Comparison of the relative cytotoxic effector cell capabilities and the proportions of cells bearing various surface markers in human tonsil and peripheral blood mononuclear cells

The relative cytotoxic effector cell capabilities and the proportions of cells bearing various surface markers in human tonsil and peripheral blood mononuclear cells has been studied. The peripheral blood contained a substantial proportion of monocytes (22 +/- 2.9%) compared to tonsil cell suspensions (2.5 +/- 0.3%). The percentages of T lymphocytes was significantly higher in the blood than in the tonsil (P is less than 0.01); however, the percentages of cells forming rosettes with 7S EA were not significantly different in each group (P greater than 0.5). Mitogen-induced cellular cytotoxicity by blood and tonsil mononuclear cells against Chang cells was proportional to the percentages of T lymphocytes in these cell suspensions, and both antibody-dependent and mitogen-induced cellular cytoxicity against sheep red blood cells was proportional to the percentages of monocytes in these suspensions. Tonsil mononuclear cell suspensions were incapable of mediating antibody-dependent cellular cytotoxicity against Chang cells, whereas blood mononuclear cells functioned normally. These findings are in contrast to the findings of similar percentages of Fc receptor-positive lymphocytes in blood and tonsil mononuclear cell suspensions. Previous studies have shown that the effector cells against antibody-coated Chang cells are Fc receptor-positive lymphocytes. These studies show that in the case of cytotoxicity mediated by an Fc receptor-bearing lymphoid cell, there may be a clear discrepancy between the relative proportions of Fc-bearing lymphoid cells in different organs and the relative levels of cytotoxicity.     

Natural killer cells in guinea pig spleen bear Fc receptors

The phenomenon of natural cytotoxicity or spontaneous cell-mediated cytotoxicity (SCMC) was investigated in guinea pigs and compared with two other in vitro cytotoxicity reactions: mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC). The same xenogeneic target cells were employed in all three cytotoxicity assays. Organ distribution studies revealed that SCMC effector cell activity was present in spleen and peripheral blood but not in thymocytes. Bone marrow cells possessed low levels of SCMC effector cell activity. The organ distribution of effector cell activities for MICC and ADCC paralleled that for SCMC. Studies of the cell surface characteristics of the SCMC effector cell revealed that spleen cells nonadherent to antigen-antibody but not to antigen-F(ab')₂ antibody-coated surfaces possessed markedly reduced SCMC effector cell activity. In addition, spleen cells depleted of Fc receptor-bearing cells by EA rosetting also possessed diminished SCMC effector cell activity, while cell populations enriched in Fc receptorbearing cells by EA resetting possessed enhanced SCMC effector cell activity. These fractionation techniques had similar effects on MICC and ADCC effector cell activity. Depletion of adherent spleen cells including macrophages by nylon wool column passage resulted in a population of cells with enhanced SCMC, MICC, and ADCC effector cell activity. Thus, in guinea pig spleen the effector cells mediating SCMC were shown to belong to a population of nonadherent Fc receptor-bearing lymphocytes possessing several cytotoxic capabilities including the potential of mediating MICC and ADCC.     

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.     

Langerhans cell-containing epidermal cells are not cytotoxic in antibody-Dependent - and mitogen-induced - cellular cytotoxicity

Langerhans cells (LC) constitute a small (2–6 %) subpopulation of mammalian epidermal cells (EC) and are the only EC that bear Fc-IgG receptors and that share several other cell-surface characteristics and functional capabilities with cells of the monocyte-macrophage series. Since Fc-IgG receptor-positive macrophages are potent effector cells in antibody-dependent cellular cytotoxicity (ADCC), we determined whether LC-containing EC populations are also able to mediate ADCC.In the ADCC assay employed, BALB/c LC-containing EC at different effector/target cell ratios consistently failed to lyse bovine red blood cell (BRBC) targets which were either trinitrophenyl(TNP)-modified or not modified and reacted with an appropriate antibody: a purified rabbit IgG anti-BRBC, or a mouse or rabbit anti-TNP antiserum. In addition, LCcontaining EC did not mediate mitogen-induced cellular cytotoxicity using purified phytohemagglutinin. BALB/c spleen cells, used as a positive control, gave substantial specific release in all experiments performed.     

Effect of morphine and beta-endorphin on human Fc receptor-dependent and natural killer cell functions.

Interactions between opiates and the human immune system have important clinical implications. To further evaluate these interactions, we studied in vitro and in vivo effects of morphine sulfate (morphine) and beta-endorphin (Bend) on antibody-dependent cell cytotoxicity (ADCC), natural killer cell cytotoxicity (NKCC), and effector cell expression of antibody Fc receptors. Morphine and Bend had no potent in vivo or in vitro effect on FcR expression nor did they have a significant in vitro effect on ADCC by monocytes or polymorphonuclear cells. Bend enhancement of NKCC in vitro was inhibited by coincubation of effector cells with morphine. After taking 90 to 150 mg of oral morphine, study volunteers demonstrated a significant decrease in ADCC by peripheral blood mononuclear cells. The same individuals demonstrated a consistent increase in NKCC and no change in the expression of Fc receptors. Effector cells from these individuals responded normally to in vitro incubation with interferon-gamma (IFN-gamma).     

Contribution of lymphocytes bearing Fcgamma receptors to PHA-induced cytotoxicity.

Lymphocytes participating in PHA-induced lysis of chicken erythrocytes were characterized by means of cell fractionation methods. Selective depletion of, or enrichment in, E-rosetting cells indicated that the effector cell population was heterogenous, consisting of both T and non-T lymphocytes. Most effector cells, however, were shown to bear Fcgamma receptors detected by the formation of erythrocyte-antibody (EA) rosettes, but to lack C3 receptors. This distribution of effector cells among tonsils, peripheral blood and thoracic duct lymph paralleled that of EA-rosette forming cells but not that of T or B cells. Addition of aggregated IgG resulted in a moderate decrease of PHA cytotoxicity. However, almost complete inhibition was achieved within a few hours of contact between effectors cells and insoluble immune complexes. The results support the hypothesis that cytotoxic capacity is associated with the presence of Fcgamma receptors on the cell surface.     

Evidence of ''K''-Cell Killing by Alloactivated, Fc-Receptor-Bearing Cytotoxic T Lymphocytes

Upon in vivo alloactivation of Ig-anti-Ig column-purified splenic 'T' cells in lethally irradiated allogeneic recipients, a variable proportion of donor-derived cytotoxic T lymphocytes (CTLs) are able to bind IgG antibody-coated erythrocytes through surface Fc receptors (FcR) and form rosettes. The use of fractionation procedures based on the ability of these cells to form rosettes has enabled us to separate FcR-positive CTLs from FcR-negative CTLs and to examine the ability of these two cell populations to perform as effector cells in direct T-cell-mediated killing and in antibody-dependent cellular cytotoxicity. A series of experiments, either by direct isolation of the two cell populations or by deletion of the FcR-positive population by filtration through complexed immunoglobulin columns (Ig-anti-Ig), has shown both populations to be efficient in direct T-cell mediated cytotoxicity against the relevant target cell. The striking difference between the two populations is the exclusive ability of the FcR-positive population to function as effector cells in antibody-dependent cellular cytotoxicity (ADCC). Purification steps before in vivo alloactivation of our responding cells for the removal of 'B' cells and FcR-bearing cells with 'K'-cell activity, followed by procedures to remove phagocytic and adherent cells in the resulting immune spleen cell preparation and, finally, b y velocity sedimentation of the rosetting and nonrosetting blasts from the small lymphocyte population, has resulted in a population of FcR-positive cells 98% positive for the Thy 1.2 alloantigen. These fractionation steps and immunofluorescence criteria of purity strongly favor the contention that the ADCC activity within the FcR-positive T-cell population is indeed a property of the CTL itself.     

人IFN-α对人外周血单个核细胞ADCC活性的调节

本文以鸡红细胞为靶细胞,采用~(51)Cr释放法,检测正常成年人外周血单个核细胞的ADCC活性,并研究干扰素对这一活性的调节作用。发现Namalva细胞干扰素对正常成年人ADCC 活性有明显促进作用,其促进幅度依赖于所用干扰素的剂量和处理时间,抗人 IFN-α可抑制该促进作用。人白细胞干扰素亦能明显促进正常人ADCC活性,而鼠干扰素对人 ADCC活性则无明显促进作用。     

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

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.     

The Role of Viral Glycoproteins in Mumps Virus-Mediated Antibody-Dependent Cellular Cytotoxity in Vitro

Treatment of human peripheral blood lymphocytes with live or UV-inactivated mumps virions enhances antibody-mediated cellular cytotoxicity (ADCC), reflected by increased target cell lysis in a 51Cr-release assay or an increased number of plaque-forming cells on monolayers of bovine erythrocytes (Eb) in the presence of anti-Eb antibodies. Virus treatment of the Eb targets causes a similar enhancement. The role of viral glycoproteins in ADCC enhancement was investigated by using a panel of monoclonal antibodies raised in mice against mumps virions. Most of the lymphocytes bound mumps virions, as ascertained by indirect immunofluorescence. A high proportion of virus-treated lymphocytes also formed rosettes with Eb. Anti-HN antibodies inhibited rosetting to various degrees. Although antibodies with high haemagglutination inhibition titres were most efficient inhibitors, antibodies without this serological activity were also inhibitory. Anti-F antibodies were only weakly inhibitory, and anti-NP antibodies had no effect. Anti-HN antibodies also abrogated target cell lysis in the 51Cr-release assay and effector cell recruitment in the ADCC plaque assay by inhibiting virus-mediated Eb-lymphocyte interactions both at the target cell and at the effector cell level. Anti-F or anti-NP antibodies were only weakly or not at all inhibitory. The results suggest that virus-mediated enhancement of ADCC is caused by the HN glycoprotein, primarily (although perhaps not exclusively) by its improvement of the effector cell-target cell contacts necessary for the efficient execution of target cell lysis.     

Distribution of Cells Binding Erythrocyte-Antibody (EA) Complexes in Human Lymphoid Populations

Cells bearing Fc receptors were studied using a rosette technique with chicken erythrocytes coated with rabbit antibodies (EA). By comparison with other markers and by selective depletion of T and B cells it was demonstrated that EA rosettes were formed by about 10% of the monocytes, 7% of the T lymphocytes, and 45% of the B cells. In addition, 26% of EA-rosette-forming cells (EA-RFC) in the peripheral blood carried on other markers than C3 receptors. Marked differences were found in the percentages of EA-RFC in cell suspensions from various lymphoid organs: peripheral blood, 15.2%; spleen, 26.6%; bone marrow, 30.9%; lymph nodes, 2.8%; thymus, 0.3%; and tonsils, 0.5%. It was concluded that Fc receptors were present on subsets of different lymphocyte subpopulations; their presence could not be regarded as a specific marker of one of these subpopulations.     

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.     

Identity of effector cells participating in the reverse antibody-dependent cell-mediated cytotoxicity.

Our previous work has shown that antibody-coated mouse spleen cells express enhanced cytotoxic activity against some Fc-receptor-bearing target tumour cells by a mechanism which appears to be similar to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction with reversed polarity of the antibody bridge (R-ADCC). In this report we have shown that (i) the levels of basal natural killer (NK), ADCC and R-ADCC cytotoxic activities in mouse spleen cells are strongly correlated with each other, (b) simultaneous induction of ADCC and R-ADCC reactions does not result in an additive cytotoxic response, and (iii) YAC cells which do not bear Fc receptors and are highly sensitive to lysis by NK cells, can specifically and competitively inhibit the ADCC and R-ADCC reactions. These results suggest that the R-ADCC reaction may be mediated by the same effector cell population as mediates NK and ADCC reactions against tumour target cells.     

Which Fc receptor-bearing cells are detected with the Ripley assay?

Mononuclear cells were isolated from peripheral blood and analysed with T and B markers (E rosettes and SIg) and on contaminating monocytes and PMN. The suspensions contained 63.3 +/- 4.8% T lymphocytes, 11.4 +/- 3.8% B lymphocytes, 9.0 +/- 5.5% null lymphocytes, 15.1 +/- 3.8% monocytes and 1.1 +/- 0.6% PMN. Of all cells, 27.6 +/- 12.1% formed EA rosettes with OR1R2 red cells coated with anti-CD Ripley. In preparations fixed after cytocentrifugation, the EA rosette-forming cells were studied with regard to esterase activity. The proportion of cells with detectable Fc receptors was further studied in purified T lymphocyte and in monocyte suspensions. Finally, EA rosettes were isolated by gradient centrifugation and the rosette forming cells studied with conventional T and B markers. All procedures gave corresponding results: on average 11-14% of the T lymphocytes and nearly 100% of the null cells formed EA rosettes, while only 2% of the B lymphocytes had detectable Fc receptors. Of the purified monocyte and PMN populations, on average 72 +/- 10.5 and 14.5 +/- 5.4%, respectively, formed EA rosettes. Thus, the Ripley assay, representing an important marker for null lymphocytes, cannot be regarded as specific for this population of white blood cells.     

Some parameters of the erythrocyte-antibody-complement (EAC) rosette system for recognition of bovine B-cells

A population of bovine lymphocytes formed EAC rosettes with sheep RBCs sensitized with rabbit antiserum and several species of complement. Mouse complement gave best results. Horse anti-sheep RBC serum and bovine anti-rabbit RBC serum did not support rosette formation. Optimum working concentrations of antiserum (1 : 400) and complement (1 : 20) were determined by ‘checkerboard’ titration. The occurrence of E (direct) rosettes, EA rosettes (using RBCs sensitized with rabbit antiserum but no complement), EAC rosettes and cells carrying surface immunoglobulin (sIg) (observed by a direct fluorescent antibody test) in blood and organs was observed. In a series of 33 blood samples from animals 1 week to 26 months of age the mean occurrence of these markers was: E (tested in foetal calf serum) = 2.9%; E (in CF buffer with 0.1% gelatin) = 4.3%; EA = 19.4%; EAC = 29.2%; sIg = 27.7%. The distribution of these markers in lymphoid organs of four calves and two adult heifers was examined. E rosettes occurred mostly among thymus and lymph node lymphocytes, while blood, lymph nodes and spleen contained many cells forming EA and EAC rosettes and carrying sIg. Only a few bone marrow cells carried surface markers. Cells from peripheral blood of two heifers and spleen and lymph nodes from a 3 months old calf were fractionated on nylon wool columns and contaminating macrophages and platelets were removed by treatment with carbonyl iron and adenosine diphosphate. These treatments showed that EAC and sIg cells usually occurred in the same population (presumably B-cells) and E rosette-forming cells occurred in a second, distinct population (T-cells). EA rosettes were not confirmed to B- or T-cells and their occurrence varied between organs. It was concluded that in general the EAC rosette and sIg are markers for B-cells but that probably many cell types can form EA rosettes.     

Characteristics of complexes for arming and inhibiting effector cells for antibody-dependent cell-mediated cytotoxicity.

The size of IgG aggregate effective in the inhibition or arming of human effector cells for antibody-dependent cell-mediated cytotoxicity (ADCC) was investigated using heat- and alkalipolymerized rabbit IgG or purified antibody fractionated by gel filtration. In contrast to the inhibition of ADCC against chicken erythrocytes, which was marked when effector cell were pre-incubated with high molecular weight aggregates (19S or greater), small polymers were most effective in arming for cytotoxicity against antigen-coated chicken red cells. Our data also demonstrate that while the cytotoxic potential of armed cells is short-lived and rapidly lost during culture at 37 degrees C but not 4 degrees C, the reduced capacity of these cells to kill antibody-coated targets is not altered by similar incubation at 37 degrees C. The differences in the size of aggregate active in arming and inhibition, and the stability of the two phenomena are compatible with the hypothesis that large aggregates may cause more cross-linking and redistribution of effector cell Fc receptors than small polymers of IgG.     

Enhancement of human ADCC with interferon.

We have studied the ability of a purified preparation of leucocyte interferon (IF) to augment human antibody-dependent cell-mediated cytotoxicity (ADCC). The IF preparation was added to cultures of the ADCC assay consisting of peripheral blood mononuclear target cells, a sensitizing human antiserum specific for the HLA antigens expressed on the target cells, and effector cells obtained from the peripheral blood of healthy human subjects. Increased cytolysis was found at each of four effector to target cell ratios but could be demonstrated only at concentrations of the sensitizing antiserum of 2.5% and lower. Removal of monocytes from the effector cell population did not decrease the level of cytotoxicity, and isolated monocytes were unreactive, suggesting that K cells (the subpopulation of lymphocytes mediating ADCC) were susceptible to augmentation. Since it is widely accepted that IF can augment spontaneous cytotoxicity mediated by natural killer (NK) cells, our results provide further evidence that human NK and K cells are the same or similar subpopulations or that the mechanisms regulating the activities of these cells are similar. Furthermore, our findings suggest that IF treatment can improve the discriminatory power of the ADCC assay in detecting transfusion or transplantation related immunity.     

Identification of lymphocyte subpopulations in E-RFC-enriched and E-RFC-depleted cell fractions of fresh and cryopreserved lymphocytes.

Fresh lymphocytes and frozen-stored lymphocytes were separated into E-RFC-enriched and E-RFC-depleted cell fractions by density gradient centrifugation of sheep red blood cell (SRBC) rosette-forming cells (E-RFC), since the ability to form rosettes is primarily a T cell characteristic. Subpopulations of lymphocytes were identified, demonstrating the presence of cell surface markers: T cell specific antigens (T+), receptors for SRBC on T cells (E-RFC), Fc-receptors (FcR) for IgG type antibodies, and surface Ig (sIg). Our results indicate that, although the E-RFC-depleted fraction contains virtually no cells capable of binding SRBC, there is still a considerable proportion of T cells present in that fraction, as detected with the anti-T cell antiserum. Moreover, data are presented to indicate that the E-RFC-enriched fraction does not consist exclusively of T lymphocytes. Since this separation procedure is used frequently for the identification of the nature of effector cells in cell-mediated (CMC) and antibody-dependent cellular cytotoxicity (ADCC) assays, the identification of T cells in purified lymphocyte fractions by means of SRBC rosette formation may lead to a false conclusion as to the nature of the effector cells.     

ADCC analysis with the Coulter counter: a highly sensitive, ultramicro assay suitable for clinical and experimental application

Antibody-dependent cellular cytotoxicity (ADCC) is an in vitro immune mechanism implicated in several in vivo phenomena such as transplant rejection, tumor immunity and parasite elimination. We developed a method for detecting ADCC using the Coulter Counter and the Coulter Channelyzer that circumvents many of the disadvantages associated with existing assays for ADCC. Effector mononuclear cells were incubated with chicken red blood cell (CRBC) targets and anti-target antibody for 1-1, 5h. Killing was quantified by the Coulter Counter on the basis of size differences between effector and target cell nuclei. Using a 4 microliter total volume we were able to detect cytotoxic levels of 55% when as few as 5000 effector cells were incubated with an equal number of target cells. This method for the detection of ADCC may be suitable for clinical and research application.     

Immunological responsiveness of frozen-thawed human lymphocytes.

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells.