A sensitive method for the detection of beta-galactosidase in transfected mammalian cells.

A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.

Stable indicator cell lines exhibiting HIV-1 tat function.

Since HIV tat function is essential for the HIV infectious cycle, it represents an important possible target of therapeutic intervention for HIV infection. Stable human cell lines were derived that express high levels of beta-galactosidase under the combined control of the transacting HIV-1 tat gene product and the cis-acting HIV-1 LTR. The tat gene product induces LTR-linked gene expression approximately 1000-fold in this system. The high level of expression of beta-galactosidase under HIV tat and LTR control in stable cell lines allows rapid spectrophotometric quantitation of beta-galactosidase enzymatic activity from fewer than 5000 cells seeded in a microtiter plate well. Such cell lines provide a virus-free system for the high-capacity screening of compounds for the ability to interfere with HIV tat-mediated transactivation of gene expression.

Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity.

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

Semiautomated colorimetric assay for in vitro screening of anticancer compounds.

An in vitro tetrazolium dye (MTT) reduction technique was modified and evaluated for use in the large-scale screening of anticancer compounds by examining the activity of ten clinically used drugs against 16 different human and murine cell populations. Cell populations included colon and mammary adenocarcinomas, melanomas, leukemias, and freshly isolated normal cells. Cell lines were grown in microtiter plates for 18-20 hours prior to a 72-hour continuous exposure to the drugs. Cultures were initiated at cell densities which maximized both the difference in dye reduction and the number of cell doublings between the beginning and end of the drug exposure period. Drug potency, expressed as the 50% inhibitory concentration (IC50), was comparable whether the effect on cell doublings or dye reduction was determined. There was good agreement between this method and the more labor-intensive, conventional method of counting trypan blue dye-excluding cells in a hemacytometer. Implemented as a large-scale, high-capacity system, our adaptation of the MTT technique is a rapid, sensitive, reproducible first-line screening device for detecting anticancer compounds with cytostatic or cytocidal activity.

Cytotoxic activity of normal and Herpesvirus saimiri-transformed nonhuman primate cells.

Owl monkey mononuclear cells were separated from peripheral blood by centrifugation on Ficoll gradients, removal of adherent cells, and subsequent separation on discontinuous Percoll gradients. Lymphocytes recovered from the various fractions were tested for cytotoxic reactivity immediately after isolation. Low-density cells, enriched in large granular lymphocytes (LGL), demonstrated cytotoxic activity against the human natural killer-susceptible cell lines MOLT 4 and K562. In addition, IL-2-independent T-cell lines which had been obtained by immortalization with the primate herpesvirus Herpesvirus saimiri showed cytotoxicity, even after prolonged culture in vitro, similar to that demonstrated by fresh LGL. Cytotoxic activity of these lines was regulated by IL-2 in a fashion which appeared to be independent of the growth-promoting effects of this lymphokine. These results indicate a function for IL-2 beyond its role in supporting cellular proliferation. Cytotoxic activity could also be demonstrated in culture fluids from one of these cell lines (70N2). In addition, these results indicate the usefulness of immortalized cell lines (like 70N2) as a potential source for studies of the biochemical characterization and purification of supernatants containing cytotoxic factors.

Stimulation of in vitro immunoglobulin production by interferon-alpha.

The effect of various natural and recombinant DNA-derived human interferon-alpha (IFN-alpha) on immunoglobulin (Ig) production by human B cells was investigated. The cell populations examined included peripheral blood mononuclear cells (PBMC) and highly purified B cell and helper T cell populations obtained by negative selection by using monoclonal antibodies and a fluorescence-activated cell sorter. In the presence of all forms of IFN-alpha tested, IgG and IgM production by PBMC increased twofold to fourfold. This increase was noted in the absence of pokeweed mitogen (PWM), was not affected by depletion of monocytes, required that IFN-alpha was present early in the culture period, and reached maximal levels around 500 U/ml IFN-alpha. Both IgG and IgM production were affected, but the magnitude of the IgM response was greater. The augmentation of Ig production was noted with the recombinant DNA-derived subtype, IFN-alpha F, two analogs, IFN-alpha Con1 and IFN-alpha Con2, as well as with buffy-coat-derived (leukocyte) IFN-alpha. The recombinant DNA-derived forms of IFN-alpha appeared to differ in their ability to augment Ig production. In the presence of PWM, IFN-alpha Con1 failed to increase Ig production by PBMC. In contrast to these results with PBMC, IFN-alpha Con1 increased the Ig production of purified B cells 10- to 20-fold in the presence of PWM. This increase reached maximal levels around 500 U/ml IFN-alpha Con1. Although purified B cells responded to IFN-alpha and PWM, maximal responses occurred in the presence of low numbers of helper T cells. Cell dilution experiments suggested that the effect observed with purified B cells was the result of the interaction of B cells with residual cells, e.g., helper T cells, remaining in the preparations.

Transformation of owl monkey T cells in vitro with Herpesvirus saimiri.

Owl monkey peripheral blood mononuclear cells were treated with phytohemagglutinin and expanded in interleukin 2 (IL-2)-containing medium. The cells were then exposed to Herpesvirus saimiri (HVS strain S295C)-infected owl monkey kidney monolayer cells. Four to 6 weeks later, the lymphocytes showed increased clumping and cell growth and the ability to grow in the absence of IL-2. Control lymphocyte cultures not exposed to HVS eventually died out at approximately 6-8 weeks, even in the presence of IL-2. Although infected lymphocytes grew continuously in the absence of IL-2, their growth was enhanced by addition of IL-2 to the cultures. Natural killer cell-like cytotoxicity and gamma-interferon release were also enhanced by IL-2. All cultures were positive for HVS antigens, infectious centers, or DNA. The reactivity of monoclonal antibodies to cell surface markers suggested that the resultant cell lines were comprised of activated T cells. The properties of the in vitro-transformed cells were similar to those of cells established from HVS-induced owl monkey tumors. Our results suggest that infection of T lymphocytes with HVS results in decreased dependence of T cells upon exogenous IL-2 for growth.

A monoclonal antibody recognizing human Thy-1: distribution on human and non-human primate haematopoietic cells.

A monoclonal antibody designated 'antibody 390' (Ab 390) with anti-human Thy-1 reactivity was prepared by the hybridoma technique from the splenocytes of BALB/c mice immunized with human fetal brain. This antibody was shown to have anti-human Thy-1 reactivity because (1) it precipitated a molecule with a molecular weight of about 24,000 daltons, (2) it had a pattern of reactivity similar to that of previously described anti-human Thy-1 antibodies and (3) purified human Thy-1 antigen specifically inhibited binding of Ab 390 to a known antigen-positive cell line. It was the intent of this study to investigate the distribution of Thy-1 on normal and malignant haematopoietic cells in humans and non-human primates. We show here that Ab 390 did not react with human peripheral blood leucocytes, bone marrow cells or splenocytes by immunofluorescence but did react with subcapsular and cortical fetal thymocytes by peroxidase-antiperoxidase immunohistology. A section of fetal spleen demonstrated staining of connective tissue and blood vessels and rare reactive lymphocytes. Adult spleen contained Thy-1-positive cells surrounding the white pulp and in the marginal zone, but single-cell suspensions of splenocytes did not react with Ab 390. Ab 390 was tested against a variety of fresh human leukaemia cells and human cell lines and was shown to react with only the acute lymphoblastic leukaemia T cell lines RPMI 8402 and HPB-MLT. Non-human primate studies revealed reactivity with a number of T cell lines from New World primates (cotton-topped and red-bellied marmosets) and peripheral blood granulocytes (owl monkey). Our studies support previous findings that suggest that human Thy-1 may be a marker for early T lymphocytes in man, and its distribution on non-human primate T cell lines suggests the same for certain species of non-human primates. Not consistent with the distribution on human cells was the demonstration of Ab 390 reactivity with owl monkey granulocytes.

Binding of human lymphocyte-specific monoclonal antibodies to common marmoset lymphoid cells.

Commercially available monoclonal antibodies which bind to human lymphocyte subsets were screened for their ability to bind to lymphoid cells from the common marmoset Callithrix jacchus. Anti-Leu-5 and T11 were the only pan T-cell antibodies which reacted strongly. None of the antibodies which bind human lymphocytes of the helper/inducer subpopulation reacted with C. jacchus cells and only one antibody, T8, specific for the cytotoxic/suppressor subset, bound to the marmoset cells. The two antibodies tested which bind human B cells, B1 and anti-HLA-DR, were also reactive with marmoset cells. The cellular specificity of the T11, T8, and B1 antibodies was determined by dual binding studies on the fluorescence-activated cell sorter. The B1 antibody bound only Ig+ cells and all Ig+ cells were B1+. The T11 and T8 antibodies bound only to Ig- marmoset lymphoid cells and, as in the human, all T8+ marmoset cells were also T11+. Thus, using these monoclonal antibodies in the common marmoset one can identify three populations of lymphoid cells: (1) T11+, T8+ cells; (2) T11+, T8- cells; (3) B1+ cells.

Characterization of a human blood monocyte subset with low peroxidase activity.

Two human monocyte subsets from the peripheral blood of healthy donors have been isolated in greater than 90% purity by countercurrent centrifugal elutration and human serum albumin gradients and their functional capabilities have been assessed. We have demonstrated that one subset ("regular" monocytes, RM) showed intense cytoplasmic peroxidase staining and contained substantial peroxidase activity. In contrast, another subset ("intermediate" monocytes, IM) stained poorly for peroxidase and had low peroxidase activity. By electron microscopic analysis combined with peroxidase localization, it was found that IM had fewer peroxidase-positive granules per cell than did RM. IM coelutriated with some lymphocytes and by cell sizing analysis were shown to be slightly smaller than RM. Functional and cytochemical analysis of these subsets indicated that IM had less activity than RM in assays such as accessory cell function for mitogen-induced T lymphocyte proliferation and antibody-dependent cellular cytotoxicity, and that fewer IM expressed OKM1 antigen and pokeweed mitogen (PWM) receptors on their membranes than did RM. The subset of IM not bearing either the PWM receptor or the OKM1 antigen had very low peroxidase activity. IM also were found to have a greater sensitivity to polyriboinosinic and polyribocytidilic acid (100 micrograms/ml)-induced secretion of interferon. There was no significant difference in the phagocytic capability, the percentage of Fc receptor-positive cells, 5'-nucleotidase activity, DR antigen expression, or the responsiveness to migration inhibitory factor of IM as compared with RM. Furthermore, it was found that the ratio of IM to RM increased after prolonged cytapheresis, which suggests that IM are more mobilizable than RM from the extravascular reservoirs of human monocytes.

A rapid, large scale purification procedure for gibbon interleukin 2.

Interleukin 2 (IL 2) was purified from the conditioned medium of a gibbon T cell line, MLA144, which releases IL 2 constitutively. The IL 2 was obtained free of contaminating proteins by a simple process consisting of an initial batch purification on trimethylsilyl-controlled pore glass followed by reversed phase high pressure liquid chromatography. Overall recovery of IL 2 activity ranged from 70 to 100% of initial activity and yielded 2 X 10(6) or greater units of IL 2 per 15 liters of serum-free MLA144 conditioned medium. The specific activity of purified IL 2 ranged from 0.5 to 1 X 10(8) IL 2 U/mg protein. The purified IL 2 showed four molecular species when analyzed by two-dimensional isoelectric focusing-SDS-polyacrylamide gel electrophoresis. Each of the four molecular forms was active in the bioassay for IL 2 activity. Three molecular forms had apparent m.w. of 16,000 but different isoelectric points of 5.9, 6.3, and 6.7. One molecular form had an apparent m.w. of 15,000 and an isoelectric point of 7.2. The most abundant form of IL 2 had an apparent m.w. of 16,000 and an isoelectric point of 6.3. The purified IL 2 supported the growth of IL 2-dependent lymphocytes to a greater extent than did the same level of crude IL 2-containing MLA144 conditioned medium. The ability to purify large amounts of IL 2 by a rapid and efficient procedure will be of great help in both biochemical and immunologic studies of this lymphokine.

Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells.

The ability to grow lymphoid cells in serum-free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, beta-mercaptoethanol, L-alpha-phosphatidylcholine, and transferrin supported the long-term proliferation of a gibbon ape lymphoma T-cell line, MLA144. These cells continue to produce interleukin 2 (IL-2, T-cell growth factor) constitutively in the serum-free medium. IL-2-dependent human T cells initiated and maintained in culture in serum-free medium containing IL-2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL-2-dependent marmoset T-cell line, OH-1, also proliferated on the serum-free medium when supplemented with IL-2. Several established primate B-cell lines which do not require IL-2 for growth were able to proliferate in the serum-free medium. These B-cell lines included B95-8, an Epstein-Barr virus (EBV)-transformed marmoset cell line, HuCo/R-H, a human cord B-lymphocyte line transformed with EBV, and Namalwa, an EBV-positive B-cell line established from a Burkitt's lymphoma. B95-8 cells grown on serum-free medium showed high levels of EBV antigen-positive cells after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).

Identification of normal and transformed lymphocyte subsets of nonhuman primates with monoclonal antibodies to human lymphocytes.

Monoclonal antibodies (MAb) that detect specific lymphocyte subsets of man were tested for their reactivity with peripheral blood lymphocytes (PBL) and established cell lines of owl monkeys (Aotus trivirgatus) and marmosets (Saguinus spp). Results of these studies showed that certain determinants were conserved and that the pattern of reactivity observed in one genus (or even species) could not be used to predict the reactivity in another. On owl monkey PBL, reactivity equivalent to that on human PBL was observed with OKT11a, anti-Leu 3a, B1, and anti-HLA-DR MAb thus detecting T cell, helper/inducer T cell, B cell, and HLA-DR determinants. After neuraminidase treatment of owl monkey PBL, reactivity with anti-Leu 5 and OKT8 (T cell and suppressor/cytotoxic T cell determinants) was observed. Cell separation experiments indicated these determinants were found on lymphocytes with the same general properties as those of human origin. The use of these MAb to examine owl monkeys infected with Herpesvirus saimiri and tumor cell lines established from H. saimiri-inoculated monkeys revealed that the virus was found in OKT11a-positive, B1-negative cells. In chronically infected nondiseased animals, H. saimiri was found in anti-HLA-DR-negative cells and was restricted to anti-Leu 3a-positive cells (two animals) or was nearly equally distributed in anti-Leu 3a-positive and negative cells (one animal). Established H. saimiri tumor cell lines had the phenotype OKT11 a-positive, anti-HLA-DR-positive, B1-negative and were either anti-Leu 3a-positive or negative. Studies of interleukin 2-responsive tumor cells in short-term culture suggested the tumor was composed of virus-positive anti-Leu 3a-positive and negative populations.

Detection of antibodies to Epstein-Barr virus antigens by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) is described for the serologic analysis of antibodies to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), viral capsid antigen (VCA), and early antigen (EA). The specificity of each of the ELISAs was demonstrated by the use of well-characterized human sera shown by immunofluorescence assay to be variously reactive for antibodies to one or more of the three viral antigens studied. The ELISA for EBNA was four to 256 times more sensitive than immunofluorescence assays with all 33 EBNA-positive sera tested. The ELISAs for VCA and EA were also more sensitive than immunofluorescence assays: approximately 50% of the sera tested showed higher antibody titers. Sera that were negative for all three antigens by immunofluorescence assay were also negative by ELISA for each antigen. These ELISAs for EBV are rapid, sensitive, and objective and thus provide new and valuable methods for the detection of antibodies to EBV-related antigens.

The effect of T cell growth factor on the cell cycle of primate T cells.

The effect of T cell growth factor (TCGF) on the cell cycle of four T cell lines that differed in their response to TCGF was studied. Activated normal marmoset T cells (OH-1) were totally dependent on the addition of TCGF for long-term proliferation. In the absence of TCGF, the cells were incapable of traversing the cell cycle and became arrested in G1. The addition of TCGF to arrested OH-1 cells stimulated them to enter the S phase after a lag phase of 24 to 33 hr. The TCGF-stimulated cells reached a maximum of cells in the S phase by 12 hr after the initiation of DNA synthesis. TCGF was required for a minimum of 18 hr before cells would enter the S phase. In the absence of TCGF, activated owl monkey (8I) and human (RG) lymphocytes displayed a TCGF-sensitive block; addition of TCGF stimulated these cells to enter the S phase after 12 and 16 hr, respectively. In contrast, an owl monkey tumor-derived T cell line (OMT-1), not dependent on exogenous TCGF for proliferation, was able to progress slowly through the cell cycle without a TCGF-sensitive block. These cells responded to TCGF by showing an initial increase in cells that entered the S phase after a lag of 6 hr and a continuing movement of additional cells into S over the course of the next several hours.

Discordant Epstein-Barr virus nuclear antigen (EBNA) antibody patterns in nasopharyngeal carcinoma.

Epstein-Barr virus nuclear antigen (EBNA) preparations from three sources were tested with sera from normal individuals and patients with Hodgkin's disease, breast carcinoma, Burkitt's lymphoma, and American and Chinese nasopharyngeal carcinoma. Individual sera with discordant antibody patterns were noted in all groups. Sera from both NPC groups gave significantly higher anti-EBNA titers on cell lines converted with P3HR-1 or B95-8 virus compared with anti-EBNA titers on Raji cells. Anti-EBNA titers of Chinese NPC sera showed no correlation among the three EBNA sources, while all other groups had highly correlated titers. Cross-absorption experiments present evidence for more than one antigenic determinant on EBNA. These results suggest an additional parameter for distinguishing Chinese NPC from other EBV-related disorders.

Surface markers of primate B and T lymphoid cell lines identified by antibodies to human and simian lymphocyte antigens.

Simian B and T lymphoid cell lines were shown to maintain surface markers found on mature lymphocytes in vivo. The T lymphoid cell lines expressed Ia-like antigens on their surfaces, further suggesting that they represent mature, activated T cells. These Ia antigens show a structural similarity to Ia on human cells although some diversity exists. The Ia antigen expressed on T lymphoid cell lines was shown to be very similar to those on B lymphoid cell lines. Owl monkey and marmoset T lymphoid cell lines were also shown to express a VH immunoglobulin-related determinant, a marker which is thought to be associated with T cell antigen receptor. Owl monkey and marmoset T cell lines express a surface antigen which identifies the sheep erythrocyte receptor on human T cells and some of these lines express an antigen found on human helper T cells. It is noteworthy that substantial conservation of surface components has occurred within primate evolution such that monoclonal antibodies to human Ia, OKT-11a and Leu 3a markers can be used to type lymphocytes of lower primates.

Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen.

Five murine hybridoma lines that produce monoclonal antibodies against Epstein-Barr virus membrane antigen (MA) were established. Immunoprecipitation experiments demonstrated that three of the antibodies precipitated both the 236,000 (236K) MA and the 212K MA. The other two antibodies precipitated the 86K MA. Antibodies against the 236K-212K MA and the 86K MA mediated complement-dependent cytolysis of Epstein-Barr-virus-infected cells. The antibodies against the 86K MA neutralized both the B95-8 and P3HR-1 viruses.

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin.

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.

Spontaneous release of a factor with properties of T cell growth factor from a continuous line of primate tumor T cells.

A continuous lymphoid cell line had been previously established from a gibbon with spontaneous lymphosarcoma. This cell line, designated as MLA144, when tested after several years in culture was shown to release spontaneously a factor biologically and biochemically similar to human T cell growth factor (TCGF). Conditioned media (CM) from MLA144 cells support growth and DNA synthesis of T cells from humans, several other species of primates, and also from mice and rabbits. The activity in the MLA144 CM is resistant to 60 degrees C and to low and high pH, has a m.w., as determined by gel filtration, of 21,500, elutes from DEAE-cellulose at 0.04 to 0.06 M sodium phosphate buffer, pH 7.6, and has an isoelectric point of about 6.45. Surface-marker analysis of MLA144 cells by rosetting techniques indicates that they are T cells lacking in the receptor for the Fc portion of IgG. The release of TCGF by MLA144 cells should have practical value in terms of ease of TCGF production and should be of great help in the facilitation of studies on the cell biology and molecular biology of TCGF production.