T cell growth factors from adult T cell leukemia virus-transformed cell lines

Some characteristics of T cell growth factors derived from adult T cell leukemia virus (ATLV)-transformed cell lines, MT 1 and MT 2 were analyzed. MT 1 cells release significant interleukin 2 (IL 2) activity into the culture medium, which showed the same elution pattern of gel filtration and isoelectric focusing of IL 2 from lectin-stimulated normal human lymphocytes. This activity was also detected in the cell extract of MT 1. In contrast, MT 2 cell line did not produce IL 2 activity, but non-IL 2 type growth factor was observed. The significance of these factors from MT cell lines is discussed from the viewpoint of 'autokine' in ATLV-transformed cells.     

Human T lymphotropic virus type-I (HTLV-I) immortalizes human T cells in vitro--its implication in the pathogenesis of adult T cell leukemia (ATL).

HTLV-I is the first human retrovirus that was isolated from a patient with T-cell malignancy in 1980 in the United States. HTLV-I is detected in most patients with adult T cell leukemia (ATL) and healthy carriers, who are frequently found in the southwestern parts of Kyushu and Shikoku Districts. HTLV-I-infected cells express IL-2 receptors, and HTLV-I-infected T cell lines can be established from most of ATL patients in culture in the presence of IL-2. Furthermore, these IL-2 dependent T cell lines often begin to proliferate in the absence of IL-2 and to not respond to IL-2, despite IL-2 receptors on their cell surface, thus mimicking ATL cells in vivo. These findings suggest that HTLV-I is an etiological agent of ATL. In this mini-review, the T cell immortalizing activity of HTLV-I in vitro, with special reference to the evolution of ATL cells based on our results, is described.     

Production of and response to interleukin 1 by cloned human oligodendroglioma cell lines

Clones and subclones of the human oligodendroglioma line TC620 were characterized with respect to surface and cytoplasmic markers as well as for ability to produce and respond to immunoregulators of inflammation such as products of arachidonic acid metabolism, interleukin 1(IL1) and tumor necrosis factor (TNF). Clone 1.0 and its subclone 1.3 both resembled immature oligodendroglia or precursor-type cells. Both clones were 100% positive for surface galactocerebroside (Gal C), though distinct with respect to predominance of surface gangliosides and lectin receptors. Both clones responded to IL1 beta and IL1b by proliferation and both constitutively released IL1 alpha. The parental line also produced some IL1 but did not proliferate in response to IL1. The IL1 alpha produced could be detected in supernatants as well as cell lysates of TC620 1.0 and 1.3. Neither clone produced prostaglandin E (PGE), leukotriene B4 (LTB4), or tumor necrosis factor (TNF) but IL1 alpha production by 1.3 could be influenced by exogenous PGE and TNF. Response to IL1 beta and IL1 alpha could be specifically inhibited by anti IL1 alpha or beta respectively. The clones also responded to autologous conditioned supernatants. This response was partially inhibited by anti IL1 alpha but not anti IL1 beta, indicating that the factor in the supernatant was IL1 alpha-like.     

Growth of AKR T cell leukemia lymphoblasts in medium containing interleukin 2 (IL-2)

Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteristics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quantify IL-2 activity.     

Growth of AKR T cell leukemia lyphoblasts in medium containing interleukin 2 (IL-2)

Summary Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteriztics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect, to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quatify IL-2 activity. Supported by Grants CA-20484 and CA-26245, from the National Cancer Institute, Department of Health and Human Services and grants from the Briggs and Stratton Corporation Foundation, the Evan and Marion Helfaer Foundation, the Kearney and Trecker Foundation, the MACC Fund, the Miller Foundatione and the Stackner Family Foundation. This study represents partial fulfillment for a Ph.D. in Biology from the Marquette University, Milwaukee, Wisconsin.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.     

Production of cytotoxic factor(s) in human T cell lines transformed by a human retrovirus

Human T cell lines, MT-2, TCL-Ter, TCL-Haz, and TCL-Kan which were transformed by a human retrovirus, constitutively produced cytotoxic factor(s) (CF) in the culture supernatants. In these cell lines, MT-2 produced the largest amount of CF. The amount of CF produced by MT-2 was 9-10 or 3-4 times larger than that produced by a human B cell line, RPMI 1788, or normal peripheral blood leukocytes stimulated with mitogens and phorbol ester. The kinetics of the production by MT-2 was similar in media with and without serum. The activity was stable at 56 degrees C for 30 min but was lost at 80 degrees C for 30 min and at pH 2 for 20 hr. On gel filtration, the molecular weight of the factor produced by MT-2 was approximately 90,000. On isoelectric focusing, the activity was recovered in the fraction at pH 6.5-7.0.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.     

Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

Immortalization of antigen specific, helper T cell lines by transformation with the radiation leukemia virus (RadLV)

Antigen-specific immune T lymphocytes of male C57BL/6 mice were enriched in vitro on monolayers of antigen-pulsed syngeneic macrophages. The cells were treated in vitro with RadLV and inoculated intrathymically into irradiated female C56BL/6 animals. Thymomas arising in the inoculated recipients were characterized as donor- (male) type according to their karyotype. In vivo and in vitro cell lines were established from the primary lymphomas, two of which (designated ROT/6.1 and ROT/6.2) were capable of providing antigen- (carrier) specific help in normal or preimmunized mice. None of the lymphomas could induce antigen-specific DTH reaction. Five months after their establishment, ROT/6.2 alone retained its carrier specificity. ROT/6.2 consisted mainly of Lyt-1+ cells, whereas the ROT/6.1 population was more heterogeneous and contained Lyt-1+, Lyt-2+, and Lyt-3+ cells. The carrier specificity of the latter may have been lost due to selection against the specific helper cells during prolonged passages.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

Production of interleukin 3 and gamma-interferon by an antigen-specific mouse suppressor T cell clone

An interleukin 2 (IL 2)-dependent, keyhole limpet hemocyanin (KLH)-specific, mouse suppressor T cell clone, 3D10, was found to produce interleukin 3 (IL 3) and gamma-interferon (IFN-gamma) in response to T cell mitogens Con A and PHA. Different from KLH-specific suppressor factor (TsF) that was spontaneously released into the medium when cultured in IL 2-containing conditioned medium, the production of IL 3 and IFN-gamma was induced by mitogenic stimuli. IL 3, IFN-gamma, and TsF were separable by gel filtration through a Sephadex G-100 column, being recovered in fractions of m.w. 25,000 to 30,000, 45,000 to 50,000 and 60,000 to 70,000, respectively. On the other hand, minimum size of IL 3 and IFN-gamma were shown to be about 25,000 and 20,000, respectively, by determining the lymphokine activities contained in the extracts from slices of SDS gels. These results indicate that IFN-gamma was present as a homodimer or hetero-complex with another carrier protein(s), whereas IL 3 was present as a monomeric form. A highly positive correlation (a correlation coefficient r = 0.96) between the titers of IL 3 and IFN-gamma produced by seven subclones derived from 3D10 was obtained, suggesting that IL 3 and IFN-gamma are induced by a process with a common mechanism. 3D10 also produced IL 3 and IFN-gamma when cultured with its specific antigen, KLH, in the presence of antigen-presenting cells. When Con A-stimulated 3D10 cells were labeled with L-[35S]methionine, we found that at least three proteins, with m.w. of 35,000, 25,000, and 20,000, were specifically released into medium by the stimulation. The latter two may be IL 3 and IFN-gamma described above, respectively, because of the similarities in m.w.     

Production of high levels of interleukin 1-like activity by the SPI-802 human leukemia cell line with E receptors

The SPI-802 human leukemia cell line, which possesses E receptors and used to have natural killer activity, has been demonstrated to produce high levels of interleukin 1 (IL-1)-like activity. SPI-802 supernatants prepared in 1% serum-containing cultures with lipopolysaccharide stimulation, like similarly prepared adherent-cell-derived IL-1, enhanced phytohemagglutinininduced mouse thymocyte proliferation. When adherent-cell IL-1 gave 50% maximum activity at a reciprocal dilution of 20, SPI-802 supernatant gave it at 200, indicating the production of high levels of IL-1-like activity by the cell line. SPI-802 supernatant promoted the production of interleukin 2 (IL-2) by the Jurkat-F1884 T-cell line: Levels of IL-2 activity obtained with 15% SPI-802 supernatant were almost equivalent to those obtained with 50% adherent-cell IL-1 as estimated by the maximum proliferation of IL-2-dependent cytotoxic T cells. SPI-802 supernatant by itself exhibited no IL-2 activity. Major IL-1-like activity of SPI-802 supernatant was present in fractions from AcA54 columns corresponding to M, 12,000–20,000 and 60,000–70,000 and resolved on isoelectrofocusing into two distinct species with pI values of 5.0 and 7.0, being consistent with the results of adherent-cell IL-1. The SPI-802 cell line having E receptors is an ideal source of a soluble factor with the biological and biochemical Characteristics of human IL-1.     

Human B lymphoblastoid cell lines provide an interleukin 1-like signal for mitogen-treated T lymphocytes via direct cell contact

The B lymphoblastoid cell lines (B-LCL) 8392, SB, 1788, and Daudi provide accessory cell activity for mitogen-treated T cells, whereas the T lines MOLT-4, 8402, CEM, and HSB do not provide this function. Direct cell contact is required for the accessory cell activity, and active lymphocyte growth factors could not be detected in the supernatants of the B-LCL. The B-LCL also present alloantigens to responding T cells, and this response is independent of additional accessory cells. The target for the B-LCL is the responding T cell itself, rather than a minor contaminating population of endogenous accessory cells. This conclusion is based on the finding that, under culture conditions in which T cells do not proliferate in response to PHA, accessory cell activity of the B-LCL is maintained. Paraformaldehyde- or glutaraldehyde-treated B-LCL retain their accessory cell activity at levels of these agents that completely eliminate metabolic activity of the B-LCL, as determined by incorporation of leucine, thymidine, and uridine into macromolecules. This treatment eliminates alloantigen presentation by the B-LCL. T cells treated with IO-4 or with monoclonal anti-T3 antibodies fail to respond to highly purified IL 1, and respond minimally to supra-optimal concentrations of IL 2. Nevertheless, these cells respond maximally to the accessory cell activity of the B-LCL. The IO-4 treated cells or cells exposed to anti-T3 also proliferate in response to TPA. Together, our data suggest that the B-LCL provide an IL 1-like signal for mitogen-treated T cells via direct cell contact, in the absence of detectable soluble IL 1.     

Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Superinduction of T lymphocyte-endothelial cell (EC) binding by treatment of EC with interleukin 1 and protein synthesis inhibitors

We have previously reported that marked enhancement of the in vitro binding of lymphocytes to endothelial cell (EC) monolayers is observed after stimulation of the EC with interleukin 1 (IL 1). To determine whether new protein synthesis was required for this effect of IL 1, EC were incubated with IL 1 in the presence of cycloheximide or puromycin. Three different effects of these protein synthesis inhibitors on T-EC binding were observed. First, preincubation of the EC with both IL 1 and an inhibitor blocked the increase in binding if the inhibitor was present during both the preincubation and the 1 hr duration of the T-EC binding assay, suggesting that new protein synthesis is required for the enhancement of T-EC adhesion by IL 1. Second, preincubation of the EC with low doses of the inhibitors (0.1 to 1 microgram/ml) in the absence of IL 1 consistently increased T-EC binding, even if the inhibitors were present during the T-EC adhesion assay; in addition, the inhibitors additionally increased the stimulatory effect of IL 1 if the EC were washed free of the inhibitor before the assay step. The binding-enhancing effect of low concentrations of cycloheximide could be inhibited by an antibody to the CDw18 complex on the T cell, suggesting an up-regulation of the ligand on the EC involved in CDw18-dependent T cell adhesion. Third, higher concentrations of the inhibitors (3 to 10 micrograms/ml) were toxic for the EC in the presence of IL 1, possibly due to the combined blocking effect of IL 1 and inhibitors on EC protein synthesis.     

Lysis of human T cell leukemia virus infected T and B lymphoid cells by interleukin 2-activated killer cells

The human T cell leukemia (HTLV-1) retrovirus is the etiologic agent for adult T cell leukemia. Interleukin 2 (IL-2) activated killer (AK) cells have been shown to lyse freshly explanted tumor cells in vitro and have been used as a form of adoptive immunotherapy for the treatment of cancer. In this report, the ability of AK cells to lyse HTLV-1-infected targets was examined. Normal lymphocytes, when cultured in recombinant IL-2 for periods of 3 to 7 days, killed infected T and B cell lines. The precursor for these AK cells resided in the CD-16 antigen-positive subset (i.e., natural killer (NK) cells). Resting T cells, NK cells, or unfractionated lymphocytes did not lyse the infected targets. However, when isolated NK cells were incubated for 24 hr in IL-2, suboptimal cytolysis was induced whereas activation of NK cells with a four pulse of IL-2 was insufficient to generate effector cells. The results of performing cold target inhibition studies with Epstein-Barr virus-infected B cell lines and HTLV-1-infected T and B cell lines suggest that there are discrete subsets (i.e., clonotypic) in the AK population that preferentially lyse a given virally infected cell line. Thus to consider AK cells as true polyspecific killer cells may be inaccurate. Alternately AK cells may express a number of different receptors with variable affinities for the Epstein-Barr virus- and HTLV-1-infected cell lines. In addition, it was shown that HTLV-1-infected B cells are relatively resistant to AK cell-mediated lysis. These results clearly indicate that AK cells but not resting NK cells kill HTLV-1-infected cells.     

Heterogeneity in response to interleukin 2 and interleukin 2-producing ability of adult T cell leukemic cells

To examine the possibility of heterogeneous mechanisms in the proliferation of adult T cell leukemia (ATL) cells, leukemic cells from 13 patients, nine acute-type and four chronic-type ATL, were examined for the production of interleukin 2 (IL 2) with or without mitogenic stimulation and their response to recombinant IL 2 when exogeneously added. The leukemic cells were classified into four groups, as follows. Group 1 (two patients): Cells of this group produced IL 2 messenger RNA, secreted IL 2, and proliferated when cultured in mitogen-free medium. The spontaneous proliferation of the cells in mitogen-free medium was inhibited by anti-Tac/IL 2 receptor and anti-IL 2 monoclonal antibodies. Moreover, the thymidine incorporation by the cells was enhanced in response to exogeneously added recombinant IL 2 and IL 2 produced by themselves. These results indicate that the ATL cells of this group proliferate with autostimulation by IL 2. Group 2 (seven patients): Cells of this group did not secrete IL 2 when cultured in mitogen-free medium, but the cells showed response to exogeneously added recombinant IL 2 and proliferated in culture. These results indicate that the ATL cells of this group proliferate by a paracrine mechanism. Group 3 (one patient): Cells of this group secreted IL 2 in mitogen-free medium. However, the spontaneous proliferation of these cells in vitro was very low, and the response to recombinant IL 2 was also very low. Group 4 (three patients): Cells of this group did not secrete IL 2 in mitogen-free medium. Spontaneous proliferation and the response to recombinant IL 2 were also very low. The clinical feature of all patients of Groups 1 and 2 was acute-type, and that of Groups 3 and 4 was chronic-type. Thus, we conclude that heterogeneous mechanisms exist in the proliferation of leukemic cells, and that growth rate in mitogen-free medium and response to IL 2 of the cells may have a significant relationship to the clinical feature, acute- or chronic-type.