Bone marrow-derived lymphoid cells (B cells): functional depletion with cobra factor and fresh serum.

Treatment of rat spleen cells with cobra factor and fresh rat serum provided a simple, rapid means of functionally eliminating complement receptor lymphocytes. Cells able to differentiate into plaque-forming cells in a syngeneic, irradiated host were diminished, but cells able to induce a graft-versus-host reaction were not diminished. There was no effect on plaque-forming cells from an immune spleen.

A direct comparison of procedures for the detection of mycoplasma in tissue culture.

Mycoplasma contamination of cell cultures has been shown to perturb a number of immunologic parameters. Because such contamination is almost always introduced in the laboratory, the immunologist requires a procedure to screen his cell lines frequently for mycoplasma. Two procedures recently described for the detection of mycoplasma in cell cultures, the uridine-uracil incorporation procedure and a direct fluorescent assay, were compared with the standard procedures of agar culture and transmission electron microscopy. The results with uridine-uracil incorporation were totally non-concordant with those of any of the other 3 procedures and, moreover, were inconsistent through serial assays on the same cell culture. In contrast, the direct fluorescent assay, using the fluorochrome Hoechst 33258, yielded consistent results in full agreement with the agar culture data. Since the fluorescent assay is rapid and has discriminatory capability at least equivalent to that of agar culture, it would appear to be the method of choice for routine screening of cell cultures for mycoplasma.

Cell-mediated cytotoxicity and serum-mediated blocking: evidence that their associated determinants on human tumor cells are different.

The preceding paper showed that patients with gliomas may have lymphocyte-mediated cytotoxic activity (LMC) directed against at least two determinants on the glioma cell surface. The present study showed that serum from patients with gliomas could block this LMC. The blocking activity, however, was specific for different determinants on the glioma cell than those to which the LMC was directed. Blocking activity was specific for tumor cells homotypic to those of the serum donor. It was effective, however, in blocking the cytotoxic activity against these cells of lymphocytes from patients with tumors either homotypic or heterotypic to that of the serum donor. Likewise, although patients with glioblastomas or melanomas had LMC against fetal glial cells, sera from such patients were unable to block the LMC against these fetal glial targets. The specificity of the blocking activity was confirmed by absorption of the sera with various normal and neoplastic cells. These studies have thus shown an immunologic functional dichotomy among different determinants on the glioma cell surface.

Multiplicity of the tumor-directed immune response. Implications for immunotherapy.

The host may generate several different types of immune response to a tumor. Some of these responses are advantageous to the host; others are not. There is evidence that several tumor-associated antigens, perhaps stimulating different facets of the immune response, are expressed by the tumor cell. We discuss the implications of this concept for immunotherapy.

Radiolabeling of immunoglobulin without loss of antibody activity--use of 14C-phenylisothiocyanate with human cytotoxic antibody.

Radioiodination of immunoglobulins, particularly human cytotoxic IgG, induced a marked loss of antibody activity. Phenylisothiocyanate (PITC) readily reacts with alpha-amino groups and the epsilon-amino groups of lysines to form phenylthiocarbamyl derivatives. Since PITC is commercially available with 14C, 3H, and 35S substitutions, it provided an alternative means for labeling immunoglobulin which preserved antibody activity. There were approximately 80 PITC binding sites per IgG molecule, and 14C-PITC was bound with an efficiency in excess of 80%. With as many as 40 PITC molecules bound per IgG molecule, full cytotoxic activity was retained by both anti-HLA isoantisera and human anti-melanoma autoantisera. Even with 70 to 80 molecules of PITC bound per IgG molecule, over 80% of the antibody activity remained. Radiolabeling of antibody with 3H, 14C, or 35S-substituted PITC will greatly facilitate studies of antibody binding, particularly to cell surface antigens.

A blood test for multiple sclerosis based on the adherence of lymphocytes to measles-infected cells.

The increased capacity of lymphocytes from patients with multiple sclerosis to adhere to human epithelial cells persistently infected with measles virus has provided an accurate blood test for multiple sclerosis. When lymphocytes from affected patients were mixed with measles-infected human epithelial cells, the lymphocytes formed rosettes around a mean (+/-S.E.) of 69.2 +/-1.7% of the measles-infected cells. In contrast, lymphocytes from controls, either healthy or with other neurologic and non-neurologic diseases, formed rosettes around a mean of only 28.2+/-2.1% of the measles-infected cells. Of greater importance was the complete absence of overlap between multiple sclerosis and control values, thus indicating the diagnostic potential of the rosetting phenomenon. The severity, duration and activity of the disease had no effect on the degree of rosette formation.

Spontaneous cytotoxicity against virus-infected cells: relationship to NK against uninfected cell lines and to ADCC.

Cellular immunoadsorption and cold cell competition assays were used to examine the specificity of NK. Lymphocytes nonadherent to monolayers of cells persistently infected with Sendai virus (SV-HEp2) were almost totally depleted of NK against the uninfected tumor cell lines K562, MOLT-4, and HSB, as well as against SV-HEp2, when assayed immediately after separation. Lymphocytes adherent to SV-HEp2 monolayers were also partially depleted of NK against these targets. After overnight incubation, the nonadherent cells remained relatively depleted of NK against all targets, compared to controls. The adherent cells displayed a variable amount of activity, depending on the donor; in all cases, however, substantial NK activity was observed in this fraction against all targets, infected and uninfected. Exogenous interferon failed to augment the activity of the nonadherent lymphocyte fraction. Cellular immunoadsorption on monolayers of K562 or HSB was similarly found to partially deplete NK against SV-HEp2, as well as against the homologous cells. Cold cell inhibition studies showed that SV-HEp2 and, to a lesser extent, HEp2, could inhibit the lysis of K562; similarly, K562 could partially inhibit the lysis of SV-HEp2. We concluded that infected and uninfected target cells are lysed by overlapping populations of NK effector cells. In addition, the ADCC activity of SV-HEp2-adherent and nonadherent lymphocyte fractions was examined immediately after separation. Whereas the nonadherent lymphocytes were depleted of both ADCC and NK, the adherent lymphocytes were partially depleted of NK yet contained high levels of ADCC activity.

Cytogenetic engineering in vivo: restoration of biologic complement activity to C5-deficient mice by intravenous inoculation of hybrid cells.

Splenic macrophages were identified as at least one source of C5 elaboration in normal mice. Hybrid cells were formed from splenic macrophages from C5-deficient mice and either kidney cells from mice with normal amounts of C5 or chicken erythrocytes. These hybrids elaborated C5 in vitro. C5-Deficient mice inoculated with these hybrid cells developed, in their serum, antigenically active mouse C5, as well as both hemolytic and biologic complement activity. These studies demonstrate the feasibility of genetic "repair" in mammals.