The use of Salmonella schottmulleri for mapping and separation of human lymphocyte subpopulations.

Human lymphocyte subpopulations (B cells, B1, B2, T1, T2, T3, and T4 cells; our denomination) have been previously identified and isolated by bacterial adherence and functional differences between them have been demonstrated. Here we examined the binding properties of Salmonella schottmulleri to human lymphocytes in peripheral blood smears and found that it binds to more lymphocyte subpopulations, namely B, T1, T2 and T3 cells, than any bacteria previously tested. Thus, using only four bacteria: Salmonella schottmulleri, Brucella melitensis, Arizona hinshawii and Bacillus globigii we identified in blood smears B cells, two B and four T cell subpopulations. When we used gelatin-coupled monolayers of Sal. schottmulleri to isolate lymphocyte subpopulations, we showed that the nonadherent (T4) cells could be efficiently separated from the adherent cells. Furthermore, we tested the isolated subpopulations for natural killing (NK) activity and for antibody-dependent cell-mediated cytotoxicity (ADCC). Using both NK and ADCC assays, we observed a significantly higher cytotoxic activity in the nonadherent cell population than in the unseparated or adherent cell populations. Also the nonadherent cells contained most of the lymphocytes that have receptors for the Fc portion of IgG and those cells described as large granular lymphocytes. We concluded that Sal. schottmulleri is a valuable new reagent for the identification and separation of human lymphocyte subpopulations.

Identification and separation by bacterial adherence of human lymphocytes that suppress natural cytotoxicity.

Human lymphocyte subpopulations (B1, B2T1, T2T3, and T4, our denomination) have been previously identified by bacterial adherence, and differences in functions (mitogen responses, specific cytotoxicity, and natural killing activity) have been associated with some of these subpopulations. The natural killing activity (NK) was located in the T4 lymphocyte subpopulation. Here we investigated the possibility that lymphocytes capable of suppressing the NK activity of the T4 cells could be identified and isolated from one of the other lymphocyte subpopulations. Freshly isolated, monocyte-depleted human peripheral blood lymphocyte (PBL) were separated into adherent and nonadherent cells after centrifugation against various bacterial monolayers. The PBL and the resulting subpopulations of PBL were tested as effector cells in a 4-hr cytotoxicity assay against the CEM lymphoblastoid cell line. The addition of viable T2 lymphocytes to either PBL or T4 lymphocytes resulted in a significant decrease in NK activity, whereas no decrease was seen when T1, T1T3, or killed T1T2 cells were added. This decrease in NK activity was not due to a simple dilution of the active NK cells, to alteration of the lymphocytes by their processing on the bacterial monolayers, or to a competition for binding to the target cells. We concluded that the T2 lymphocyte subpopulation contains the cells capable of suppressing the ability of normal human peripheral blood lymphocytes (T4 subpopulation) to perform natural killing.

The measurement of leukocyte subsets in the peripheral blood of cancer patients with solid tumors using monoclonal antibody reagents.

Monocyte and lymphocyte subsets were quantitated in the peripheral blood of normal subjects and patients with solid tumors using the monoclonal antibody reagents OKM1, BRL63D3, OKT3, OKT4, and OKT8. Percentages and numbers of cells reacting with monoclonal reagents were analyzed by indirect immunofluorescence. Correlations between leukocyte subset values and stage of disease or immunocompetence were sought. No differences from normal were seen in the percentage of OKT3, OKT4, and OKT8 cells or in the ratios of OKT4/OKT8 cells in peripheral blood mononuclear cells (PBMC) from all cancer patients, patients with localized disease, or patients with advanced disease. A significant decrease in absolute numbers of lymphocytes, OKT3 cells, OKT4 cells, and OKT8 cells was seen in the peripheral blood of patients with advanced disease reflecting the absolute lymphopenia of these patients. A significant increase was seen in the percentages of PBMC reacting with OKM1 and BRL63D3 from patients with advanced disease compared with normal donors or localized disease patients. A positive correlation was found between PHA responsiveness and absolute numbers of OKT3 and OKT4 cells. A negative correlation was found between PHA responsiveness and percentages of OKM1 cells. These data indicate that malignant disease does not alter T cell subset percentages in patient peripheral blood but may decrease their absolute numbers in association with absolute lymphopenia. On the other hand, percentages of OKM1 and BRL63D3 cells are increased in patients with advanced solid tumors in association with impaired PHA responsiveness.

The use of bacteria as markers of leukemic lymphocytes and for the isolation of natural killer cells.

Human lymphocyte subpopulations as well as leukemic lymphocytes can be identified and enumerated in blood smears by using bacteria that bind spontaneously to lymphocytes or by using bacteria to which antibodies are chemically coupled. The mechanism of natural binding of bacteria to lymphocytes was shown to involve a lectin on the lymphocyte surface and a carbohydrate on the bacteria. Also, we found that natural killer (NK) cells can be separated by negative selection using monolayers of bacteria. A subpopulation of T cells, identified by their binding of B. globigii, was shown to be suppressors for NK cells.