Effector memory alphabeta T lymphocytes can express FcgammaRIIIa and mediate antibody-dependent cellular cytotoxicity

Human memory T cells are comprised of distinct populations with different homing potential and effector functions: central memory T cells that mount recall responses to Ags in secondary lymphoid organs, and effector memory T cells that confer immediate protection in peripheral tissues. In the present study we demonstrate that a proportion of effector memory T cells express FcgammaRIIIa (CD16), are perforin positive, and directly mediate Ab-dependent cytotoxicity ex vivo. This particular alphabeta T lymphocyte subset has the morphology of large granular lymphocytes, increases proportionately in vivo during reactive lymphocytosis, and can be detected in vitro among EBV-specific T lymphocytes after stimulation with EBV Ags. Consequently, during a normal immune response, amplification of these effector memory T lymphocytes that are capable of Ab-dependent cytotoxicity may have beneficial or harmful consequences depending on the presence of pathogen- or tissue-specific Abs, respectively.     

Antibody–receptor interactions mediate antibody-dependent cellular cytotoxicity

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody–receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting–MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab–receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab–receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc–polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG–Fc receptor biology.     

Development of immunomonitoring of antibody-dependent cellular cytotoxicity against neuroblastoma cells using whole blood

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a ‘real-time’ ex vivo whole-blood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement-dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.     

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Inhibition of antibody-dependent cellular cytotoxicity by autologous lymph node cells.

A population of lymph node cells that lack the usual T, B, or K cell markers was found to inhibit autologous spleen cells from mediating antibody-dependent cellular cytotoxicity (ADCC) to antibody-coated chicken erythrocytes. Inhibitor cells were not susceptible to treatment with anti-Thy 1.2 or anti-Ig and C; they did not adhere to Sephadex G-10, to nylon wool, or to monolayers of sheep erythrocytes (E) or erythrocytes plus 7S antibody (EA). After a brief (4-min) exposure to 45 degrees C, the ability to inhibit was lost whereas other cellular responses remained intact. ADCC mediated by nonadherent splenic effector cells (presumptive K cells) was highly susceptible to inhibition. Possible mechanisms for and implications of lymphocyte-mediated inhibition of ADCC are discussed.     

Herpes simplex virus type 1 Fc receptor protects infected cells from antibody-dependent cellular cytotoxicity.

Recent studies indicate that the herpes simplex virus type 1 (HSV-1) Fc receptor (FcR) can bind antiviral immunoglobulin G by participating in antibody bipolar bridging. This occurs when the Fab domain of an immunoglobulin G molecule binds to its antigenic target and the Fc domain binds to the HSV-1 FcR. In experiments comparing cells infected with wild-type HSV-1 (NS) and cells infected with an FcR-deficient mutant (ENS), we demonstrate that participation of the HSV-1 FcR in antibody bipolar bridging reduces the effectiveness of antibody-dependent cellular cytotoxicity.     

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC).

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.     

Differences in antibody-dependent cellular cytotoxicity against tumor cells in in vitro differentiating mononuclear phagocytes from bone marrows of normal and inflamed mice

Mononuclear phagocytes, differentiated in vitro from bone marrow cells of mice inflamed in vivo with either Corynebacterium parvum or thioglycollate, expressed a higher activity in antibody-dependent cellular cytotoxicity (ADCC) against tumor cells, than those of normal mice. A good correlation between the cytolytic activity and chemiluminescence activity of the different mononuclear phagocyte populations was observed. The ADCC activity of BMDMP from normal mice was inhibited by exogenous prostaglandin E₂ (PGE₂) to a higher extent than that of BMDMP of inflamed mice. When the three BMDMP populations were cultured in the presence of aspirin (without exogenously added PGE₂), the ADCC was significantly increased. The three populations gave identical high values. This suggests that the differential ADCC activity of BMDMP from normal and inflamed mice is due to their differential response to endogenous prostaglandins. PGE₂ showed also a differential effect on the mononuclear phagocyte-forming capacity of bone marrow macrophage precursor cells from normal and inflamed mice. Bone marrow precursor cells from inflamed mice showed a higher resistance to the suppressive effect of PGE₂ (10^?5M) on mononuclear phagocyte-forming capacity than those of normal mice which were totally suppressed. It is concluded that the observed differential properties of the three bone marrow-derived mononuclear phagocyte populations originate at the level of bone marrow precursor cells and that, therefore, the similar functional differences observed in inflammation-induced peritoneal macrophage populations, observed by our and other groups, stem at least partly from differences at the level of bone marrow precursor cells.     

Micro-scission of YAC tumor cells during antibody-dependent cellular cytotoxicity mediated by human neutrophils

The cellular events accompanying neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) directed against YAC erythroleukemic target cells have been studied by time-lapse fluorescence-intensified microscopy. The YAC plasma membrane and cytosol were labeled with the fluorescent probes diC18Icc and eosin Y, respectively. Fluorescently labeled and IgG-opsonized YAC cells were incubated at 37 degrees C while observed by optical microscopy. During temporal studies of neutrophil-YAC conjugates, the cytosol of YAC cells accumulated in tubular and spherical compartments of the neutrophils' vacuolar apparatuses. To distinguish between several possible mechanisms of target cytosol uptake, diC18Icc-labeled YAC cells were observed during identical conditions. The membrane label diC18Icc was found to accumulate within neutrophils in an identical fashion. At roughly 30 min, 25 and 38% of neutrophils in apparent conjugates had internalized tumor cell cytosol or plasma membrane, respectively, within a vesicular compartment. The IgG-dependent uptake of eosin Y and diC18Icc by neutrophils was diminished by exposure to 2.5 mM sodium azide. When cells were exposed to 5.5 mM sodium azide, 1 mM iodoacetamide, or 4 degrees C, conjugate formation and uptake of eosin Y or diC18Icc were abolished. An artifactual accumulation of eosin Y or diC18Icc in neutrophils was further ruled out by control studies. Non-specific exchanges of eosin Y and diC18Icc labels of YAC cells with tannic acid-treated red blood cells (RBCs) and normal neutrophils were studied. Since hemoglobin binds tightly to eosin Y, RBCs can easily detect eosin Y leakage. No exchange of eosin Y or diC18Icc from YAC cells into bound tannic acid-treated erythrocytes was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of effector cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies

We examined purified human large granular lymphocytes, peripheral monocytes, and T cells for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) with murine monoclonal antibodies. We also evaluated the effects of pretreatment of cells with interleukin 2 and interferon to augment ADCC activity. MB3.6, a murine monoclonal antibody directed against the GD3 ganglioside, induced high levels of ADCC. This ADCC was mediated predominantly, if not completely, by human killer cells (large granular lymphocytes) whereas other effector cell populations demonstrated no significant cytotoxic activity in 6- or 18-hr assays. The IgG2a an anti-melanoma antibody 9.2.27 generated low or no ADCC with most normal donors or melanoma patients. IL 2 was a very potent booster of ADCC activity. Interferon alpha also was effective, whereas interferon gamma did not augment but rather inhibited reactivity. We tested a large panel of antibodies of various isotype against colon carcinoma cells and found that gamma-3 isotype antibodies more frequently generated ADCC and produced higher levels of cytotoxic activity than did IgG1 or IgG2 antibodies. It appears that a variety of parameters can affect ADCC reactions, including the type of effector cell and its level of activation, the isotype of the antibody, and properties of the target cell line such as its susceptibility to lysis.     

Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst.

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.     

A subset of CD16-natural killer cells without antibody-dependent cellular cytotoxicity function

The low affinity IgG receptor, CD16 (Fc gamma RIII), is expressed on almost all peripheral blood natural killer (NK) cells. A small subset of CD3- CD16- CD56+ NK cells, representing less than 1% of peripheral blood lymphocytes, expands during in vivo IL-2 treatment. To analyze this CD16- NK cell subset in more detail, NK clones have been generated. One of them (TNK2) has been used to study the function of these cells in more detail. It is demonstrated that TNK2 exerts normal NK activity and displays large granular lymphocyte morphology. Since this clone lacks CD16 expression, antibody-dependent cellular cytotoxicity cannot be exerted. CD16 monoclonal antibodies fail to induce cytotoxic activity against NK-resistant target cells. These studies reveal that the lack of CD16 detection is not due to the modulation or the stage of activation of these NK cells. TNK2 is representative of this small subset of peripheral blood NK cells, expanded during IL-2 treatment, which does not express Fc gamma RIII and therefore cannot perform antibody-dependent cellular cytotoxicity.     

Inhibition of antibody-dependent cellular cytotoxicity by protein A from Staphylococcus aureus.

Protein A, a cell wall constituent of several strains of Staphylococcus aureus, binds strongly to the Fc portion of immunoglobulins. This investigation demonstrated that such binding can inhibit antibody-dependent cellular cytotoxicity (ADCC). The degree to which ADCC was inhibited depended upon the relative concentrations of protein A and anti-target cell antiserum. Protein A also inhibited the formation of rosettes between antibody-coated sheep red blood cells and lymphoid cells with Fc receptors. We, therefore, conclude that protein A inhibits ADCC by preventing the binding of antibody-coated target cells to Fc receptors on cytotoxic effector cells.     

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.     

Gamma delta T cells from HIV+ donors can be expanded in vitro by zoledronate/interleukin-2 to become cytotoxic effectors for antibody-dependent cellular cytotoxicity

Background aimsImmunotherapy using γδ T cells capable of mediating antibody-dependent cellular cytotoxicity (ADCC) is a promising anti-human immunodeficiency virus (HIV) strategy. Approved aminobispohsphonate drugs, for example zoledronate (Zometa), stimulate γδ T cells in cancer patients, where they may promote direct tumor killing. Knowing that γδ T cells are modulated during HIV disease, documenting their responses and potential for controlling HIV is important. We investigated whether zoledronate/interleukin (IL)-2 could expand cytotoxic Vδ2 cells from HIV⁺ donors and whether these cells functioned in ADCC.MethodsPeripheral blood mononuclear cells from uninfected controls and HIV⁺ individuals receiving anti-retroviral therapy were treated with isopentenyl pyrophosphate (IPP) or zoledronate plus IL-2 to expand the Vδ2⁺ subset. Immunophenotyping and functional analyzes (cytotoxicity or cytokine expression) allowed us to compare cell properties from individual donors and to compare the responses to each stimulating agent.ResultsZoledronate stimulated a greater expansion of Vδ2 cells in HIV⁺ individuals compared with phosphoantigen IPP, and these cells expressed CD16. CD56 expression (a marker for cytotoxic cells) was lower on zoledronate-expanded cells, consistent with significantly lower cytotoxicity against the Daudi tumor cell line. Cells expanded with either zoledronate or IPP were active in ADCC, were similar in terms of interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression, and degranulated in response to Fc receptor cross-linking.ConclusionsZoledronate causes ex vivo expansion of Vδ2 cells from HIV⁺ individuals. Despite lower expression of CD56 and decreased direct cytotoxicity, these effectors were potent in ADCC. Zoledronate/IL-2- expanded cells have potential for immunotherapy to activate Vδ2 cells in HIV patients and enhance ADCC.     

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

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