Steven L. Shore

E-rosette receptors induced by phytohemagglutinin on human K cells expressing T-cell surface antigens

Abstract Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity: 1. General requirements of the reaction and temporal relationship between lethal hits and cytolysis

Abstract The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a 51 Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of 51 Cr release with an initial lag phase of at least 10 min, whereas 51 Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by 51 Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to 51 Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells. The cytotoxic reaction was completely inhibited by 10 m M EDTA and did not occur in a Ca 2+ - and Mg 2+ -free medium. Neither Ca 2+ nor Mg 2+ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca 2+ - and Mg 2+ -dependent events in the lytic pathway.

Lysis of antibody-coated human red cells by peripheral blood mononuclear cells: Altered effector cell profile after treatment of target cells with enzymes

Abstract Type O Rh positive human red blood cells (HRBC), native or treated with one of three enzymes (papain, trypsin, or neuraminidase), were labeled with 51Cr and then sensitized with anti-Rh immune globulin. These cells served as targets in antibody-dependent cellular cytotoxicity (ADCC) for unfractionated human mononuclear cells (MC), MC depleted of monocytes by adhesion to plastic, and MC enriched for monocytes. Enzyme-treated HRBC were lysed with greater efficiency in ADCC than native HRBC. This was explained by the finding that the enzyme modified HRBC were lysed both by lymphocytes and monocytes, whereas native HRBC were lysed only by monocytes. The lysis of native HRBC was strongly inhibited by small amounts of human serum or free IgG. In contrast, the lysis of enzyme-treated HRBC was considerably more resistant to inhibition by human serum or free IgG. The enhanced lysis of enzyme-treated HRBC could not be the result of increased binding of antibody to the target cells, since augmented lysis was observed both for HRBC sensitized before neuraminidase treatment as well as for HRBC sensitized after neuraminidase treatment. These results suggest that the surface charge on target cells plays a critical role in determining which classes of leukocytic effector cells are active in ADCC systems.

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity: II. Reversibility of the heat inactivation of K cell killing and relationship of Fc receptor capping to lysis

Abstract The heat inactivation of human blood mononuclear cells active in antibody-dependent cellular cytotoxicity (ADCC) is largely reversed after 24 hr in culture at 37 °C. The reactivation process is inhibited by actinomycin D, cycloheximide, and emetine but not by mitomycin-C, indicating that recovery requires RNA and protein synthesis but not DNA synthesis. The ability of lymphocytes to cap surface immunoglobulin (SIg) and IgG-Fc receptors (FcR) was also studied. As with ADCC effector cell activity, both SIg and FcR capping were abolished by heating, and the kinetics of inactivation was similar to that of the inactivation of ADCC effector activity. In addition, the heat inactivation of capping was reversible in culture and followed kinetics of reactivation similar to that of K cell reactivation. These results suggest the participation of heat-labile proteins at or near the surface of the effector cell, which are also apparently involved in the capping of surface receptors. Presumably these heat-labile proteins are membrane-associated enzymes, but they may also be cytoskeletal structures such as microfilaments or microtubules whose heat-sensitivity is currently unknown. The mounting of lethal hits may involve the same membrane machinery which is responsible for capping or a capping process itself.

Antibody-Dependent Cell-Mediated Cytotoxicity to Target Cells Infected with Herpes Simplex Viruses

Two major mechanisms have been described by which lymphoid cells damage or destroy target cells bearing foreign antigens on their surface. The first of these mechanisms, cell-mediated cytotoxicity (CMC), is important in allograft rejection and tumor immunity (4) and is carried out by sensitized T lymphocytes from specifically immunized hosts. The second mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), has as yet an undefined role in vivo and is conveyed by the action of mononuclear effector cells on target cells sensitized with antibody to surface membrane antigens (7,15). Unlike CMC, the effector cell in ADCC is found in nonimmunized, as well as immunized hosts. Clearly not a T lymphocyte, in some ADCC systems it appears to be a K cell (3,22), a nonphago-cytic lymphoid cell with an Fc receptor, but no readily detectable surface immunoglobulin. A variety of target cells have been employed in vitro to demonstrate ADCC; these have included xenogeneic (8,14), allogeneic (9,10), and various tumor cells (16), as well as cells passively coated with antigens (2,21). We recently demonstrated for the first time ADCC using viral-infected target cells (17,19).

Antibody-dependent cellular cytotoxicity to virus-infected target cells: Role of nylon wool-adherent T cells as effectors

Abstract This study was designed to elucidate whether populations of human blood lymphocytes other than non-T, non-B, Fc receptor-positive K cells can mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells acutely infected with type 1 herpes simplex virus. With appropriate technical precautions, a subset of E rosette-positive, nylon wool-adherent T cells were found to be effective in killing antibody-coated target cells. Thus, there appears to be at least two relatively distinct populations of lymphocytes in human peripheral blood that function as effectors in ADCC: one that consists of E rosette-negative cells, the other of E rosette-positive cells. These findings suggest a possible relationship between “classical” E rosette-negative K cells and E rosette-positive T cells.

Preformed frozen sucrose gradients—A new laboratory aid

Abstract A useful method for preparing large numbers of virtually identical gradients of sucrose has been described. This technique involves storage of preformed gradients at −60°C for future use after a 90 min thaw at room temperature. The method is applicable to salt-containing sucrose gradients with certain limitations, noted above.

968 LYSIS OF VARICELLA ZOSTER VIRUS (VZV) INFECTED CELLS BY ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC)

Human foreskin fibroblasts acutely infected with VZV were exposed to VZV antibody-positive human serum and human mononuclear cells (MC) in a 6-hr 51Cr release assay. Specific lysis could be detected by 60 min and was proportional to the effector cell:target cell ratio. The reaction was temperature dependent, proceeding optimally at 37°C. Only VZV antibody-positive serum mediated the reaction, and uninfected targets were not lysed. MC alone had little or no lytic activity. Median antibody titers of 1000 were noted in the serum of normal adults without a history of zoster. The antibody was of the IgG class as indicated by its adsorption with S. aureus containing protein A, its presence in high titer in immune serum globulin and zoster immune globulin, and its quantitative placental transfer. A requirement for effector cell IgG-Fc receptors was shown by blocking of ADCC by protein A and inhibition of effector cell activity by heat-aggregated gamma globulin. Lymphocytes were more effective as killer cells than were monocytes or polyorphonuclear leukocytes. ADCC was able to lyse cells earlier in the VZV infectious cycle than antibody-dependent complement-mediated lysis. These results are the first demonstration of ADCC against VZV-infected cells and suggest a mechanism whereby IgG antibody aids in prophylaxis of infection or reduces the severity of exogenous reinfection.

CHARACTERIZATION OF THE EFFECTOR CELL IN AN ANTIBODY DEPENDENT CELL MEDIATED CYTOTOXICITY SYSTEM

Publisher Summary This chapter discusses the characterization of the effector cell in an antibody-dependent cell-mediated cytotoxicity system. A study described in the chapter utilized herpes simplex virus (HSV) type-I-infected Chang liver cells coated with human anti-HSV antibody as targets and human mononuclear cell preparations as effectors. Human peripheral blood mononuclear cells (MC), after Ficoll/Hypaque separation, were further purified by a variety of techniques, including carbonyl iron depletion, plastic adherence, nylon wool adherence, and E rosette depletion, alone and in combination. Evaluation of membrane markers was done at all stages of the separation procedures. Plastic adherence routinely removed 80–90% of the latex positive monocytes from the MC preparations but did not lead to depletion of the ADCC activity, suggesting that the major effector cell was not a monocyte. Carbonyl iron depletion experiments also suggested that the effector cell was not a monocyte, though this technique is even more efficient in removing latex-positive cells.