B-cell stimulatory factor 1 and not interleukin 2 is the autocrine growth factor for some helper T lymphocytes

Clonal expansion of T lymphocytes of the helper/inducer class is generally thought to be mediated by an interleukin 2 (IL-2)-dependent autocrine mechanism. Thus, T cells stimulated by antigens or mitogenic lectins secrete IL-2 and, under appropriate conditions, express membrane receptors for IL-2, and the specific hormone-receptor interaction induces cellular proliferation. Recent studies indicate that B-cell stimulatory factor 1 (BSF-1) is secreted by T cells and is capable of stimulating T-cell proliferation. We now report that BSF-1 and not IL-2 is the sole autocrine growth factor for certain cloned lines of inducer T lymphocytes. On stimulation by the lectin concanavalin A, anti-receptor antibody, or specific antigen with antigen-presenting cells, such clones secrete a lymphokine that stimulates DNA synthesis by the "IL-2 indicator line," HT2, but is identified as BSF-1 by specific inhibition with monoclonal antibodies. The proliferative response of such BSF-1-secreting clones to receptor-mediated signals is dependent on BSF-1 and not IL-2. These results demonstrate a function of BSF-1 and confirm the existence of a previously unknown autocrine pathway of T-cell activation.     

Autocrine growth stimulation of a human T-cell lymphoma line by interleukin 2.

The ability of tumor cells to produce and to respond to their own growth factor (autocrine secretion) may be of importance for their growth. We describe a human tumor cell line regulated by an autocrine secretion of the growth factor interleukin 2 (IL-2). This T-lymphocyte cell line, IARC 301, was established from a patient with a T-cell lymphoma in the absence of any added specific growth factor. It constitutively expresses biologically functional high-affinity cell-surface receptors for IL-2 as shown by the binding of both radiolabeled purified IL-2 and monoclonal antibodies to IL-2 receptors. In addition, it synthesizes IL-2, which is bound to cell surface receptors. Monoclonal antibodies directed against either IL-2 or the IL-2 receptor block IARC 301 cell growth. These findings demonstrate that the proliferation of this tumor cell line is mediated by an autocrine pathway involving endogenous IL-2 production and its binding to cell surface receptors.     

B-cell-stimulatory factor 2 (beta 2 interferon) functions as a second signal for interleukin 2 production by mature murine T cells.

Purified peripheral murine T cells, in the presence of concanavalin A, can be activated to produce interleukin 2 (IL-2) through stimulation either with a previously described murine lymphokine designated T cell-activating factor (TAF) or with a cloned human lymphokine that has been called beta 2 interferon, B-cell-stimulatory factor 2, hybridoma growth factor, inducible 26-kDa protein, or hematopoietic colony-stimulating factor 309 by different investigators. We and others propose the designation interleukin 6 (IL-6) for the latter molecule. Our experiments demonstrate that either murine TAF or human IL-6 can restore the ability of purified T cells to proliferate in response to Con A or antibodies against the T-cell antigen receptor. Most if not all of the proliferation can be blocked by antibodies against the alpha chain of the IL-2 receptor. Furthermore, highly purified CD8- T cells can be activated by IL-6 in the presence of Con A to secrete IL-2. We propose that IL-6 and murine TAF are important "second signals" in primary antigen-receptor-dependent T-cell activation. Whether or not murine TAF is a homologue of human IL-6 remains to be determined.     

Interleukin 7 is a T-cell growth factor.

Interleukin 7 (IL-7) is a 25-kDa cytokine which was purified and its corresponding cDNA was cloned based upon its ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also function as a costimulator with Con A for the proliferation of T lymphocytes by inducing the production of interleukin 2 (IL-2). We demonstrate here that IL-7 in combination with phorbol 12-myristate 13-acetate can directly drive the proliferation of purified T cells and that this response is not inhibited by cyclosporine A or by antibodies to IL-2 and IL-4. Stimulation of T cells with phorbol myristate acetate and IL-2, IL-4, or IL-7 prepared T cells to respond to any of the three lymphokines. Although T cells activated in vitro by anti-CD3 or allogeneic cells failed to proliferate when challenged with IL-7, T cells primed in vivo to the same stimuli demonstrated a significant proliferative response when restimulated in vitro with IL-7. IL-7 can, therefore, function both as a growth factor for T cells in an IL-2-independent manner and as a competence factor for the induction of lymphokine responsiveness. The ability to induce IL-7 responsiveness via stimulation of the T-cell receptor complex in vivo, but not in vitro, raises the possibility that IL-7 may play a role in T-cell growth and differentiation in vivo.     

Phorbol ester induces expression and function of interleukin-2 receptors on a human leukemic cell line with a T-cell precursor phenotype

We show here that a human leukemic cell line, PER-117, bearing the markers of a T-cell precursor phenotype, can be induced to express receptors for interleukin-2 (IL-2). These IL-2 receptors could be demonstrated to mediate a physiologic response to the lymphokine for which the high-affinity form of the IL-2 receptor appears to be essential. The phenotype of PER-117 cells corresponds to the earliest identifiable stage of T-cell differentiation, which is defined by the lack of the T3-T-cell receptor complex and the presence of the 40 Kd protein recognized by monoclonal antibodies of the CD7 group. Further evidence for the clonality and T-cell lineage of this cell line was obtained by analysis of rearrangements of genes for the T-cell receptor (TCR) beta chain and for the immunoglobulin heavy-chain (IgJH) genes. PER-117 cells could be shown to have rearranged TCR beta genes but no rearrangement of the IgJH genes. Cell line PER-117 provides a model to investigate the requirements for induction of IL-2 receptors in a cell expressing the first T-cell-specific marker and may help to elucidate the role of IL-2 during thymic differentiation and in the uncontrolled proliferation of T-cell leukemias.     

Only high-affinity receptors for interleukin 2 mediate internalization of ligand

Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.     

T-Cell lymphoma model for the analysis of interleukin 1-mediated T-cell activation.

Several laboratories have recently demonstrated that the requirement for macrophages in mitogen-induced production of murine T-cell interleukin 2 (IL-2; formerly referred to as "T-cell growth factor") could be circumvented by using the macrophage-derived peptide interleukin 1 (IL-1; formerly referred to as "lymphocyte-activating factor"). Using two cloned T-cell lymphomas, we investigated the mechanism through which IL-1 exerted its effect on IL-2 production. One of the cell lines used (LBRM-33 5A4) produces large concentrations of IL-2 upon mitogen stimulation, whereas the second (LBRM-33 1A5) is incapable of producing IL-2 in response to mitogen. It was observed that addition of purified IL-1 to nonproducer 1A5 cells converted them to a state in which subsequent mitogen stimulation triggered production of IL-2. The concentration of IL-2 produced by IL-1 treated 1A5 cells was equivalent in magnitude to that generated by mitogen-stimulated 5A4 cells (500-1000 units/ml, or approximately 1000 times the concentration of IL-2 contained in conventional preparations of murine mitogen-conditioned medium). The observations that (i) brief exposure to IL-1 was sufficient for 1A5 cell conversion to IL-2 production and (ii) IL-1 could actively be absorbed from culture medium by live or fixed 1A5 cells led us to propose the existence of IL-1 receptors on responsive 1A5 cells. On the basis of these experiments, we have postulated that IL-1 mediates its effect on immune reactivity (enhancement of thymocyte mitogenesis and induction of antibody and cytotoxic T cell responses) by maturation of a subset of immature T cells to the point where they are capable of IL-2 production. Subsequent release of IL-2 after ligand activation allows for clonal expansion of activated T cells which mediate particular effector functions.     

Keratinocyte-derived T-cell growth factor: a T-cell growth factor functionally distinct from interleukin 2.

T-cell growth factor, more recently termed interleukin 2 (IL-2), is the product of activated T lymphocytes and is considered the principal trophic factor for T lymphocytes. The activity of IL-2 preparations is assessed by the degree to which they support the growth of various IL-2-dependent cell lines. We report that murine epidermal epithelial cells (keratinocytes) produce and release a factor that supports the growth of the helper-T-cell-derived, IL-2-dependent cell line HT-2. This substance, keratinocyte-derived T-cell growth factor (KTGF), does not support the growth of an IL-2-dependent cell line derived from cytotoxic T cells (line CTLL-2). This differential effect on IL-2-dependent cell lines is unique to KTGF. KTGF has an apparent molecular weight of 25,000-35,000 and has properties similar to those of conventional IL-2 by reversed-phase and gel-filtration HPLC analysis. However, even highly purified KTGF fails to stimulate the proliferation of CTLL-2 cells. The observation that epidermal epithelium produces a trophic factor for T lymphocytes may help explain the basis for preferential proliferation of T cells in the microenvironment of skin in certain dermatologic disorders. Further, it suggests that different IL-2-dependent T-cell lines may have distinct growth requirements and that non-lymphocyte cell types may produce factors capable of maintaining the growth of T cells.     

Leukemia-derived growth factor (non-interleukin-2) produced by murine lymphoma T-cell lines.

Autocrine growth factor activity was found in supernatants of AKR T-cell lymphoma lines cultured in serum-free medium. This factor was designated leukemia-derived growth factor (LDGF). Active supernatants stimulated the growth of the AKR murine T-cell lymphoma line SL 12, its cloned derivatives, and all other murine T-cell lymphoma lines tested. Growth factor activity in conditioned medium was found to be different from interleukin 2 (IL-2) and several other known growth factors. LDGF was able to stimulate growth of the human leukemia T-cells MOLT4f, and the LDGF from MOLT4f cells stimulated the mouse cells. Because mouse T-cell lymphoma lines produced and respond to this factor, it may support the continued proliferation of these cells and could be responsible for their malignant in vivo properties.     

Early precursor thymocytes can produce interleukin 2 upon stimulation with calcium ionophore and phorbol ester

T-cell precursors were stimulated with a conventional T-cell mitogen or with the calcium ionophore A23187 in order to determine whether pre-T cells acquire the ability to produce interleukin 2 (IL-2) before they acquire the ability to respond to antigen or mitogenic lectins. Immature T cells were obtained by eliminating mouse thymocytes that expressed the Lyt2 and L3T4 cell surface proteins. The remaining Lyt2-, L3T4- cells were stimulated for IL-2 production by using concanavalin A (Con A) or A23187, together with phorbol 12-myristate 13-acetate (PMA). We found that these "double-negative" thymocytes were unresponsive to Con A plus PMA but produced substantial amounts of IL-2 when stimulated with A23187 plus PMA. In contrast, both stimulation regimens induced more mature T-lymphocyte populations to produce IL-2. This implies that developing T cells acquire the ability to make IL-2 upon induction before they acquire the ability to be triggered by Con A. Day-15 fetal and cortical thymocytes were also tested for their ability to make IL-2. Both populations failed to synthesize this growth factor, even when stimulated with A23187 and PMA. For cortical thymocytes, this result, together with the finding that A23187 plus PMA fails to activate these cells, suggests that this population is immunologically inert rather than immature. On the other hand, the inability of day-15 fetal thymocytes to produce IL-2 indicates that these T-cell precursors are developmentally distinct from adult Lyt2-, L3T4- thymocytes, which they phenotypically resemble.     

Differential regulation of T-cell growth by IL-2 and IL-15

Although interleukin 2 (IL-2) and IL-15 signal through the common gamma chain (gammac) and through IL-2 receptor beta-chain (CD122) subunits, they direct distinct physiologic and immunotherapeutic responses in T cells. The present study provides some insight into why IL-2 and IL-15 differentially regulate T-cell function by revealing that these cytokines are strikingly distinct in their ability to control protein synthesis and T-cell mass. IL-2 and IL-15 are shown to be equivalent mitogens for antigen-stimulated CD8(+) T cells but not for equivalent growth factors. Antigen-primed T cells cannot autonomously maintain amino acid incorporation or de novo protein synthesis without exogenous cytokine stimulation. Both IL-2 and IL-15 induce amino acid uptake and protein synthesis in antigen-activated T cells; however, the IL-2 response is strikingly more potent than the IL-15 response. The differential action of IL-2 and IL-15 on amino acid uptake and protein synthesis is explained by temporal differences in signaling induced by these 2 cytokines. Hence, the present results show that cytokines that are equivalent mitogens can have different potency in terms of regulating protein synthesis and cell growth.     

T-cell lines established from human T-lymphocytic neoplasias by direct response to T-cell growth factor.

Long-term growth of lymphoblastoid T cells from tissue samples from six of six patients with cutaneous T-cell lymphoma (CTCL) and six of six patients with acute T-lymphoblastic leukemia (ALL) has been achieved by using partially purified mitogen-free human T-cell growth factor (pp-TCGF). One cell line, CTCL-2, is now independent of added growth factor; the others continue to show absolute dependency on its presence. All lines have been in continuous culture for at least 4 months and some for > 1 year. They are erythrocyte-rosette positive and are negative for Epstein-Barr virus nuclear antigen. Most of the lines are negative for Fc and complement receptors and for surface immunoglobulin except that CTCL-1 and CTCL-2 have some cells positive for these cell surface markers. Results of histochemical studies on these cell lines are similar to the known patterns for fresh cells from their disease of origin. Cell line CTCL-3 has an abnormal karyotype, but no detectable chromosomal abnormalities were found in the other lines, consistent with the karyologic features of their clinical sources. Because T cells from normal donors do not respond to pp-TCGF unless the cells are first "activated" by a lectin mitogen such as phytohemagglutinin or an antigen, the direct response to pp-TCGF of T cells from patients with T-cell neoplasias suggests that the cell lines represent a transformed neoplastic cell population. Although some of the cell lines may be normal T cells activated by the malignant cells, the morphologic and histochemical properties of the cell lines, the abnormal karyotype of CTCL-3, and the independent growth of CTCL-2 support the conclusion that most of these cell lines are of malignant origin.     

Acquired erythropoietin responsiveness of interleukin-2-dependent T lymphocytes retrovirally transduced with genes encoding chimeric erythropoietin/interleukin-2 receptors

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TILs) causes regression of some human tumors. However, the sustained proliferation and antitumor activity of TILs requires the coadministration of potentially toxic amounts of interleukin-2 (IL-2). In an effort to overcome the requirement by T cells for IL-2, we have introduced alternative growth factor receptors that use the relatively nontoxic cytokine erythropoietin (Epo) as a ligand. In our model system, the coexpression of chimeric receptors consisting of the extracellular portion of the Epo receptor (EpoR) and the intracellular portions of the IL-2 receptor subunits, beta and gamma, conferred Epo responsiveness on a T-cell line. By contrast, cells expressing the wild- type EpoR did not proliferate in response to Epo. This suggested that Epo binding caused the activation of an IL-2 signal pathway mediated by the chimeric receptors. This approach can be used to minimize toxicity and potentially improve cancer immunotherapy with TILs.     

Evidence for different interleukin 1 receptors in murine B- and T-cell lines.

Previous studies have shown that binding of interleukin 1 (IL-1) to its receptor and intracellular processing of the IL-1/IL-1 receptor complex appear to be different in B- and T-lymphocyte cell lines. In this study we used a B-lymphoid cell line, 70Z/3, and T-lymphoid cell line, EL-4 6.1 C10, to explore further the differences that exist between IL-1 receptors on cells of B and T lineage. We show that a monoclonal antibody against the IL-1 receptor on EL-4 cells does not bind to the IL-1 receptor on 70Z/3 cells. This finding suggests that there are structural differences in the extracellular domains of the IL-1 receptors on the two cell lines. Furthermore, affinity crosslinking showed that the molecular mass of the IL-1 receptor on EL-4 is 87 kDa, whereas that of 70Z/3 is significantly lower (66 kDa). Activation of phospholipid/Ca2+-dependent protein kinase, protein kinase C, by phorbol 12-myristate 13-acetate (PMA) greatly reduced the number of IL-1 binding sites on 70Z/3. But, in sharp contrast, PMA had no effect on surface IL-1 receptor expression on EL-4 cells despite having an equally potent effect in activating protein kinase C. The different effects of protein kinase C suggest that the cytoplasmic domains of the IL-1 receptors in 70Z/3 and EL-4 may also be different. Lastly, a probe containing the entire coding region of the murine T-cell IL-1 receptor hybridized under high stringency conditions with mRNA from EL-4 cells but not with mRNA from 70Z/3 cells. Taken together, the observations made in this study suggest that major structural differences exist between the IL-1 receptors on B and T lymphocytes.     

T-cell ontogeny after autologous bone marrow transplantation: failure to synthesize interleukin-2 (IL-2) and lack of CD2- and CD3-mediated proliferation by both CD4- and CD8+ cells even in the presence of exogeneous IL-2

T cells generated during a second round of ontogeny after autologous bone marrow transplantation (ABMT) represent a unique model of early T- cell ontogeny in an autologous situation. Since grafted bone marrows were pretreated in vitro with the cyclophosphamide derivative ASTA Z 7557, circulating T cells had to be regenerated from reinfused hematopoietic progenitor cells. The T-cell population derived from 25 patients post-ABMT was phenotypically characterized: an increase in CD8+ cells, a low percentage of CD4+ cells, and a median of 12% CD56+ (NKH1+) cells were found. When the T cells were stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), defective interleukin-2 (IL-2) secretion was observed. In addition, proliferative responses of the T cells after activation through the antigen-receptor- dependent CD3 pathway, through the CD2 dependent alternative T-cell pathway, and by the lectin PHA were investigated. Despite the presence of CD2, CD3, alpha/beta chains of the T-cell receptor, and CD25+ IL-2 surface receptors, abnormal proliferative responses were obtained even in the presence of exogeneous IL-2. In experiments where the T-cell population was separated into CD4+ cells and CD8+ cells, both the CD4- and CD8+ subsets were unable to respond to activating and proliferating signals. Thus, T cells at early stages of ontogeny not only possess an intrinsic defect in IL-2 synthesis but, in addition, were unable to express functional IL-2 receptors in response to mitogenic stimuli.     

Chemical reduction of oxidized human lymphocytes inhibits interleukin 2 production but not induction of interleukin 2 responsiveness.

Treatment with neuraminidase (NA) plus galactose oxidase (GalOxase) does not cause stimulation of human thymocytes. However, stimulation can be achieved by addition of exogenous interleukin 2 (IL-2). The IL-2-induced stimulation was inhibited with anti-Tac antibody, indicating that NA/GalOxase-oxidized cells can serve as inducers of functional IL-2 receptors on IL-2-responding T cells. The induction of IL-2 receptors by the oxidized cells was not inhibited by subsequent reduction with borohydride, since the cells could still be stimulated with IL-2. The presence of IL-2 receptors was also confirmed by flow cytometry using indirect immunofluorescence. Peripheral blood lymphocytes can be stimulated by NA/GalOxase treatment, and the conditioned medium from this treatment can support the growth of an IL-2-dependent line. This stimulation can be inhibited with borohydride and restored with IL-2. The conditioned medium derived from the borohydride-reduced cells cannot support the growth of the IL-2-dependent line, indicating that borohydride inhibits the oxidation-induced IL-2 production. The results suggest that NA/GalOxase-oxidized sites can be modified chemically without losing the potential to induce IL-2 receptors.     

Phorbol myristate acetate-stimulated production of interleukin 2 by T-cell lymphoma and constitutive production by derived hybridomas

To isolate a stable tumor cell line source of IL-2 (TCGF), 19 murine T-cell lines and their derivatives were screened for both constitutive and mitogen-stimulated IL-2 production. The cloned subline of a mouse thymic lymphoma EL-4 designated as EL-4TF could be stimulated with PMA to produce 80 U/ml of IL-2. A TK- EL-4TFR mutant line has been selected from the EL-4TF cell population by treatment with 5-BrdUrd. The EL-4-TFR cells were stimulated with PMA and fused with the cells of thymic lymphoma BW5147. The resulting BH3 hybrid cell population was repeatedly cloned and tested for constitutive IL-2 production: two of the BH3 hybridoma clones were found to produce IL-2 constitutively. The IL-2 of EL-4TF origin was found to support permanent in vitro growth of IL-2 dependent, tumor-specific T killer cell line CTLL when present in culture medium at a concentration of at least 0.1 U/ml. Since the EL-4TF-derived IL-2 preparations were contaminated with PMA, it was of interest whether PMA alone has a growth-promoting activity in CTLL cell cultures. Permanent cultivation of CTLL cells in an IL-2-free medium containing PMA was not possible. However, both mitogenic and co-mitogenic effects of PMA on CTLL cells were observed.     

Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes

Glucocorticoid (GC)-induced apoptosis is a well-recognized physiologic regulator of murine T-cell number and function. We have analyzed its mechanisms in human mature T cells, which have been thought to be insensitive until recently. Peripheral blood T cells showed sensitivity to GC-induced apoptosis soon after the proliferative response to a mitogenic stimulation, and were also sensitive to spontaneous (ie, growth factor deprivation-dependent) apoptosis. CD8+ T cells were more sensitive to both forms than CD4+ T cells. Acquisition of sensitivity to GC-induced apoptosis was not associated with any change in number or affinity of GC receptors. Both spontaneous and GC-induced apoptosis were increased by the macromolecular synthesis inhibitors, cycloheximide (CHX) and puromycin. A positive correlation between the degree of protein synthesis inhibition and the extent of apoptosis was observed. Interleukin-2 (IL-2) IL-4, and IL-10 protected (IL-2 > IL-10 > IL-4) T cells from both forms of apoptosis in a dose-dependent manner. Our data suggest that spontaneous and GC-induced apoptosis regulate the human mature T-cell repertoire by acting early after the immune response and differentially affecting T-cell subsets.