Continuous T-cell Lines Research Articles

Longitudinal immune monitoring of patients receiving intratumoral injection of a MART-1 T-cell receptor-transduced cell line (C-Cure 709)

Adoptive transfer of tumor-specific lymphocytes is a promising strategy in the treatment of cancer. We conducted intratumoral administration of an allogeneic irradiated continuous T-cell line (C-Cure 709) expressing an HLA-A2-restricted MART-1-specific T-cell receptor (TCR) into HLA-A2(+) melanoma patients. The C-Cure 709 cell line is cytotoxic against MART-1(+) HLA-A2(+) melanoma cell lines and secretes several immune stimulatory cytokines upon stimulation. Anti-tumor immune responses against the commonly expressed tumor antigen (Ag) MART-1 were longitudinally analyzed in peripheral blood by fluorescence-activated cell sorting (FACS) before and after intratumoral injection of C-Cure 709. No treatment-induced increase in Ag-specific T-cell frequencies was observed in peripheral blood, and the phenotype of MART-1-specific T cells was very stable during the treatment. Interestingly, despite a very stable frequency of MART-1-specific T cells over the course of treatment, clonotype mapping revealed that the response was in fact highly diverse and dynamic, with new clonotypes emerging during treatment. Only a few clonotypes were recurrently detected in consecutive samples. One MART-1-specific T-cell clone disappearing from peripheral blood was later detected in a metastatic lesion. Sequence analyzes of the CDR3 region revealed conserved structural characteristics in the MART-1-specific TCR used by T-cell clones.

Expression of CD10 by Human T Cells That Undergo Apoptosis Both In Vitro and In Vivo

AbstractThis study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

Expression of CD10 by Human T Cells That Undergo Apoptosis Both In Vitro and In Vivo

This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

FIV infection of IL-2-dependent and -independent feline lymphocyte lines: Host cells range distinctions and specific cytokine upregulation

We have analyzed the ability of three molecular clones of feline immunodeficiency virus (FIV) and an ex vivo variant to infect nine distinct specific-pathogen-free feline cell lines in tissue culture. The purpose of these studies was to elucidate mechanisms by which host cells regulate the level of virus infection and expression and to assess host cell cytokine responses to virus infection. Cells used for the analyzes included four IL-2-dependent continuous T-cell lines (104-C1, 104-C7, MCH5-4 and DB FeTs) which arose from long-term passage, followed by limiting dilution cloning of peripheral blood mononuclear cells (PBMCs); two IL-2-independent T-cell lines (104-C1DL and MCH5-4DL) which originated from two of the IL-2-dependent lines, 104-C1 and MCH5-4; respectively; Crandell feline kidney cells (CrFK); G355-5 brain-derived glial cells; and the T-cell lymphoma line, 3201. Cells were infected with FIV-PPR, FIV-34TF10, FIV 34TF10orf2rep, and a variant arising from FIV-PPR during ex vivo passage on 104-C1DL cells, termed FIV-PPRglial. Infection of the IL-2-dependent T-cell line, 104-C1, by FIV-PPR resulted in the specific and distinct upregulation of cytokine expression. In particular, these cells doubled their expression of the pleiotropic cytokines, interleukin-4 and interleukin-12 after FIV infection. Interferon-γ production also increased after infection with FIV whereas, TNFα expression remained constant. Also, a marked upregulation of MHC class II expression was noted post infection of MCH5-4 and 104-C1 cells with FIV-PPR. Similar results were obtained after infection with FIV-34TF10orf2rep, indicating that the upregulation of cytokine expression is not an isolate-specific phenomenon. Changes in cytokine and class II expression are similar to various reports for the in vivo cytokine alterations in FIV, SIV and HIV infections. The ex vivo infection of these cell lines offers a manipulable system to examine the mechanism(s) by which lentiviruses alter cytokine expression.

Purine ribonucleotide content in infected HIV-RT + and HIV-RT − lymphoblastoid cell lines

We have studied the purine nucleotide metabolism in the following cell lines: a), H9 (continuous human T-cell line) and H9/HTLV-III (H9 cell line, infected with RT+ HIV-I virus); b), A3.01 (human lymphoblastoid cell line CD4+) and 8E51 (line A3.01 permanently transfected with RT-HIV-I virus). Purine metabolism was studied by evaluating the content of the most important ribonucleotides (AMP-GMP-IMP-NAD-ADP-GDP-ATP-GTP) and their ratios. We determined several differences between the cell lines before and after viral infection. All nucleotides except triphosphates were reduced in H9/HTLV-III with respect to H9 cells; in 8E51, however, triphosphates were markedly reduced, while monophosphates increased with respect to A3.01 uninfected cells. Also the ratios exhibited different behaviors, for example the total adenine nucleotides total guanine nucleotides ratio (sigma A/sigma G) was enhanced in H9/HTLV-III cells with respect to H9 and unaltered in 8E51 with respect to A3.01 cells. We may conclude that the HIV-I virus strongly influences the purine nucleotide metabolism of the host cells and that the changes are different when induced either by RT+ or RT- virus.

Common clonal chromosome aberrations in cytokine-dependent continuous human T-lymphocyte cell lines

Antigen-mediated T-cell proliferation is a transient phenomenon. Like other somatic cells, T lymphocytes generally show replicative senescence in vitro. However, we here show that cytokine-dependent continuous (immortal) T-cell lines can be established from skin biopsy specimens of inflammatory skin diseases. Continuous growth can be obtained by culturing T cells in medium supplemented with interleukin-2 and interleukin-4, but without antigen or antigen-presenting cells added. Loss of the T-cell antigen receptor complex is observed in some of the continuous T-cell lines. Most T-cell lines develop clonal chromosome aberrations during continuous growth. Aberrations for chromosomes 1, 2, 8, 16, and 18 are most commonly observed.

Common clonal chromosome aberrations in cytokine-dependent continuous human T-lymphocyte cell lines

Antigen-mediated T-cell proliferation is a transient phenomenon. Like other somatic cells, T lymphocytes generally show replicative senescence in vitro. However, we here show that cytokine-dependent continuous (immortal) T-cell lines can be established from skin biopsy specimens of inflammatory skin diseases. Continuous growth can be obtained by culturing T cells in medium supplemented with interleukin-2 and interleukin-4, but without antigen or antigen-presenting cells added. Loss of the T-cell antigen receptor complex is observed in some of the continuous T-cell lines. Most T-cell lines develop clonal chromosome aberrations during continuous growth. Aberrations for chromosomes 1, 2, 8, 16, and 18 are most commonly observed.

Levels of free purine nucleotides in continuous T-cell lines infected with virus HIV-1

The purine nucleotide content of H9 lymphoblastoid cell lines shows significant variations after infection with HIV-1 virus. demonstrating; the influence of the virus on purine metabolism in the infected cell.

Phenotypic and genetic polymorphisms among human herpesvirus-6 isolates from North American infants

Fifteen human herpesvirus-6 (HHV-6) isolates from North American infants with primary infection manifest as febrile or roseola ( exanthem subitum) like illnesses were characterized phenotypically on the basis of their in vitro growth in continuous T-cell lines and primary human mononuclear cells and by their reactivity with monoclonal antibodies. All isolates replicated efficiently in primary human cord blood mononuclear cells, but five distinct patterns of viral replication in human cells lines were observed. Two of the HHV-6 isolates from infants were found to replicate in HSB-2 cells, a property associated with so-called group A viruses, which had previously been isolated only from adults. These same isolates also reacted with a panel of A-specific monoclonal antibodies. Genomic characterization of viral isolates using well-characterized restriction site polymorphisms indicated that these two isolates contained a mixture of both A- and B-type genomes, in different proportions. These data suggest that not all HHV-6 isolates can be categorized into one of two broad groups and that such segregation of HHV-6 isolates may in fact be misleading.

Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates

Fifteen human herpesvirus-6 (HHV-6) isolates from normal donors and patients with AIDS, systemic lupus erythmatosis, chronic fatigue syndrome, collagen-vascular disease, leukopenia, bone marrow transplants, Exanthem subitum (roseola), and atypical polyclonal lymphoprolfferation were studied for their tropism to fresh human cord blood mononuclear cells, growth in continuous T cell lines, reactivity to monoclonal antibodies, and by restriction enzyme banding patterns. All isolates replicated efficiently in human cord blood mononuclear cells, but mitogen stimulation of the cells prior to infection was required. The ability to infect continuous T-cell lines varied with the isolates. Isolates similar to GS prototype infected HSB2 and Sup T1 cells and did not infect Molt-3 cells, whereas isolates similar to Z-29 infected Molt-3 cells but not HSB2 and Sup Ti cells. Some of the monoclonal antibodies directed against the HHV-6 (GS) isolate showed reactivity with all isolates tested, but others only reacted with HHV-6 isolates similar to the GS isolate and not with those similar to Z-29 isolate. Restriction enzyme analysis using EcoRI, BamHI, and HindIII revealed that HHV-6 isolates from roseola, bone marrow transplant, leukopenia, and an HIV-1-positive AIDS patient from Zaire (Z-29) were closely related but distinct from GS type HHV-6 isolates. Based on the above findings, we propose that, like herpes simplex virus types 1 and 2, the 15 HHV-6 isolates analyzed can be divided into group A (GS type) and group B (Z-29 type).

C-type particles are inducible in Se-Ax, a continuous T-cell line from a patient with S�zary's syndrome

Se-Ax is a continuous mature T-cell line that we have established from a patient with Sézary's syndrome. An important finding was that the Se-Ax cell line required human serum for initial growth. Here we show that transfer of the permanent cell line to a medium deficient of human serum induces production of C-type retroviral-like particles with a unique morphology. Ultrastructurally, these particles are 120 nm in diameter with hexagonal shape, and show a small, centrally located round core of 30 nm. They are observed only extra-cellularly; typical budding, however, is not found. Both by serological testing and molecular analysis we substantiate the morphological conclusion that these particles do not represent common human or animal retroviruses. The inducibility of the particles may be a hint as to a possible endogenous origin.

Unique p24 epitope marker to identify multiple human immunodeficiency virus variants in blood from the same individuals

The human immunodeficiency virus (HIV) was isolated from the blood of 192 of 410 seropositive individuals. Original isolations were made in peripheral blood mononuclear cell (PBMC) cultures, and only one-fifth of the HIV isolates could be adapted to replicate in continuous T-cell lines. Of the 192 HIV isolates, 42 had the characteristic p24 antigen marker of the acquired immunodeficiency syndrome-associated retrovirus type 2 strain of HIV (HIV ARV-2) and 150 resembled the human T-cell lymphotropic virus type III strain of HIV (HIV HTLV-III). Significantly, primary PBMC cultures from two patients yielded multiple variants. When these variants were exposed to continuous T-cell lines, only one of them continued to replicate. The remaining variants were lost and could not be reisolated following passage back into PBMC cultures. We conclude the following from these studies: PBMC cultures are more efficient at isolating HIV than continuous T-cell lines are; some patients harbor more than one genetic variant of HIV in the blood at the same time; and continuous T-cell lines are likely to yield only a portion of the HIV variants originally present in the blood.

Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus.

We have developed a continuous human T-cell line (A3.01) for the study of acquired immunodeficiency syndrome (AIDS)-associated retrovirus that mimics normal peripheral blood lymphocytes in susceptibility to viral cytopathic effect without the need for cell activation or conditioned medium. Following infection, substantial quantities of virus are produced during a 3- to 5-day period; the associated killing of cells can be monitored in a microtiter assay as a function of virus input. Southern blot hybridization of infected cellular DNAs indicated that no gross alteration occurred in the restriction maps of the proviral DNA during the transfer of virus to and its passage in A3.01 cells. This cell system offers an alternative to other AIDS retrovirus cell systems because it permits the monitoring of viral cytopathic effects.

Studies on the liver sequestration of lymphocytes bearing membrane-associated galactose-terminal glycoconjugates: Reversal with agents that effectively compete for the asialoglycoprotein receptor

The removal of “effete” glycoproteins from the circulation represents a proposed physiologic role for the hepatocyte asialoglycoprotein receptor. Our experiments support the hypothesis that this receptor may also be directly involved in the removal from the circulation of cells bearing asialoglycoconjugates. We report that the enhanced liver localization of neuraminidasetreated lymphocytes can be competitively inhibited by the coinjection of asialofetuin (ASF). Fetuin itself was without effect. Competitive inhibition of the liver receptor allowed normal localization to lymphoid tissues of the enzyme-treated lymphocytes, a condition which persisted as long as free ASF was present in the circulation. Our studies support the concept that cell surface carbohydrates play an important role in the tissue distribution of circulating lymphocytes. The process of thymocyte maturation, bone marrow transplantation, and the adoptive immunotherapy with continuous T-cell lines represent conditions where recirculation potential may be influenced by the presence of galactose terminal glycoconjugates.

An insight into the T-cell recognition systemusing antigen-specific functional T-cell lines

Summary For a better understanding of the molecular nature of the antigen-specific T-cell recognition system, continuous T-cell lines specific to the synthetic polypeptide (T,G)-A-L, have been established from murine and human activated T cells. These lines are constitutive secretors of antigen-specific T-cell replacing helper factors. Lines prepared from (T,G)-A-L activated C3H.SW (high-responder) T cells were cloned and characterized. The cells and their secreted products were identical in their fine antigenic specificity, as tested using synthetic polypeptides closely related to (T,G)-A-L. The secreted T-cell helper factor was shown to possess MHC determinants as well as V-region determinants or, more specifically, idiotypic determinants. Using the fluorescence-activated cell sorter (FACSII), we demonstrated the expression of cross-reactive idiotypic markers on the monoclonal helper T cells. Gel analysis of the twice affinity-purified eluate of a (T,G)-A-Lcolumn revealed the existence of iodinated bands with a molecular weight of 15 kd and 17 kd, in addition to a diffuse band of high molecular weight. The specific helper activity of the factors was associated with a 65–75% ammonium sulfate precipitate. Gel electrophoresis experiments indicated that both a high (less than 67 kd) and a low (15–17 kd) molecular weight fraction contained the biological activity of the factor. The two fractions were also shown to be synthesized by the T-cell lines, as indicated by internal labelling experiments using 35 S-methionine. A (T,G)-A-L-specific long-term T-cell line of human origin which constitutively secreted a helper factor, was established from antigen-activated T cells of a donor previously determined to be a responder to (T,G)-A-L. In addition to the antigenic specificity of the factor, it was found to possess V H determinants in addition to the relevant HLA-DR determinants. The latter finding was confirmed by the demonstration that the factor reacted with monoclonal anti-Ia antibodies. Thus, the molecular characterization of the active products of the antigen-specific T-cell lines of murine and human origin will hopefully lead to elucidation of the T-cell receptor.

Delayed hypersensitivity induced by anti-T-cell-line antisera is enhanced by pertussigen and not restricted by histocompatibility genes.

Anti-T-cell-line antisera were raised by repeatedly injecting mice with syngeneic, antigen-specific, delayed hypersensitivity (DH) inducing cells grown as continuous T-cell lines in vitro. Many of these antisera could induce antigen-specific DH responses which, in some cases, were rather slight. For example, the DH reaction to azobenzenearsonate induced in A/J mice by an A/J antiserum produced against the syngeneic azobenzenearsonate-specific cell line AA3 was only a weak response. Administration of pertussigen, a protein purified from Bordetella pertussis, markedly enhanced this response and prolonged its duration. Histologically the reaction showed the classical mononuclear infiltration of DH. It could be transferred to naive mice by lymph node cells that were anti-Thy-1.2 sensitive, and the transfer was restricted by genes of the major histocompatibility complex (MHC). The induction of DH by antiserum, however, was not influenced by MHC or non-MHC gene products.

A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor; partial characterization of the receptor.

T-cell growth factor (TCGF or interleukin-2) is an inducible glycoprotein hormone of molecular weight 15,000 (ref. 1) synthesized and secreted by T lymphocytes following activation with antigen or mitogen2,3. TCGF is required for proliferation and expansion of T cells following antigen encounter4,5 and to maintain them in long-term culture in vitro6–9. Full expression of the human immune response requires both the induction of TCGF synthesis and the formation of specific TCGF membrane receptors10,11. Monoclonal antibodies binding TCGF have been prepared12,13. In contrast, antibodies specific for the TCGF membrane receptor have not been identified, nor has the receptor been characterized. We have prepared a monoclonal antibody, termed anti-Tac14,15, which appears to bind to the human membrane receptor for TCGF. In support of this, we now demonstrate that anti-Tac suppresses TCGF induced proliferation of T cells and blocks binding of radiolabelled TCGF to cells from a cloned human continuous T-cell line. Also we have partially purified and characterized the putative TCGF receptor. This receptor is a glycoprotein with a molecular weight (Mr) of 47,000–53,000.

Macrophage activation factors derived from a continuous T-cell line

Macrophage activation factors derived from a continuous T-cell line

Flow cytometry analysis of T cells and continuous T-cell lines from autoimmune MRL/l mice.

The study of spontaneous autoimmunity in mouse models affords an opportunity to determine the cellular basis of the immune dysregulation observed in this disease. Recently, a new mouse strain, MRL/Mp-Ipr/Ipr (MRL/l) has been developed which carries an autosomal recessive gene (lpr) that results in massive lymph node enlargement concomitant with the development of several autoantibodies. The interest in this strain lies in the possibility that the defect in T-cell regulation of the immune response is manifested at a different level from that in the NZB mouse. It has been reported that the proliferating population of lymphoid cells in the nodes of these mice are T cells, but that many of them are devoid of Lyt surface antigens. We have accordingly initiated several lines of research with these mice, including quantitative flow cytometry characterization of Lyt antigen expression of cells in the lymph nodes of the mice. In an approach to isolate and study the properties of these cells, we have also established continuous cell lines from the lymph node cells of MRL/l mice, using techniques similar to those used to establish continuous lines of antigen-activated cytotoxic T cells and helper T-cell populations.

Continuous cytotoxic T-cell lines.

Recently, it was reported that it was possible to maintain normal human T lymphocytes in continuous exponential proliferative culture with the aid of a T-cell growth factor (TCGF) derived from lectin-stimulated peripheral blood mononuclear cells (Morgan et al., 1976; Ruscetti et al., 1977). Subsequently, we (Gillis and Smith, 1977; Gillis et al., 1978a; Baker et al., 1979) and others (Rosenberg et al., 1978; Strausser and Rosenberg, 1978; Nabholz et al., 1978) found that antigen-specific cytolytic T cells could be selected, maintained, and cloned in the presence of TCGF (IL2).* The discovery that such cytolytic T-lymphocyte lines (CTLL) were completely dependent upon TCGF (IL2) for their growth allowed for the development of a rapid, sensitive microassay for this mitogenic factor (Gillis et al., 1978b). This development, in turn, provided the means to perform detailed experimentation directed toward a definition of the biochemical and biological characteristics of TCGF(IL2). The results of these experiments, summarized in this chapter, suggest that the ability to culture functional monoclonal T cells will provide the means leading to an increased understanding of the T-cell immune response, in much the same way that the ability to culture immunoglobulin-secreting plasmacytoma cells has contributed to our understanding of the B-cell immune response.KeywordsConditioned MediumMixed Lymphocyte CultureRetard Tumor GrowthMurine Spleen CellSyngeneic Tumor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.