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Histocompatibility antigens on the surface of human lymphoblastoid cells were quantified by a microadsorption technique. During the course of measles virus infection, no quantitative or qualitations in surface HLA antigens were observed. In contrast, infection with poliovirus type 1 or vesicular stomatitis virus, or treatment with puromycin (50 microgram/ml) resulted in a significant decrease in surface HLA. These experiments suggest that an inhibition of host protein synthesis rather than the insertion of virus-specificied antigens into the membrane results in a net decrease in amounts of this cell surface antigen. The HLA antigens also appear to be both functionally and structurally distinct from measles virus surface antigens. Pretreatment of cells with HLA-directed antibody did not prevent the infection of these cells by measles virus, thus HLA antigens appear unrelated to the measles virus receptor site on the plasma membrane. Electron microscopic studies revealed that measles virus maturation occurs at membrane sites devoid of demonstrable HLA. Furthermore, HLA antigens could not be detected on the surfaces of mature infectious virions.

Prospective recipients of kidney transplants were tested for lymphocytotoxicity; from these we selected 102 sera that lacked cytotoxic antibodies against peripheral lymphocytes from at least 80 unrelated subjects. To detect humoral sensitization, we then reacted these with 17 cultured human lymphoid cell lines having different HL-A phenotypes. Cytotoxic antibodies reacting with these cultured cells were now detected in some of the sera. These antibodies were not directed against HL-A antigens, yet mediated lysis of target cells in the presence of rabbit but not of human or guinea pig complement. Furthermore, they activated the classical pathway of the rabbit complement system. Later, a significant association was found between occurrence of cytotoxic antibodies and rejection of the transplant. Thus, cultured human lymphoid cells, because of their great susceptibility to complement-mediated lysis, appear to be useful in detecting humoral sensitization in candidates for kidney grafts.

Cultured human lymphoid cells sensitized with human histocompatibility (HL-A) antibodies were able to activate the human complement system in vitro. Some HL-A alloantisera selectively activated the alternate complement pathway while other antisera activated only the classical pathway. A third group of alloantisera was equally able to initiate complement action by way of either pathway. The mechanism of complement activation did not correlate with the HL-A antigen present on the cells or the HL-A specificity of the alloantisera, indicating that the antigenic determinants or distribution on the cell surface play on direct role in selecting the pathway of activation. In this completely homologous system the alternate pathway was found to have the same cytolytic potential as the classical pathway. Thus, an altered or damaged membrane is not a prerequisite for the production of cytolytic damage by the alternate pathway. A complete understanding of the mechanism of interaction of membrane bound antigens and antibodies with the complement system may provide a versatile tool for the investigation of membrane antigen expression.

Immunochemical studies revealed the presence of the fourth component of complement (C4) on surfaces of human lymphoid cells. Antiserums to C4 inhibited the mixed lymphocyte reaction and the mitogenic response to phytohemagglutinin, suggesting a role for membrane-associated C4 in the afferent phase of immune recognition phenomena.

Studies of human peripheral lymphocytes (HPL) from healthy subjects and of human lymphoblastoid cell lines (HLC) revealed spontaneous formation of rosettes with Macaca speciosa monkey red blood cells (MRBC). The percentage of HPL forming rosettes with MRBC paralleled the percentage of HPL exhibiting markers ascribed to human B cells. The MRBC-rosetting cells were positive for membrane-bound immunoglobulin (MBIg). Furthermore, by employing a mixed rosette method, lymphocytes carrying both EAC3dmo and FITC-MRBC were encountered. In contrast, no lymphocytes carrying both SRBC and FITC-MRBC were observed. Ninety-six per cent of purified B cells obtained at the interface after interaction of HPL with SRBC and Ficoll-Isopaque centrifugation formed rosettes with MRBC. In contrast, only 8% of an enriched population of T cells obtained at the interface after interaction with EAC3dmo and Ficoll-Isopaque centrifugation formed MRBC-rosettes. Incubation of HPL with MRBC and subsequent centrifugation on Ficoll-Isopaque resulted in a highly enriched T cell population at the interface. Several lymphoblastoid cell lines with B cell characteristics, but not two cell lines with T cell characteristics, formed spontaneous rosettes with MRBC. Inhibition experiments showed that the structure on the lymphoid cell surfaces mediating the adherence of MRBC was independent of the MBIg as well as the recptors for IgGFc, C3b, and C3d. It appears that rosetting with MRBC will provide a new detection marker for human cells classified as B type and a technique for obtaining an enriched T cell population.

In patients with different types of solid tumors who had no detectable metastasis and had received no radio- and chemotherapy the levels of Ia-like antigen bearing lymphocytes was determined by indirect immunofluorescence staining of peripheral lymphocytes with xenoantisera to framework determinants of Ia-like antigens. The level of Ia-like antigen bearing lymphocytes was increased in patients with urinary bladder carcinoma, was decreased in patients with carcinoma of the gastrointestinal tract and was within normal ranges in patients with melanoma, carcinomas of the breast, lung and genital tract. In patients with carcinomas of the gastrointestinal tract as well as in patients with carcinoma of lung and genital tract the levels of T lymphocytes measured by rosetting with sheep red blood cells, was reduced while it was not different from normal in other groups of patients. Determination of the level of lymphocyte subpopulations may provide information about the immunocompetence status of patients with solid tumors.

Four out of 8 thrombocytopenic patients without detectable cytotoxic antibodies to human peripheral lymphocytes contained cytotoxic antibodies to cultured human lymphoid cells. The presence of such antibodies was associated with reduced survival of infused allogeneic platelets in these patients. The antibodies reacted in a distinct fashion with a panel of cultured human lymphoid cells and are directed to B cell antigens. Cytotoxic antibodies to cultured human lymphoid cells did not react in vitro with platelets suggesting that the antibodies play no significant role in the accelerated destruction of infused allogeneic platelets, although their presence predicts it. Therefore, screening of sera with cultured human lymphoid cells appears to be a useful test in addition to those now used to select patients for transfusion of allogeneic platelets. Sera from polytransfused patients without cytotoxic antibodies to peripheral lymphocytes may be a useful source of antibodies to B cell antigens.

Based on reports that both human and murine lymphoblastoid cell lines can effect lysis in antibody-dependent (ADCC) and mitogen-induced (MICC) cell-mediated cytotoxicity, 10 human lymphoblastoid cell lines were investigated for their ability to act as effector cells in these lytic reactions. Four cell lines promoted MICC and ADCC of chicken erythrocytes. MICC was inhibited by subjecting the effector cells to gamma-irradiation, and ADCC was partially inhibited by treating antibody-coated target cells with Protein A from Staphylococcus aureus. The latter finding suggests that antibody plays a role in the cytotoxic reaction. The lytic activity of cultured human lymphoid cells in both MICC and ADCC reactions was, however, quite variable for unknown reasons. Because of this instability human lymphoblastoid cell lines are not a suitable source of effector cells for studying the biological and molecular properties of structures responsible for the lytic reaction.

The interaction of histocompatibility (HL-A) antibodies and complement with synchronized human lymphoid cells in continuous culture has been investigated. The sensitivity of cultured lymphoid cells to HL-A antibody-mediated lysis in the cytotoxic test, the extent of activation of the complement system, the degree to which labeled complement components are bound, and the ability of these cells to absorb HL-A alloantibodies do not vary significantly during the cell growth cycle. The constancy of histocompatibility antigen expression throughout the growth cycle of cultured cells suggests that these cell surface markers are an essential part of membrane cytoarchitecture and could well play a critical role in determining the normal function of the cell membrane.