Establishment of a Long-Term Cultured Suppressor T-Cell Line Specific for Guanosine

Induction of suppressor cells specific for guanosine in C57BL/6 mice with syngeneic spleen cells coupled with guanosine was demonstrated. Spleen cells from mice that had been tolerized by the injection of guanosine-coupled syngeneic spleen cells (G-SC) were repeatedly stimulated with mitomycin C-treated G-SC (MMC-G-SC) in vitro. These cells effectively suppressed the secondary in vitro response to guanosine-keyhole limpet haemocyanin (G-KLH) but did not suppress the response to sheep erythrocytes (SRBC). From these cell populations a long-term-cultured suppressor T-cell line specific for guanosine was established by using interleukin-2 and MMC-G-SC. This cell line suppressed the response to G-KLH but did not suppress the response to trinitrophenyl-KLH and SRBC. The suppressive effect was completely eliminated by treatment with anti-Thy 1.2 and complement.     

Persistence of Cells Capable of T-Cell Growth Factor Production in Long-Term Human T-Lymphoblast Cultures

Human T-lymphoblasts maintained in culture for 1-2 months in T-cell growth factor conditioned medium continue growth when placed in fresh culture medium in the presence of phytohemagglutinin and irradiated adherent cells. In the absence of lectin, no growth occurs. Neither long-term cells nor irradiated adherent cells plus lectin produce soluble factors that stimulate long-term cell growth, but small amounts of this biologic activity can be detected in the culture medium when the two cell sets are mixed together. The data indicate that human long-term cell populations retain the ability to make enough TCGF to stimulate proliferation, but require physical contact with monocytes in the presence of phytohemagglutinin for this response to occur.     

Biochemical Characterization of the T-Cell Mitogen Derived from Mycoplasma arthritides.

A biochemical procedure is described to purify the T-cell mitogen in the supernatant of cultured Mycoplasma arthritidis organisms. The mitogenic material was bound on an affigel blue column. The eluate of this column was then acylated at 0 degrees C for 1.5 h and subsequently chromatography on a Sepharose Cl 6B and a Superose 12HR column were performed. SDS-PAGE showed a major band at MW 26,000 and some minor bands at 50,000. With this material biological tests were performed, including induction of lymphoproliferation and interferon induction in murine spleen cell cultures. Purified Mycoplasma arthritidis supernatant (MAS) vigorously stimulated spleen cell cultures of A/J, CBA, C3H/He, and DBA/2 mice, whereas a low-grade but definitive response was observed in C57BL/6 spleen cells. Cultures of Balb/c nu/nu mice, in contrast to those of their euthymic littermates, were non-reactive. When induction of interferon was tested, a marked response to purified MAS was observed in CBA and C3H/HeJ spleen cell cultures, whereas C57BL/6 spleen cells were non-reactive.     

Detection of Antigen-specific Cellular Immune Response by the in Vitro Production of T-Cell Growth Factor

The amount of T-cell growlh factor (TCGF) produced in vitro in antigen-stimulated lymphocyte cultures was measured by induction of growth in cell dependent on the factor. The method distinguishes eight herpes-simplex-seronegative subjects from ten seropositive ones, and these results correlated with those of the lymphocyte blast transformation (LBT) test. The LBT responses and TCGF amounts produced in cultures with purified tuberculin (PPD) were also in good correlation (r = 0.833, P<0.001). Considerable-levels of the factor were already present 12 h after the beginning of the culture. The method offers rapid quantification of cell-mediated immunity to specific antigens.     

Regeneration and Tolerance Factor: a Correlate of Human Immunodeficiency Virus-Associated T-Cell Activation

Human immunodeficiency virus (HIV) infection causes extensive phenotypic alterations in lymphocytes. Cellular markers that are normally absent or expressed at low levels on quiescent cells are upregulated throughout the disease course. The transmembrane form of regeneration and tolerance factor (RTF) is expressed at negligible levels on resting T cells but is quickly upregulated following in vitro stimulation and activation. Recently, we reported that expression of RTF was significantly higher in cells from HIV-seropositive (HIV⁺) individuals than in cells from HIV-seronegative (HIV⁻) individuals. Because T cells from HIV⁺ individuals express markers reflecting chronic activation, we hypothesized that these in vivo-activated cells would coexpress RTF. Flow cytometry was used to assess RTF expression on activated (CD38⁺ and HLA-DR⁺) CD4⁺ and CD8⁺ T cells. HIV⁺ individuals had higher percentages of RTF⁺ CD38⁺ (P < 0.0001) or RTF⁺ HLA-DR⁺ (P = 0.0001) CD4⁺ T cells than HIV⁻ individuals. In HIV⁺ individuals, increased percentages of CD4⁺ T cells that were RTF⁺, RTF⁺ CD38⁺, and RTF⁺ HLA-DR⁺ correlated inversely with the absolute number and percentage of CD4⁺ T cells and correlated positively with plasma β₂-microglobulin concentrations. HIV⁺ individuals had higher percentages of CD8⁺ T cells that were RTF⁺ CD38⁺ (P = 0.0001) or RTF⁺ HLA-DR⁺ (P = 0.0010). In HIV⁺ individuals, increased percentages of CD8⁺ T cells that were RTF⁺ HLA-DR⁺ correlated inversely with the percentage of CD4⁺ T cells, and high percentages of CD8⁺ T cells that were RTF⁺ CD38⁺ correlated positively with plasma β₂-microglobulin levels. These findings strongly suggest that increased RTF expression is a correlate of HIV-associated immune system activation.     

Coexpression of the Genes for Platelet-Derived Growth Factor and Its Receptor in Human T-Cell Lines Infected with HTLV-I

Abstract

Several HTLV-I-infected T-cell lines express the two genes encoding platelet-derived growth factor (PDGF). Therefore, we examined the question of a possible self-stimulatory mechanism of proliferation involving PDGF in these cells. Using a nucleic acid probe and an antibody specific for the PDGF-B receptor (PDGFR-B), we examined four established human T-cell lines infected with HTLV-I, as well as several noninfected T-cell lines for expression of the PDGF-B receptor. Previous reports indicate that lymphocytes do not display receptors for PDGF; our results show that two noninfected T-cell lines (HUT-78, CCRF-HSB-2) expressed the canonical 5.5-kb PDGFR mRNA, and two HTLV-I-infected T-cell lines (C10/ MJ, MT-2) expressed a novel PDGFR mRNA of 4.8 kb. Concomitantly, the cell lines expressing PDGFR mRNA also synthesize PDGFR proteins immunoprecipitated by the antibody to PDGFR-B. Differences were observed in the molecular weight of PDGFR molecules immunoprecipitated from uninfected and HTLV-infected T-cells. Immunofluorescence studies demonstrated that the PDGFR-B proteins are localized primarily on the cell external membrane. The results suggest that the HTLV-I-infected T-cells acquire an autostimulatory mechanism of cell proliferation that involves PDGF.     

Induction of Concanavalin A Dose-dependent T-Cell Growth Factor Production by Insertion of T-Cell Membrane Components into the AKR Thymic Lymphoma BW 5147

Sendai virus vesicles were used as vehicles for the insertion of various cell membranes into different cell lines. The transplantation efficiency was measured by using FITC-labelled concanavalin A (Con A) or monoclonal antibodies against the T-cell marker Lyt 2 and the major histocompatibility complex product H-2Db in a fluorescence-activated cell sorter. Results indicate that it is possible to transplant mitogen responsiveness to certain cell types. Con A stimulation of T-cell membrane transplanted BW 5147 showed that it is possible to induce a mitogen dose-dependent T-cell growth factor production. Consequently this method appears to be an attractive model for further study of the properties of the membrane structures involved in mitogen triggering of cells.     

Enhancement of IgE Synthesis in the Human Myeloma Cell Line U-266 with an IgE Binding Factor from a Human T-Cell line

An IgE-binding factor(s) (IgE-BF(s] was partially purified from the supernatant of human HTLV-II carrying T-cell line MO. This IgE-BF(s) was shown to increase the IgE synthesis in the human myeloma cell line U-266, but did not affect its viability or growth. The effect of the IgE-BF(s) was dose-dependent and selective for IgE protein synthesis as beta 2-microglobulin synthesis in the U-266 and the immunoglobulin production in the U-1958 IgG-secreting human myeloma cell line were unaffected. The IgE-BF(s) increased the production of the epsilon heavy chain but not the lambda light chain production. The IgE-BF(s) was distinct from IL-1 beta, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-alpha, -beta, -gamma, M-CSF, and fragments of CD23.     

Long-Term Culture of Human Bone Marrow Stromal Cells in the Presence of Basic Fibroblast Growth Factor

Abstract

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells. Normally, large numbers of human bone marrow stromal cells are difficult to obtain. However, nanogram/ml concentrations of bFGF stimulate the growth of passaged bone marrow stromal cells both in media formulated for optimal growth of stromal cells and in a simple mixture of RPMI-1640 and 10% fetal calf serum facilitating the successive expansion of stromal cells through multiple passages. bFGF also greatly accelerates the formation of a primary stromal cell layer following inoculation of newly harvested bone marrow cells into dishes. In the presence of bFGF, the stromal cells attain high densities, lose their contact inhibition and grow in multilayered sheets. Heparin greatly potentiates the stimulatory effect of low concentrations of bFGF. The effects of bFGF are fully reversible: cells cultured in the presence of this factor for multiple passages revert to normal growth rates following trypsinization and subculture. A short (4 h) exposure of the cells to bFGF elicits profound growth stimulation. This supports the hypothesis that this factor binds to glycosaminogly-cans in the cell matrix which act as a storage reservoir for this cytokine.     

Characterization of Keratinocyte Growth Factor and Receptor Expression in Human Pancreatic Cancer

Keratinocyte growth factor (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer growth and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast growth factor (FGF) receptor type II (FGFR-2) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-2 in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-2 and examined the expression of KGF, KGFR, and FGFR-2 in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-2 immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-2 immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-2 immunoreactivity. By in situ hybridization, KGF, KGFR, and FGFR-2 were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-2 are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell growth in vivo.     

Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4⁺ and CD8⁺ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.Utilization of cryopreserved peripheral blood mononuclear cells (PBMC) for immunologic assays has dramatically increased in recent years. Cryopreserved PBMC are particularly useful in clinical trials with low end-point frequencies because they allow the immunologic assays to be performed after the conclusion of the studies, when all the end points have already been identified (1, 14, 18). In addition, the use of cryopreserved PBMC allows all assays to be performed in a single laboratory, eliminating interlaboratory variability, which has been a confounder in some studies (15). Furthermore, if changes in immunologic parameters over time are the main outcome of the immunologic studies, interassay variability may become a confounder, too. Testing all the cryopreserved PBMC per subject at one time can eliminate this confounder.The use of cryopreserved PBMC in immunological assays poses challenges, including the availability of adequate equipment (7) and the need for technical proficiency. Assays have to be adapted and validated for the use of cryopreserved PBMC (4, 6, 9, 11, 17, 19), and the quality of the frozen cells has to be monitored (5) to ensure reliable results in functional and phenotypic assays.The Cryopreservation Working Group (WG) of the Pediatric AIDS Clinical Trials Group (PACTG), which operated between 1999 and 2006, developed a series of experiments aimed at optimizing methods of PBMC cryopreservation for assays requiring viable cells, adapting immunologic assays to cryopreserved PBMC, and establishing quality control parameters for immunologic assays with cryopreserved PBMC. The group focused on highly complex functional and phenotypic assays commonly used as outcome measures in studies that address immune suppression or reconstitution. Unlike previous studies of cryopreserved PBMC in immunologic assays, which were done at single laboratories (3, 8, 13, 17), our study presents results obtained at eight laboratories, substantiating the generalizibility of our findings.     

碱性成纤维细胞生长因子对体外培养的乳兔软骨细胞中p53 mRNA表达的影响

目的 探讨在乳兔关节软骨细胞体外培养过程中,成纤维细胞生长因子（bFGF）对p53mRNA表达水平的影响及意义。方法 原代体外培养乳兔关节软骨细胞并传代,实验组加入10ng／ml的bFGF,光镜下观测各代细胞形态学变化,RT—PCR检测各代细胞中p53基因在mRNA水平上的表达。结果光镜观察体外培养的软骨细胞,对照组第4代培养细胞始出现凋亡细胞,bFGF组第7代出现少量凋亡细胞;RT—PCR检测bFGF组p53的目的基因指数（p53吸光光度值,β—actin吸光光度值）明显低于对照组（P〈0．05）。结论 在体外培养的兔关节软骨细胞中bFGF下调p53基因mRNA水平表达。     

Structure of the T-Cell Receptor in a Tiαβ, Tiγδ Double Positive T-Cell Line

The multichain T-cell receptor is composed of at least six different polypeptide chains. The clonotypic Ti heterodimer (Tiαβ or Tiγδ) is non-covalently associated with the CD3 chains (CD3γδɛζ). The exact number of subunits constituting the T-cell receptor is still not known. It has been suggested that each T-cell receptor contains two Ti dimers. To gain insight into the structure of the T-cell receptor we constructed a Tiαβ, Tiγδ double positive T-cell line which contained four functional Ti chains (Tiαβ, β, γ, and δ). The data demonstrated an absence of Ti dimers containing mixtures of chains other than the typical Tiαβ and Tiγδ combinations. Furthermore, by co-modulation experiments we demonstrated that the Tiαβ and the Tiγδ dimers were not expressed in the same T-cell receptor. Our data indicate that the T-cell receptor does not contain two Ti dimers.     

Isolation and Characterization of a Thymic Factor

A protein was isolated from bovine thymus that was shown to accelerate the appearance of hemolysin to sheep erythrocytes in neonatal mice. The isolation procedure consisted of saline homogenization, ammonium sulfate precipitation, and methanol precipitation, followed by chromatography on diethylaminoethyl-cellulose and two chromatographies on Sephadex G-150. The protein was shown to be homogeneous by polyacrylamide gel electrophoresis and by sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. The molecular weight was determined to be 79,950. Additional physical and chemical characteristics were also determined.     

Human T-Cell Hybrids Secreting Lymphokines: Effect of Different Parent Cell Clones of the Human T-Cell Acute Lymphoblastoid Leukaemia Line CCRF-CEM

We have cloned a number of cell lines from the human T-lymphocyte acute lymphoblastoid leukaemia (ALL) line CCFR-CEM, and attempted to construct functionally active human T-lymphocyte hybrids with them. Functional hybrids were generated using only one particular clone, 3H6. The activities found in the supernatants of two of these hybrids, DB1G7 and DB2D10, are described. Supernatant from DB1G7 was found to suppress strongly the migration of normal human peripheral blood mononuclear cells, while that from DB2D10 was shown to inhibit the proliferative response of human T lymphocytes to both phytohaemagglutinin and concanavalin A. There was no cross-reactivity between the two supernatants, confirming the usefulness of the human T-lymphocyte hybrid technique in dissecting human T-lymphocyte function. The successful use of 3H6 is contrasted with the failure of another clone, 2H2, to permit the production of functional hybrids. Problems relating to the use of CCRF-CEM and its clones as parent cell lines in the production of human T-lymphocyte hybrids are discussed.