Comparison of MHC antigen expression on PHA- and MLC-induced T cell lines with that on T and B lymphoblastoid cell lines by cell cycle dependency

Although it is well known that the expression of major histocompatibility complex (MHC) antigens on the surface of lymphoblastoid cell lines are cell cycle dependent, the way in which the MHC antigen expression on activated T cells varies with cell cycle phase has not previously been described. Using 11 lymphoblastoid cell lines from malignant and nonmalignant tissues (B cells, T cells, and myeloid cells) and five activated T cell lines (two cell lines activated by phytohemagglutinin and three alloreactive T cell clones), MHC antigen expression was quantitatively studied by dual-beam flow cytometry. Correlated measurements of surface antigen quantity (immunofluorescence), DNA content (Hoechst 33342), and cell size (light scatter), uninfluenced by induction synchrony and cell fixation, were performed. The data indicate that cell surface antigen quantity and cell surface area demonstrate specific values at each phase of the cell cycle when the cells are in logarithmic growth. Examining cells in logarithmic growth, it was confirmed, for all lymphoblastoid cell lines, that the quantity of MHC antigens on G2 (S + G2 + M) cells was greater than that on G1 cells. In addition, it was found, by analyzing antigen quantity and surface area, that class I antigen density in the G2 phase is 17% less than that in the G1 phase in leukemic T cell lines, and that both class I and class II antigen densities in the G2 phase were 21% less than that in the G1 phase in lymphoblastoid B cell lines. In activated T cells, class I antigen density in the G2 phase was 11% less than that in the G1 phase, while class II antigen density in the G2 phase was 12% greater than that in the G1 phase. We describe four important observations in this report. In both G1 and G2 phases, activated T cells express: quantitatively fewer class I antigens than lymphoblastoid B cell lines; similar quantity of class I antigens as that of leukemic T cell lines; and similar quantity of class II antigens as that of lymphoblastoid B cell lines. Also, class II antigens are expressed in greater density in the G2 phase than in the G1 phase in activated T cells. In contrast, lymphoblastoid B cell lines express greater density of class II antigens in the G1 phase than in the G2 phase of the cell cycle. These findings differ from previous reports, suggesting that G1 phase cells may have a more significant role than G2 phase cells as target cells for MHC restricted cytotoxic cells.     

Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient

Two IgEL and one IgGK producing cell lines have been established in vitro from peripheral blood and bone marrow of an E myeloma patient. Morphologic and immunologic studies indicate that cells of the IgE producing lines are derived from the same clone of myeloma cells which grew in vivo. They have remained essentially unaltered with a stable near-diploid karyotype and continuous production of intact IgE molecules during 18 months in culture. The IgGK producing line has all the characteristics ascribed to permanent lymphoblastoid lines and is considered to be of non-malignant lymphoid origin. The process of establishment is described and it is suggested that lymphoblastoid cells have a selective advantage over myeloma cells. Permanent myeloma cell lines can therefore probably be obtained only if cells capable of forming established lymphoblastoid lines are very few or absent in the original biopsy. The establishment of a permanent cell line continuously producing intact molecules of IgE should permit further studies on the biological role of this class of immunoglobulins.     

Tissue distribution of the Pk antigen as determined by a monoclonal antibody

Using an anti-Pk monoclonal antibody (mAb) designated CPK-1, the expression of the Pk antigen was assessed on normal human tissue from non-Pk individuals. Although the Pk antigen was detected on fibroblasts and blood vessels as previously reported, it was also found on smooth muscle cells of the digestive tract and the urogenital system. Pk was also found on glandular cells of the stomach, oesophagus and prostate. Additionally, CPK-1 reacted weakly with oesophagus squamous cells, and a small number of glomeruli and tubules in the kidney. The mechanism of expression of the Pk determinant in non-Pk individuals is discussed.     

HLA typing of Epstein-Barr virus transformed lymphoblastoid cell lines (LCL)

The application of standard tissue typing techniques to cells other than peripheral blood lymphocytes has been accompanied by the problem of extra reactions. This applies as well to Epstein-Barr virus transformed lymphoblastoid cell lines (LCL) as to leukemic cells and human spleen cells. These extra reactions are attributable to additional antibodies in the typing sera which are not apparent under standard conditions with PBLs. Two types are described: Type 1 extras, which becomes apparent after longer incubation times and are attributed to weak antibodies and type 2 extras which are apparent after shorter incubation times and are attributed to subpopulation specific or differentiation antigens. Technical modifications are proposed by which these extras can be circumvented. They include: Only start typing when cells have been cultured for 2 to 3 days. Remove dead cells by spinning over standard ficoll-hypaque or 11% triosil. Use shorter incubation times. Avoid using sera that give too many type 2 extras. In this way phenotypes can be accurately identified on LCL's obtained from kidney transplant donors and recipients. When LCL's were compared with their matching PBL, HLA phenotypes were concordant in 87% of cases for HLA-A, 90% for HLA-B, 81% for HLA-C and 70% for HLA-DR.     

Ye-1, a monoclonal antibody that cross-reacts with HLA-B27 lymphoblastoid cell lines and an arthritis causing bacteria.

A monoclonal antibody, Ye-1, was generated by immunizing BALB/c mice with Yersinia enterocolitica. This antibody also reacted with all of 12 B27 positive lymphoblastoid cell lines, but only four of 31 B27 negative ones. Three of the four reactive B27 negative cell lines were B7 positive. A B27 positive cell line which has lost the B27 expression because of experimentally-induced mutation became unreactive with the Ye-1. These findings support the possibility that there is cross-reactivity between HLA-B27 antigens and Y. enterocolitica.     

Host cell reactivation of uv- and X-ray-damaged herpes simplex virus by epstein-barr virus (EBV)-transformed lymphoblastoid cell lines

The efficacy of using an infected centers assay, employing herpes simplex virus-infected, Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) as components, to study host cell reactivation has been explored. Herpes simplex virus type 1 (HSV-1) was shown through the infected centers assay to have detectable but varying ability to lytically infect LCLs established from chromosomal breakage syndromes or closely related genetic disorders. The rate of HSV inactivation by ultraviolet (uv) irradiation was faster in LCLs established from Cockayne's syndrome than in normal LCLs, and faster still in LCLs established from xeroderma pigmentosum. These results indicate that Cockayne's syndrome, while having what appears to be quantitatively normal levels of uv-induced DNA repair replication, shows decreased ability to host cell reactivate uv-damaged HSV. In direct contrast, X-irradiated HSV showed identical survival when assayed on normal LCLs or LCLs established from ataxia telangiectasia showing increased sensitivity to X irradiation as measured by colony formation. Through the infected centers assay, it has also been possible to demonstrate low levels of multiplicity reactivation of mutagen-damaged HSV in permanently proliferating LCLs.     

Clonal analysis of a human lymphoblastoid cell line (B17) secreting antibody to N-acetyl-D-glucosamine

In this paper we analyse the clonal composition of a human lymphoblastoid B-cell line secreting IgM/k antibody to N-acetyl-D-glucosamine, the immunodominant sugar of Group-A-streptococcal carbohydrate. Besides non-antibody secreting cells, the line consists of two clonotypes of antibody-secreting cells: B17 cells producing over 90% and F6 cells producing less than 10% of the antibody in the supernatant. The proportions of B17 and F6 cells in the cell line seem to be similar to the proportion of antibodies in the supernatant. F6 cells can be isolated by cloning and maintained as stable lines, whereas this is more difficult with B17 cells. The results suggest that upon establishment of the line, at least two N-acetyl-D-glucosamine-specific B cells were immortalized and coexist together as independent clonotypes. Although F6 cells seem to have a slight tissue culture advantage, they represent the minor clonotype in the B17 cell line.     

Differential cyst(e)ine requirements in human T and B lymphoblastoid cell lines

In an effort to find exploitable metabolic differences between human T and B lymphoblasts, we have compared the ability of lymphocytes of varying phenotype to grow in cystine-deficient medium. Only 6 of 12 human lymphoblastoid cell lines tested were able to utilize homocysteine thiolactone or cystathionine in place of cystine for growth. This difference in growth requirements was unrelated to the rate of cell division, the presence of Epstein-Barr viral genetic material, or whether or not the cell lines derived from benign or malignant tissues. Rather, all B lymphoblastoid cell lines grew in homocysteine thiolactone- or cystathionine-containing medium, while the T and non-T, non-B lymphoblastoid cell lines did not. Normal human peripheral blood T and B lymphoblasts did not respond to mitogens in the homocysteine thiolactone or cystathionine medium, but developed the ability to utilize these cysteine precursors after stimulation with concanavalin A, protein A, or Epstein-Barr virus. The differences in cysteine requirements among T and B cell lines may reflect a fundamental difference in de novo protein-synthesizing capacity of the two cell types.     

Cell surface phenotyping and cytokine production of Epstein-Barr Virus (EBV)-transformed lymphoblastoid cell lines (LCLs)

Epstein-Barr Virus-transformed B lymphoblastoid cell lines (EBV-LCLs) are routinely used for the in vitro expansion of T cells. However, these cell lines are reported to produce the cytokine IL-10, which is inhibitory for T cells. We, therefore, characterized a panel of 37 EBV-LCLs for a variety of cell surface markers, for secretion of various cytokines including IL-10 and for immunoglobulin production. These cell lines were derived from normal donors or patients with nonsmall cell lung cancer, acute myelogenous leukemia, melanoma or colon cancer. Overall, 26 lines were positive for CD19 and CD20, and 11 were negative for both. All of the lines were strongly HLA-DR+, while CD40 expression was variable. Twenty-four (65%) were both CD23+ and secreted immunoglobulin, and 33 expressed kappa and/or lambda light chains. Additionally, all of the EBV-LCLs were negative for T cell (CD3), NK cell (CD16, CD56), monocyte (CD14) and granulocyte (CD66b) surface markers. Some level of IL-10, IL-6, IL-12p40 and TNF-alpha cytokine production was detected in 33, 18, 19 and 12 EBV-LCLs, respectively. Together, these data reflect the heterogeneity of EBV-LCLs, which cautions their use nondiscriminately in various immunologic assays.     

Study of the antigen common to continuous epithelial cell lines and human gastric mucosa

An antigen common to continuous human epithelial cell lines (CHC) and gastric mucosa, described by the writers previously, was studied. The antigen was found in one other cell line (MDA-MV-231), derived from carcinoma of the human breast, not contaminated by Hela cells. The antigen described was found in exophytically growing adenocarcinomas of the stomach and in the mucosa of a stomach affected by cancer at a distance of 10–12 cm from the site of the lesion. The antigen was not found in endophytically growing carcinomas of the stomach or in areas close to a gastric ulcer. The antigen is not a glycoprotein, for glycoprotein fractions obtained with the aid of 1.2 M perchloric acid from homogenate of normal gastric mucosa and extract E16b were inactive in the immunodiffusion test with a sensitive serum. The antigen described has the electrophoretic mobility of ₂-₁-globulin. The antigen is interesting because its presence or absence may perhaps help to establish the initial type of cell from which a gastric carcinoma developed and to identify more precisely the histological form of a gastric carcinoma.Laboratory of Sarcoma-Leukemia Viruses and Laboratory of Clinical Biochemistry, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 12, pp. 744–747, December, 1978.     

Expression of membrane-bound IgM and HL-A antigens on lymphoblastoid cells in different stages of the cell cycle

The expression of membrane-bound IgM and HL? A on human lymphoid cells in different stages of the cell cycle was determined by cytophotometric techniques. Asynchronously proliferating cells were pulse labeled with [³H]thymidine. IgM and HL? A were then stained directly and indirectly with fluorescein-conjugated antibodies and quantitated on the single cell level by microspectrofluorometry. The same individual cells were analyzed further by dry mass and Feulgen DNA determinations using microinterferometry and microspectrophotometry, respectively. Cells in the S phase were subsequently identified by [³H]thymidine autoradiography. The amounts of membrane-bound IgM and HL? A were both found to increase continously throughout interphase. Thus, in absolute terms, the IgM and HL? A content was maximal in G2, minimal in the G1 and intermediate in the S phase. However, since the total proteins (dry mass) increased in the same manner, the IgM (and HL? A)/total protein ration remained constant during the cell cycle. For geometric reasons, the total cell surface area increases less than the cell volume (mass) during interphase. This might indicate that the density of IgM and HL? A on the membrane is greater in the G2 than in the G1 phase.     

The definition of an MB related specificity by a monoclonal antibody

A number of monoclonal antibodies have been described that react with monomorphic and polymorphic Ia-like specificities on human B cells, but it is not clear whether these react with HLA-DR encoded molecules or with the products of other closely linked genes within the MHC, such as MB, MT, or DC 1 antigens. A monoclonal antibody is described herein (MC-26.1) which detects a new Ia-like specificity as shown by B-cell reactivity, chemistry, family studies, including recombinant family and coprecipitation studies. The latter studies showed that specificity detected by the MC-26.1 antibody coprecipitated with determinants detected by conventional anti-MB3 and MT3, but not with HLA-DR4 antisera. The determinant was polymorphic but not related to any of the known MB or MT specificities. However, cross-reactions were demonstrated by the variability of reaction on different individuals. Coprecipitation studies indicate that the antigen defined by MC-26.1 coprecipitates with MB and MT specificities suggesting that the antibody defines a new specificity on the MB3 and MT3 molecules.     

A monoclonal antibody to common acute lymphoblastic leukemia antigen (CALLA) and its expression on several human tumor cell lines.

We describe a newly-made murine monoclonal antibody to the common acute lymphoblastic leukemia antigen (CALLA), named SHB-10. The antigen detected by SHB-10 has a molecular weight of about 105 kDa. This antibody is very similar to that of conventional anti-CD10 Ab on indirect flowcytometric analysis using lymphoid malignant cell lines and peripheral lymphocytes of acute lymphoblastic leukemia (ALL) patients. The binding of anti-CD10 to Daudi cell and peripheral lymphocytes of ALL patients is blocked by SHB-10. Thus this monoclonal antibody is thought to detect the CALLA. The distribution of antigen detected by SHB-10 on several cell lines of neuroectodermal tumor and lymphoid malignancy was analysed and a slight difference in their cell surface expression is observed when compared with that by conventional anti-CD10. Further biochemical analysis is now under way for a better characterization of this antigen.     

Effects of mycoplasma contamination on immunoglobulin biosynthesis by human B lymphoblastoid cell lines.

The synthesis and secretion of immunoglobulin M (IgM), as well as the relative ratio of membrane and secretory mu heavy chain (mu m and mu s, respectively), were evaluated in mycoplasma-contaminated B lymphoblastoid cell lines. The ratio of mu m to mu s was drastically lowered in infected cultures, and mu s chains could now combine with light chains. A 50 to 100% increase in IgM synthesis occurred in contaminated cultures, and small amounts of IgM were detectable in the culture media. These molecules possessed mu chains typical of secreted IgM. Reexpression of mu on the surface of B lymphoblastoid cells was substantially delayed in mycoplasma-contaminated cultures. Thus, mycoplasma contamination alters the synthesis and expression of a specific differentiated gene product (immunoglobulin) in B cell lines; such changes could significantly affect the interpretation of data on immunoglobulin synthesis by B cells with different phenotypes. This system may also provide a means of studying how mycoplasma infection alters specific gene expression in B cell lines.