Simultaneous development of antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity in irradiated mice reconstituted with bone marrow cells

Spleen cells from irradiated, bone marrow-reconstituted mice were tested for their ability to mediate antibody-dependent cellular cytotoxicity against P815 target (ADCC-P815), ADCC against sheep red blood cells (ADCC-SRBC), and natural killer (NK) activity judged as YAC-1 lysis at different times after bone marrow reconstitution. Donor-derived ADCC-P815 effectors were found to appear in the spleens 10-12 days after bone marrow reconstitution simultaneously with the appearance of donor-derived NK cells. NK cells recently derived from bone marrow are known to express the Thy-1 antigen; the phenotype of the "early" ADCC-P815 effectors was found to be the same as that of NK cells, i.e., Thy-1+, asialo-GM1+. These data suggest that ADCC-P815 effector cells belong to the NK cell population. ADCC-SRBC, in contrast to ADCC-P815 and NK activity, was already high on Day 7 after bone marrow reconstitution. However, it was mediated partly by recipient-derived effectors. ADCC-SRBC effectors were characterized to be different from ADCC-P815 effectors.     

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.     

Natural killing and antibody-dependent cellular cytotoxicity: characterization of effector cells by E-rosetting and monoclonal antibodies

Recent investigations have indicated that the OKM1 hybridoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKM1+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKM1+ and E- OKM1+ cells; the E+ OKT3+ cell possessed essentially no ADCC or NK activity; (2) that E+ OKM1+ cells mediated more NK activity on a per cell basis than E- OKM1+ cells; this was verified by separating OKM1+ cells on a cell sorter into E+ and E- with the OKT11 monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKM1+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM1+ (both E-receptor positive and negative) and (b) OKM1- E-receptor positive.     

Influence of cytokines on HIV-specific antibody-dependent cellular cytotoxicity activation profile of natural killer cells

There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate "educated" KIR3DL1(+) NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate "uneducated" KIR3DL1(+) NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy.     

Lenalidomide enhances antibody-dependent cellular cytotoxicity of solid tumor cells in vitro: influence of host immune and tumor markers

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student’s t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D–NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.     

Inhibition of human natural killer (NK) activity and antibody dependent cellular cytotoxicity (ADCC) by lipomodulin, a phospholipase inhibitory protein

A highly purified preparation of lipomodulin, a phospholipase-inhibitory protein from rabbit neutrophils treated with glucocorticoids, inhibited NK and antibody-dependent cellular cytotoxicity (ADCC) activities of human peripheral blood lymphocytes in a dose-dependent manner. The presence of lipomodulin during the early period of the cytotoxicity assay was necessary to obtain maximal inhibition. The inhibition of NK or ADCC activity by lipomodulin was greater when effector cells were treated with lipomodulin than when target cells were incubated with lipomodulin. As lipomodulin did not block binding of effector cells to target cells, our results suggest that lipomodulin inhibits the cytolytic phase of NK and ADCC activities after binding to target cells, and imply that phospholipase(s) may be involved in NK and ADCC activities.     

Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.     

Natural killer and antibody-dependent cellular cytotoxicity in cervical carcinoma patients

Summary Natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC) was measured in 62 untreated cervical carcinoma patients and 25 normal healthy women, using a short-term chromium release assay. A significant reduction in NK and ADCC activity was observed in disseminated disease than in localized disease, when compared with normal donors. The majority of the patients received radiotherapy and both NK and ADCC activity recovered after therapy. Furthermore, interferon- was demonstrated to augment NK activity of peripheral blood mononuclear cells from healthy donors as well as patients. Also large granular lymphocytes separated on Percoll density gradient were the same in number in both the populations studied, although in cervical cancer there seemed to be a defect in killing activity.     

Alterations in antibody-dependent cellular cytotoxicity during the course of HIV-1 infection. Humoral and cellular defects

HIV-1-specific cell-mediated cytotoxicity (CMC) is a form of antibody-dependent cellular cytotoxicity (ADCC) in which HIV-1-specific antibodies arm NK cells directly to become cytotoxic for targets bearing HIV-1 antigenic determinants. This non-MHC-restricted cytotoxic activity is present in early stages of disease and declines markedly with disease progression. To understand the cellular and humoral factors contributing to the reduction in this activity, the conditions under which maximal arming of cells occurs was examined in vitro. With the use of a large patient cohort, a strong positive correlation was found between the capacity of a serum to direct lysis in standard ADCC assays and its ability to arm NK cells. Patients with minimal HIV-1-specific ADCC-directing antibodies exhibited low levels of CMC and were unable to arm normal effector cells in vitro. The lack of sufficient ADCC-directing antibodies was found to be one cause of defective CMC in some patients. Unlike asymptomatics, only a weak positive correlation was found between arming and ADCC with sera from AIDS patients, indicating that a factor other than absolute HIV-1 specific antibody titer was responsible for decreased CMC in this patient population. Another group of patients was found to have diminished CMC despite the presence of antibodies in the serum that were fully capable of arming normal effector cells to become cytotoxic for gp120-expressing targets. When compared with those of normal individuals, lymphocytes from seropositive patients mediated significantly reduced levels of cytotoxicity in ADCC and arming assays with the use of a high titered HIV-1-specific serum. In both assay systems, the magnitude and frequency of dysfunction in antibody-dependent cytolysis was found to be greater among AIDS patients than among asymptomatic individuals. The demonstration of both cellular and humoral defects in the ability of seropositive individuals to manifest ADCC reactivities strongly suggests that HIV-1 infection may significantly compromise the effectiveness of this potentially important cytolytic reactivity in vivo.     

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.     

Antibody-dependent cell-mediated cytotoxicity in simian immunodeficiency virus-infected rhesus monkeys

With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.     

Within peripheral blood mononuclear cells, antibody-dependent cellular cytotoxicity of rituximab-opsonized Daudi cells is promoted by NK cells and inhibited by monocytes due to shaving

Treatment of chronic lymphocytic leukemia patients with anti-CD20 mAb rituximab (RTX) leads to substantial CD20 loss on circulating malignant B cells soon after completion of the RTX infusion. This CD20 loss, which we term shaving, can compromise the therapeutic efficacy of RTX, and in vitro models reveal that shaving is mediated by effector cells which express Fc gammaRI. THP-1 monocytes and PBMC promote shaving, but PBMC also kill antibody-opsonized cells by antibody-dependent cellular cytotoxicity (ADCC), a reaction generally considered to be due to NK cells. We hypothesized that within PBMC, monocytes and NK cells would have substantially different and competing activities with respect ADCC or shaving, thereby either enhancing or inhibiting the therapeutic action of RTX. We measured ADCC and RTX removal from RTX-opsonized Daudi cells promoted by PBMC, or mediated by NK cells and monocytes. NK cells take up RTX and CD20 from RTX-opsonized B cells, and mediate ADCC. PBMC depleted of NK cells show little ADCC activity, whereas PBMC depleted of monocytes have greater ADCC than the PBMC. Pre-treatment of RTX-opsonized B cells with THP-1 cells or monocytes suppresses NK cell-mediated ADCC, and blockade of Fc gammaRI on monocytes or THP-1 cells abrogates their ability to suppress ADCC. Our results indicate NK cells are the principal cells in PBMC that kill RTX-opsonized B cells, and that monocytes can suppress ADCC by promoting shaving. These results suggest that RTX-based immunotherapy of cancer may be enhanced based on paradigms which include infusion of compatible NK cells and inhibition of monocyte shaving activity.     

Fc-engineered anti-CD33 monoclonal antibody potentiates cytotoxicity of membrane-bound interleukin-21 expanded natural killer cells in acute myeloid leukemia

BackgroundQualitative and quantitative defects in natural killer (NK) cells have been noted in patients with acute myeloid leukemia (AML), providing rationale for infusion of donor-derived NK cells. We previously showed that decitabine enhances expression of NKG2D ligands in AML with additive cytotoxicity when NK cells and Fc (fragment crystallizable region)-engineered CD33 monoclonal antibody (CD33mAb) was used. We conducted a phase 1 study evaluating decitabine and haploidentical NK cells in relapsed AML. Using patient samples from this study, we evaluated whether ex vivo donor-derived expanded NK cells with or without CD33mAb was effective in decitabine-treated AML.MethodsBone marrow aspirates were collected from patients at pre- and post-NK cell infusion. NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying membrane-bound IL-21 (mbIL-21). Patient samples were used to test in vitro activity of mbIL-21 NK cells ± CD33m Ab-dependent cellular cytotoxicity (ADCC) and AML patient derived xenograft (PDX) mice were developed to test in vivo activity.ResultsUpon incubation with primary AML blasts, mbIL-21 NK cells showed variable donor-dependent intra-cellular interferon-γ production, which increased with CD33mAb-coated AML. ADCC assays revealed mbIL-21 NK cells effectively lysed primary AML blasts with higher activity on CD33mAb-coated AML. Importantly, CD33mAb-dependent enhanced cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post-decitabine treatment. In vivo infusion of mbIL-21 NK cells in AML PDX mice, treated with CD33mAb, reduced the tumor burden.DiscussionThese data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of CD33mAb in AML.     

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion, removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells.     

TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.     

Cytotoxicity from cultured cells: analysis of precursors involved in generation of human cells mediating natural and antibody-dependent cell-mediated cytotoxicity.

Natural cell-mediated cytotoxicity of human peripheral blood lymphocytes natural killer (NK) against K-562 and antibody-dependent cellular cytotoxicity (ADCC) against Chang cells, as measured in a 4-hr ⁵¹Cr release assay, were both completely removed by depletion of Fc receptor-positive (FcR+) cells. After in vitro culture for 7 days, however, NK- and ADCC-like activities spontaneously regenerated. The nature of precursor cells was studied by examination of lymphocyte subpopulations required for generation of this cytotoxicity. After depletion of FcR+ cells from PBL, the following subpopulations were prepared: sheep erythrocyte rosette-forming cells (E+), surface membrane immunoglobulin-positive cells (SmIg+), and null cells (lacking E+, SmIg+, or FcR+ markers). Separate cell types or mixtures were cultured in vitro in medium containing 10% fetal calf serum for 7 days and then tested for NK and ADCC. Whereas unseparated FcR-depleted cells developed substantial cytotoxic activity, each of the subpopulations cultured alone was negative or had low activity. Addition of SmIg+ cells to other cell types had no effect; however, mixture of 80% E+ and 20% null cells resulted in optimal NK and ADCC. It is not presently clear which population the precursors were in. However, the requirement for proliferation by the null cell population but not by the E+ cells (as indicated by sensitivity to radiation and mitomycin C) suggested that the precursors for NK cells may be null cells.     

The role of apoptosis in antibody-dependent cellular cytotoxicity

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L₂C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector: target ratios (E: T) induced both release of⁵¹Cr and apoptosis. However NK cytotoxicity at higher E : T, and ADCC at all E : T, increased the level of⁵¹Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.     

HIV infection abrogates the functional advantage of natural killer cells educated through KIR3DL1/HLA-Bw4 interactions to mediate anti-HIV antibody-dependent cellular cytotoxicity

Combinations of KIR3DL1 and HLA-Bw4 alleles protect against HIV infection and/or disease progression. These combinations enhance NK cell responsiveness through the ontological process of education. However, educated KIR3DL1(+) NK cells do not have enhanced degranulation upon direct recognition of autologous HIV-infected cells. Since antibody-dependent cellular cytotoxicity (ADCC) is associated with improved HIV infection outcomes and NK cells overcome inhibition through killer cell immunoglobulin-like receptors (KIR) to mediate ADCC, we hypothesized that KIR3DL1-educated NK cells mediate anti-HIV ADCC against autologous cells. A whole-blood flow cytometry assay was used to evaluate ADCC-induced activation of NK cells. This assay assessed activation (gamma interferon [IFN-γ] production and/or CD107a expression) of KIR3DL1(+) and KIR3DL1(-) NK cells, from HLA-Bw4(+) and HLA-Bw4(-) HIV-positive and HIV-negative individuals, in response to autologous HIV-specific ADCC targets. KIR3DL1(+) NK cells were more functional than KIR3DL1(-) NK cells from HLA-Bw4(+), but not HLA-Bw4(-), healthy controls. In HIV-infected individuals, no differences in NK cell functionality were observed between KIR3DL1(+) and KIR3DL1(-) NK cells in HLA-Bw4(+) individuals, consistent with dysfunction of NK cells in the setting of HIV infection. Reflecting the partial normalization of NK cell responsiveness following initiation of antiretroviral therapy, a significant correlation was observed between the peripheral CD4(+) T-lymphocyte counts in antiretroviral therapy-treated subjects and the functionality of NK cells. However, peripheral CD4(+) T-lymphocyte counts were not correlated with an anti-HIV ADCC functional advantage in educated KIR3DL1(+) NK cells. The abrogation of the functional advantage of educated NK cells may enhance HIV disease progression. Strategies to enhance the potency of NK cell-mediated ADCC may improve HIV therapies and vaccines.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Antibody-dependent cellular cytotoxicity against HIV-coated target cells by peripheral blood monocytes from HIV seropositive asymptomatic patients

Cytotoxic effector cells like cytotoxic T cells, NK cells, monocytes/macrophages, and neutrophils can lyse directly HIV-infected or HIV-coated cells in the absence or presence of anti-HIV antibodies. Therefore, these cytotoxic mechanisms can be invoked either in the control of HIV infection at early stages of the disease or in the generalized immunosuppression observed at later stages of the disease. The relationship between anti-HIV effector mechanisms and disease, however, remains elusive. The present study investigates in HIV+ seropositive asymptomatic patients peripheral blood monocytes (PBM)-mediated antibody dependent cellular cytotoxicity (ADCC) against HIV-coated target cells in the presence of heterologous or autologous anti-HIV serum. To test for specific ADCC against HIV Ag, a T4+ CEM.TR line resistant to TNF and macrophage-mediated cytotoxicity was selected in vitro. ADCC was performed in an 18-h 51Cr-release assay using CEM.TR cells coated with inactivated HIV. Unlike PBM from normal controls, significant ADCC was observed by PBM from HIV+ seropositive patients in the presence of pooled HIV+ antiserum. The ADCC activity was specific for HIV and was dependent on the E:T ratio and the antiserum dilution used. Upon activation of PBM with rIFN-gamma, both normal and HIV+ PBM-mediated ADCC against HIV-coated CEM.TR. Furthermore, ADCC activity by PBM from HIV+ seropositive patients in the presence of their autologous serum was examined. Significant ADCC activity was observed and was dependent on the E:T ratio and serum dilution used. The findings demonstrating anti-HIV ADCC activity by PBM from HIV+ seropositive individuals and their autologous sera support the notion that monocyte-mediated ADCC may be operative in vivo.