Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbecco's modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (10⁷ cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.     

Human B-like lymphoblastoid cell lines obtained by long-term culture of normal spleen leukocytes

B-like lymphoblastoid cell lines were obtained by long-term culture of human spleen leukocytes in RPMI 1640 medium containing human plasma fraction instead of whole foetal calf serum. These cell lines, which did not form E-rosettes had membrane immunoglobulins, and expressed Epstein-Barr virus antigens. Most synthesized intracytoplasmic immunoglobulins and were shown to be diploid and to remain so after subcultures. All produced interferon upon induction with Sendai virus.     

Rabbit antiserum against Daudi-cell membrane fractions detects cell surface antigens present on subpopulations of human lymphocytes.

An antiserum was produced by immunization of rabbits with membrane preparations of a human lymphoblastoid B cell line, Daudi. After absorption with a human endometrial carcinoma cell line, this antiserum appeared to be specific for antigen(s) present on adult and fetal thymocytes as well as on tonsillar lymphocytes but absent, or present in very small amounts, on normal or phytohemagglutinin- (PHA) stimulated peripheral blood lymphocytes (PBL). When T and B cell-enriched fractions from tonsillar lymphocytes were tested with the anti-Daudi serum, the reactivity was equally distributed in each population. Among 13 human lymphoblastoid cell lines tested, reactivity was demonstrated on three out of four T cell lines, and on four out of nine B cell lines. The positive reacting B cell lines were derived from two African and two American Burkitt lymphomas. The antigen(s) described does not seem to be related either to human Ia-type antigens or to Epstein-Barr virus-associated antigens because these antigens are not present on fetal or adult thymocytes. Reciprocal absorption experiments indicate that this anti-Daudi serum detects the same antigenic structures present on certain subpopulations of T and B lymphocytes.     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

Continuous growth of human tumor cell lines in serum-free media

Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10⁻¹⁰ M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth. This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J. Hill) and grant no. CA-37138 from the National Cancer Institute.     

Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable.Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast.Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2×10⁸/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day.The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.     

Immortalization of murine leukocytes by oncogenes. II. Phenotypic characterization of transformants immortalized by v-src or Ha-ras oncogenes: expression of B220, a B-cell lineage specific antigen

In the course of study to obtain murine dendritic cell lines using oncogenic retroviruses, we have established several immortalized cell lines with characteristics different from those of dendritic cells. The transformants were mainly round nonadherent cells, capable of growing in soft agar, and negative for nonspecific esterase activity. Profiles of cell surface antigens were examined by indirect immunofluorescence technique. The cell lines were positive for Fc receptor (2.4G2), J11d (J11d.2), and B220 (RA3-3A1/6.1) antigens and negative (or dull positive in small percentages) for Ia (M5/144.15.2), IL-2 receptor (3C7), Thy-1 (B5-5), Mac-1 (M1/70.-15.11.5), and macrophage (F4/80) antigens. They were negative for both surface and cytoplasmic immunoglobulins. Several clones were established from these transformant cell lines and cell surface antigens were examined. Antigenic profiles of these clones were very similar to those of the parental cell lines. Some of these clones, however, seemed to increase their Ia antigen expression. The results suggest that the transformants originated from early B-lineage cells.     

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.     

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, β-mercaptoethanol, L-α-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).     

B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt's lymphomas

Two new cell surface antigens expressed on B lymphoblastoid cell lines (B-LCL) were defined with cytotoxic mouse monoclonal antibodies. One marker, BB-1 (for B lymphoblast antigen-1), was detected on human and nonhuman primate B-LCL, Epstein-Barr virus (EBV)-activated B cell blasts, most Burkitt's lymphomas, and Ia+ B lymphoblast-like myelomas. Polyclonal B cell activators such as pokeweed mitogen (PWM) and lipopolysaccharide (LPS) also induced the expression of BB-1 on immunoglobulin (Ig)-positive cells. In contrast, BB-1 could not be detected on normal lymphoid tissues by complement-dependent cytotoxicity and immunofluorescence (IF) assays or by analysis with a fluorescence-activated cell sorter (FACS). T cell blasts, T cell leukemias, and pre-B cell or erythroblastic leukemia cell lines were also BB-1 negative. Of particular interest was the finding that BB-1 was expressed on the Jijoye lymphoma but only marginally on a subline of Jijoye, P3HR-1, that lacks receptors for EBV and produces a defective virus incapable of transforming lymphocytes. A second lymphoblast antigen (LB-1) unlike BB-1, was present on both T and B cell blasts and virus-transformed T- and B-LCL but not on normal lymphoid tissues.     

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of the invariant chain in mouse tumor cells: relationship to B lymphoid development

We have examined 25 cultured lines of mouse tumor cells for synthesis of the Ii, an Ia-associated polypeptide, by using an anti-Ii monoclonal antibody. Six of the T lymphomas tested did not produce detectable levels of Ii or of surface Ia antigens. Three B lymphomas and two plasmacytomas that express surface Ia antigens were found to synthesize the Ii. In addition, Ii was immunoprecipitated from two of five Ia- pre-B lymphomas, two of four Ia- plasmacytomas, two Ia- myeloid tumors, and two fibroblast cell lines including LM(TK-). Because Ia antigens have so far been found only on cells that also synthesize Ii, we suggest that the Ii is a marker of those cells that in certain states of development or activation express Ia antigens.     

Synthesis and characterization of membrane immunoglobulin, Ia, and H-2 molecules of thy 1+ AKR/J lymphomas.

Three AKR lymphomas displaying B cell and T cell characteristics have been described. Because of the proclivity of normal AKR/J mice to develop T cell lymphomas, and the rarity of lymphomas with dual characteristics, the B cell markers of these tumors were studied more intensively. Fluorescence data with class-specific anti-immunoglobulin reagents demonstrated that the tumor cells stained only with class-specific anti-IgM reagents. Because of the possibility that the surface Ig was passively acquired and of reports that certain anti-mu-chain sera react with "IgT", chemical characterization of the immunoglobulin molecules was performed. Using 3H-leucine internal labeling, we showed that all three tumor lines synthesized the immunoglobulin found on their surface, and that the immunoglobulin had the chemical and immunologic characteristics most typical of monomeric surface IgM, and was composed of mu-chains and light chains. The Ia antigens found on these cells were also examined. These antigens were also synthesized by the cells and were present in the same molecular form and in the same approximate quantity as Ia antigens on normal spleen cells.     

Fibrinolytic activity associated with established lymphoblastoid cell lines and fresh peripheral blood lymphocytes of human and nonhuman primate origin.

Established lymphoblastoid cell lines and normal peripheral blood lymphocytes were examined for their ability to induce fibrinolysis, a property associated with oncogenic transformation, using a 3H-fibrin plate technique. Fibrinolytic activity showed serum preferences with dog serum being most active. Most cell lines (14/18) induced greater than 40% release, while normal lymphocytes were generally less active. Only one cell line tested released plasminogen activator into the medium. No correlation was shown between fibrinolytic activity and growth in soft agar. Normal rhesus lymphocytes showed fibrinolytic activity in B cell-enriched populations with no evidence of interaction between B cells and T cells.     

Time course study of L-tyrosine aminotransferase induction in rat liver cell lines

The enhancement of L-tyrosine aminotransferase activity by dexamethasone, an exclusive function of the liver, was serially measured at different passages of eight rat liver epithelial cell lines initiated and continuously grown in either a serum-supplemented medium or a serum-free medium. The enzyme basal activity was found to be 5.4 ± 1.8 mU for cell lines in serum and 6.8 ± 3.4 mU for cell lines without serum. Under the influence of dexamethasone (10^–6 mol/l for 5 hours) this basal level could be increased up to 2.9 fold in the presence of serum and 2.5 fold in its absence when investigations were carried out at early passages. During the following subcultures the induction ratio gradually declined and scarcely any induction could be detected after the 15th passage for cells grown in serum and after the 25th passage for cell lines grown without serum.Abbreviations SFM serum-free medium - SSM serum-supplemented medium - TAT L-tyrosine aminotransferase M.F. is a recipient of a government scholarship grant from the Grand Duchd de Luxembourg.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.     

Differential effect of antigen-nonspecific T-cell factors and lipopolysaccharide on the Ia antigens and surface immunoglobulins of BALB/c lymphoma cell lines

In order to study the mechanism of B-cell differentiation using B lymphoid tumor cells as models, we investigated the effects of antigen-nonspecific T-cell factors in combination with lipopolysaccharide (LPS) on the expression of surface markers on B lymphoid cell lines. This study demonstrated that culture supernatant from concanavalin A-activated spleen cells (CAS) gave 2- to 3.5-fold enhancement of the expression of Ia antigens. The effect of CAS was dose dependent and as little as 2% CAS gave maximum enhancement of Ia antigen expression. The CAS effect was due to concanavalin A-activated cell products and was not due to the concanavalin A. The effects of allogeneic effect factor (AEF) on Ia antigens were similar to those of CAS. In contrast to CAS and AEF, LPS did not affect the expression of Ia antigens on ×16C 8.5. LPS enhanced 1.5- to 3-fold the expression of sIgM on this cell line. The expression of sIgM was minimally affected by T-cell factors; CAS induced 20 to 70% enhancement of sIgM expression while AEF induced no significant effects. This study showed that antigen-nonspecific factors (CAS and AEF) influenced mainly Ia expression on the B-cell lymphoma, ×16C 8.5, while LPS selectively affected sIgM expression. Therefore, it was concluded that the mechanisms by which B cells are activated by T-cell factors and mitogens are different.     

Lymphoid cell line identification and the detection of cross contamination

Summary Four lymphoid cell lines, presumably recent derivatives from non-neoplastic human tonsils, were identified in actuality as established Burkitt, lymphoma and lymphoblastoid cell lines (LCL) when tested by chromosome banding and typing for HLA antigens. Additionally, the morphology, growth characteristics, tumorigenicity, expression of Epstein-Barr virus antigens and surface membrane immunoglobulins (SmIg) of the four lymphoid cell lines confirmed the correct identifications. In determining the possiblity of cross contamination, the most reliable criteria for the identification of lymphoid cell lines were found to be HLA antigenic profile, karyotype, cellular morphology, and growth characteristics. This work was supported by Contract N01 CP-53516 from the Virus Cancer Program of the National Cancer Institute, NIH, and by NIH Grants AI 13154, CA 16069, and CA 16071. Dr. Ferrone is a recipient of an American Heart Association Established Investigatorship Award. Dr. Pellegrino is a recipient of a Research Career Development Award. Dr. Glaser is the recipient of a Leukemia Society of America Scholar Award. This is publication 1786 from Scripps Clinic and Research Foundation.