When the effector cells were treated with match alone prior to being added to cytotoxicity assays, only a slight loss of lytic activity was observed

When the effector cells were treated with match alone prior to being added to cytotoxicity assays, only a slight loss of lytic activity was observed. of HIV-seropositive children, were able to mediate cell lysis when serum from an HIV-seropositive adult was added. However, effector cells from these HIV-infected children were able to mediate both ADCC against HSV-infected fibroblasts and NK cell-mediated cytotoxicity against IIIB-infected HUT78 cells. Reduced ability of PBMC from vertically HIV-infected children to mediate ADCC against HIV antigen-expressing CD4 cells may contribute to quick progression to AIDS. CD4 lymphocytes are known to be targets for human immunodeficiency computer virus (HIV) contamination in vivo. Therefore, the use of HIV antigen-expressing CD4 lymphocytes as target cells in cytotoxicity assays might yield data closely reflecting in vivo events. Previous reports indicated that peripheral blood mononuclear cells (PBMC) of HIV-infected adults are able to lyse CD4 lymphocytes expressing gp120, the major envelope glycoprotein of HIV type 1 (HIV-1) (55,57,62). The effector cells responsible for this lysis were shown to be CD16+natural killer (NK) cells, armed in vivo with cytophilic HIV-specific antibodies. Therefore, this mechanism of cytotoxicity can be classified as antibody-dependent cellular cytotoxicity (ADCC). To explore possible mechanisms for accelerated disease progression in some perinatally HIV-infected children compared to that in adults (2,4,49), we analyzed ADCC Compound 56 against HIV-1-expressing CD4 lymphocytes in children at various stages of HIV contamination. Responses were compared to those of HIV-infected adults and HIV-seronegative age-matched controls. == MATERIALS AND METHODS == == Subjects. == Subjects consisted of HIV-infected adults, monitored at the Hospital of the University or college of Pennsylvania; children with perinatal HIV contamination, monitored in the Special Immunology Clinic at The Childrens Hospital of Philadelphia; and age-matched HIV-seronegative healthy volunteers. HIV contamination was diagnosed on the basis of at least two positive PCRs and PBMC cultures for HIV. According to Centers for Disease Control and Prevention (CDC) criteria for children and adults (8,9), HIV-infected children were classified as asymptomatic with normal (P1-A) or abnormal (P1-B) immune function or as symptomatic with nonspecific findings (P2-A) or HIV-related conditions (P2-B-F), whereas adults stages were classified as asymptomatic (CDC stage A), symptomatic conditions (stage B), or AIDS-defining conditions (stage C). Patients receiving intravenous immunoglobulin were excluded from the study, since repeated administration of intravenous immunoglobulin may lead to reduced NK cell-mediated cytotoxicity (11) and might affect the ability of PBMC to mediate ADCC. This study was approved by the Institutional Review Boards of the University or college of Pennsylvania and The Childrens Hospital of Philadelphia. == Effector cells. == PBMC were separated from heparinized venous blood by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) gradient centrifugation. Monocytes were removed by adherence on plastic surfaces coated with fetal bovine serum (FBS; HyClone, Logan, Utah) as previously explained (23). PBMC were used in cytotoxicity assays within 4 h Compound 56 after the blood drawing. Experiments in which NK cells were depleted from PBMC by incubation with monoclonal antibody anti-Leu 11B (Becton Dickinson, Mountain View, Calif.), which reacts with the FcIII receptor (CD16) on NK cells, as previously described (3,39) followed by incubation with baby rabbit match (Cedarlane Laboratories, Hornby, Ontario, Canada) to destroy antibody-bound cells were performed. The surviving PBMC were used as effector cells in cytotoxicity assays. Arming of effector cells was accomplished by incubating PBMC for 12 h at 37C with undiluted heat-inactivated heterologous sera obtained from HIV-infected patients and seronegative controls (58). The cells were washed five occasions before use as effector cells in cytotoxicity assays. To elute putative cytophilic antibodies, freshly isolated PBMC were incubated at 37C for 12 h and then washed three times (57). == Target cells. == HUT78 cells, derived from a CD4+lymphoblastoid T-cell collection, uninfected and chronically infected with the HIV-1 strain IIIB (16), were kindly provided Compound 56 by J. A. Hoxie, Hospital of the Compound 56 University or college of Pennsylvania, Philadelphia. K562 cells, derived from an erythroleukemia cell collection and known to be sensitive to NK cell-mediated cytotoxicity, were used as target SERPINA3 cells in NK cell assays. FS4 cells, human embryonic foreskin fibroblasts (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), were inoculated with the NS strain of herpes simplex virus type 1 (HSV-1) (kindly provided by H. M. Friedman, Hospital of the University or college of Pennsylvania) at a multiplicity of contamination of 5.0, as previously described (37). After 6 h of incubation at 37C in 5% CO2, the Compound 56 cells were trypsinized, washed, and then stored in the vapor phase.