph node cells resulted in decreased mitogen-induced cytotoxicity. Thymic inhibitory activity was mediated only by viable cells and the phenomenon did not represent an altered shift in PHA sensitivity. This suppressive activity persisted when thymic cells from cyclophosphamide treated-animals were employed, indicating that inhibitory cells were also not selectively depleted by this drug. In antibody-dependent cellular cytotoxicity assays, thymocytes from normal or cyclophosphamide-treated animals did not alter the cytotoxicity capacity of lymph node cells.     

Sex-Associated Differences in the Antibody-Dependent Cellular Cytotoxicity Antibody Response to Measles Vaccines

In some countries, excessive non-measles-related mortality has been observed among female recipients of high-titer measles vaccines. We determined if differences in the immune response to measles vaccines underlie the excessive female mortality by measuring the measles virus (MV)-specific antibody-dependent cellular cytotoxicity (ADCC) antibody response in 65 3-year-old Gambian children immunized with Edmonston-Zagreb medium-titer (EZ) or Schwarz standard vaccines during infancy. Among the 20 females and 22 males with undetectable anti-MV antibodies at the time of immunization, females had significantly lower ADCC than males (median cytotoxicities of 1/100 serum dilutions = 8.4 and 12%, respectively; P = 0.04). This sex-associated difference was present only among the six female and seven male recipients of EZ vaccine (median cytotoxicities = 5.1 and 19.0%, respectively; P = 0.02). There were no significant sex-associated differences in neutralizing antibody activity. Decreased ADCC antibody activity may contribute to the lower survival rate observed in females receiving high-titer measles vaccination.     

HLA-C Matching Status Does Not Affect Rituximab-Mediated Antibody-Dependent Cellular Cytotoxicity by Allogeneic Natural Killer Cells

Risk of leukemia relapse after T cell-depleted hematopoietic stem cell transplantation is lower in the “HLA-C mismatched” recipient-donor combinations. This might be attributable to increased natural killing by allogeneic NK cells carrying a KIR that does not bind to HLA-C on target cells (HLA-C-uncoupled KIR). Considering a new strategy of allogeneic NK cell transfer with rituximab to treat B-cell lymphomas, however, it is unknown whether the HLA-C matching status also affects rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). To address this issue, we investigated the levels of ADCC by purified NK cells carrying an HLA-C-uncoupled KIR, where the NK cell donors had either matched or mismatched HLA-C combination with target cells. Purified NK cells carrying an HLA-C-uncoupled KIR consistently showed enhanced ADCC against target cells when NK cell donors had an HLA-C-mismatch. When NK cell donors did not have an HLA-C mismatch, it was inconsistent whether HLA-C-uncoupled KIR caused ADCC enhancement. When the levels of ADCC by whole NK cells were compared, there were substantial differences among the donors regardless of the HLA-C matching status. Subjects with HLA-C mismatch may not have an advantage when cytoimmunotherapy using allogeneic NK cells is considered in combination with rituximab.     

Inhibition of Spontaneous and Antibody-Dependent Cellular Cytotoxicity by Sera and Isolated Antiglobulin Preparations from Rheumatoid Arthritis Patients

Using sera from patients with rheumatoid arthritis and antiglobulin preparations obtained from such sera, inhibition of human lymphocyte cytotoxicity in antibody-dependent (ADCC) and spontaneous cell-mediated cytotoxicity (SCMC) reactions against an allogeneic melanoma cell line (IGR3) has been demonstrated. Fractionation of effector cell preparations indicated that Fc-receptor-bearing lymphocytes were operative in both reactions. Removal of phagocytic and adherent cells from the effector cell population resulted in more pronounced inhibition of SCMC and ADCC reactions by rheumatoid sera. The results indicate that antiglobulin preparations from human sera containing rheumatoid factor activity can effectively block the cytotoxic activity of Fc-receptor-bearing effector lymphocytes (K cells) in vitro. On analogy with the inhibition of ADCC and SCMC reactions by immune complexes and aggregated IgG, antiglobulin complexes present in the antiglobulin preparations are responsible for this effect.     

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 (HIV) 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.     

Analysis of the Adhesion Step in the Herpes Simplex Virus Antibody-Dependent Cellular Cytotoxicity System

The lysis of herpes simplex virus-infected tissue culture cells by antibody-dependent cellular cytotoxicity (ADCC) requires a preliminary step in which effector cells adhere to the immunoglobulin G antibody-coated targets. To study the adhesion step, we made use of two observations: (i) some of the mononuclear cells in human blood form rosettes with antibody-coated target cells, and (ii) most ADCC effector cells can be removed by allowing mononuclear cells to adhere to monolayers of antibody-sensitized tissue culture cells. The effect of various experimental conditions on the adhesion step was assessed in ADCC cultures both at unit gravity and after centrifugation. At unit gravity both rosette formation and monolayer adhesion were partially reduced at 4 degrees C as compared to 37 degrees C. Both were also partially inhibited in glucose-free medium containing sodium azide and 2-deoxyglucose but were unaffected in glucose-free medium containing only one of these energy inhibitors. In contrast, after centrifugation neither reaction was inhibited at 4 degrees C or in glucose-free medium with sodium azide and 2-deoxyglucose. Cytochalasin B but not colchicine suppressed both reactions. Inhibition by cytochalasin B could not be reversed by centrifugation. Both reactions were independent of extracellular Ca(2+) and Mg(2+) and were unaffected by rendering mononuclear cells cytotoxically inactive by brief heat shock. These findings indicate that the adhesion step in ADCC directed against virus-infected or uninfected tissue culture cells is only modestly dependent on effector cell energy generation, that centrifugation greatly reduces this dependence, and that microfilaments but not microtubules are necessary. The modest ambient temperature and energy requirements, independence of extracellular divalent cations, lack of sensitivity to colchicine, and relative resistance to supraphysiological temperature serve to distinguish the adhesion step from the lytic step in ADCC.     

Comparison of the Effector Cells in Human Spontaneous Cellular Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity: Differential Sensitivity of Effector Cells to in Vivo and in Vitro Corticosteroids

The effector cells mediating antibody dependent cellular cytotoxicity (ADCC) and spontaneous cellular cytotoxicity (SCC) in humans have been reported to possess similar characteristics. Multiple cell separation techniques were employed in an attempt to physically separate and distinguish the effector cells in these two types of cellular cytotoxicity. Subpopulations of mononuclear cells obtained by a variety of fractionation procedures which either enriched or depleted monocytes, lymphocytes bearing a receptor for sheep erythrocytes (SRBC), a receptor for complement (CRL) or an Fc receptor for IgG always had similar effects on both ADCC and SCC. Aggregated gamma globulin blockade of Fc receptors produced similar dose-dependent depressions of ADCC and SCC. Despite our inability to physically separate the effector cells of ADCC and SCC, administration of in vivo dexamethasone caused a relative increase in ADCC but a profound decrease in SCC. Furthermore, in vitro dexamethasone in pharmacologic and suprapharmacologic concentrations caused no change in ADCC but significantly decreased SCC. This study demonstrates that although the effector cells cannot be physically separated, ADCC and SCC are differentially sensitive to corticosteroids and are hence functionally distinct either on the basis of different subsets of effector cells with similar surface markers or different mechanisms of cytotoxicity by the same effector cell.     

Activation of Monocyte and Granulocyte Antibody-Dependent Cytotoxicity by Phorbol Myristate Acetate

We have characterized the effects of phorbol myristate acetate (PMA) on human monocyte and neutrophil oxidative metabolism and antibody-dependent cytotoxicity toward anti-D sensitized human erythrocytes (RBC) and a human lymphoblastoid cell line (CEM). Hexose monophosphate shunt activity was measured by [1-¹⁴C]glucose oxidation and target lysis by ⁵¹Cr release. PMA produced a dose-dependent stimulation of hexose monophosphate shunt activity. Neutrophils responded with higher hexose monophosphate shunt activity and at a lower PMA concentration than did monocytes. PMA increased monocyte lysis of antibody-sensitized RBC by two-thirds, but did not affect lysis of CEM targets. Neutrophils were unable to lyse either antibody-sensitized or nonsensitized RBC without the addition of PMA. When PMA was added, lysis of both targets increased markedly. Neutrophils without PMA were able to lyse a small number of both antibody-sensitized and nonsensitized CEM targets. PMA also increased neutrophil lysis of these targets. Target lysis by neutrophils from a patient with chronic granulomatous disease, cells unable to produce reactive oxygen species, was not increased by PMA. Chronic granulomatous disease monocytes, however, responded to PMA by more than doubling lysis of antibody-sensitized RBC. Hypoxia inhibited PMA augmentation of antibody-sensitized RBC lysis by neutrophils, but not by monocytes. Generation of reactive oxygen species by the xanthine-xanthine oxidase system inhibited CEM growth, but did not cause lysis, indicating that in some cases oxidative injury may be nonlytic. We suggest that PMA augments neutrophil cytotoxicity to tumor and RBC targets by stimulating reactive oxygen species-mediated lysis, but in monocytes augmentation of lysis is due to activation of a nonoxidative mechanism of lysis.     

Genetic Control of Murine Antibody-Dependent Cell-Mediated Cytotoxicity

We have observed that the intensity of the direct antibody-dependent cell-mediated cytotoxicity (ADCC) response after an inoculation of foreign tumour cells varies with the strain of mice studied. The inoculation of a human lymphoblastoid cell-line into CBA/J, BALB/c, or DBA/2 mice gives rise to a good cytotoxic response by the host K cells armed with specific antibodies. In contrast, A/J, B10.A, C57BL/6 and B10.S mice respond poorly under the same conditions. The high response is dominant in Fi hybrids between high and low responders and is also expressed among F2 backcrosses with the H-2 phenotype of low responders, suggesting that non-H-2 genes are also implicated in the regulation of ADCC. The genetic control is not exerted at the level of antibody secretion but at that of K-cell activity, since sera from high or low responders are equally effective in arming an ADCC reaction, whereas K cells from low-responder strains are less efficient than those from high-responder strains. The natural killer (NK) activity of the same strains has been screened. The results show a good correlation with some high- and low-responder strains, such as CBA and DBA/2 or A/J and SJL, respectively, but not with C57BL/6, B10.S or B10.A strains. Thus, in addition to common genes controlling both lytic functions, there are specific genetic factors influencing the balance between NK and K cells. These findings confirm the general view that NK and K cells represent only partially identical subsets.     

Damage of Liposomes in Antibody-Dependent Cell-Mediated Cytotoxicity

Lymphoid cells bearing Fc receptors are able to lyse antibody-coated animal target Cells in an antibody-dependent cell-mediated cytotoxicity reaction. The results presented here show that liposomes consisting of sphingomyelin, cholesterol, and cardiolipid and coated by cardiolipidic antibodies could be destroyed by spleen or thymus cells. No alteration of liposomes was observed when normal rabbit serum was used or when the effector cell population was depleted of cells bearing Fc receptors. The lysis of antibody-coated liposomes by effector cells could be carried out in two steps. In the first step, the fixation of antibodies on the cardiolipidic antigens could lead to a reorganization of the liposomal membrane. In the second step, the effector Fc-receptor-bearing cells might amplify this alteration of the liposomes.     

Antibody-Dependent Cell-Mediated Cytotoxicity in Various Orders of Vertebrates

By using chicken antibodies and chicken lymphoid cells, we could demonstrate antibody-dependent cytotoxicity (ADCC) against haptenated chicken erythrocytes. ADCC could be demonstrated either by treating the target cells with antibody (sensitization) or by treating the effector cells (arming). Approximately ten times higher antibody concentrations were required for arming than for sensitization. Chicken lymphoid cells did not co-operate with mammalian antibodies in either type of ADCC, and thus the situation was reciprocal to that observed earlier with mammalian lymphoid cells. In a further attempt to characterize the arming phenomenon we used biofiltered (aggregate-free) mouse antibodies for the arming of rat spleen cells. They were as efficient as nonfiltered antibodies, and this confirms that individual antibody molecules are efficient in arming.     

Antibody-Dependent and Direct Lymphocyte-Mediated Cytotoxicity in Patients on Hemodialysis

Twenty-two patients on chronic hemodialysis were tested for direct lymphocyte-mediated cytotoxicity (DCML) and antibody-dependent lymphocyte-mediated cytotoxicity (LALI) against selected panels of normal donor lymphocytes. The DCML reaction was only positive in patients who had received blood transfusions within the two or three months prior to testing. Thus this reaction has limited value in practice as a parameter for presensitization. The use of third-party effector cells and of target cells precoated with antibody in the LALI reaction was evaluated, and it is advocated that effector cells from the patient investigated and direct application of patient serum to reaction mixtures should be preferred in order to avoid false negative reactions. In patients who had once manifested lymphocytotoxins, LALI disclosed a much broader reactivity than seen with a conventional complementdependent cytotoxicity technique (CDC), whereas in this study patients who had never developed cytotoxins were also negative with the LALI technique. It is concluded that a positive LALI cross-match, in spite of a negative CDC cross-match, will be a common finding in donor-recipient combinations where the recipient has preformed cytotoxins. The effect of this on subsequent graft prognosis needs further clarification.