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.     

Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.

Interleukin 10 (IL-10), originally identified as a TH2 helper T-cell product able to inhibit cytokine production by TH1 cells, is highly homologous to BCRF1 (viral IL-10), an open reading frame in the Epstein-Barr virus genome. Here, we show that human and viral IL-10 stimulate DNA replication of B lymphocytes activated either via their antigen receptor or via their CD40 antigen. IL-4 and IL-10 display additive effects and induce a strong increase in the number of viable cells. Moreover, IL-10 induces activated B cells to secrete large amounts of IgG, IgA, and IgM, and the combination of IL-10 and IL-4 results in the secretion of the four immunoglobulin isotypes. Thus, IL-10 may play an important role in the amplification of humoral responses.     

Developmental control of lymphokine gene expression in fetal thymocytes during T-cell ontogeny.

We have used the technique of in situ hybridization to investigate the expression of lymphokine genes by immature thymocytes during intrathymic development. In 13-day fetal thymocytes a population of cells constitutively produces low levels of interleukin 2 (IL-2) and interleukin 4 (IL-4) mRNAs. A second phase of lymphokine gene expression occurs in the majority of 15-day thymocytes, and a population of cells constitutively produces both IL-2 and IL-4 mRNAs. Thymocytes at 14 days of gestation and after 16 days up until birth do not express detectable lymphokine mRNA. By contrast, the population of IL-2 receptor mRNA-producing thymocytes increases progressively up to 15 days of gestation, and expression thereafter decreases up to birth. In addition, thymocytes expressing interferon gamma mRNA were not present until just prior to birth. Our findings indicate developmental control of lymphokine and lymphokine receptor gene expression in fetal thymocytes during ontogeny.     

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.     

Interleukin 4 (B-cell growth factor II/eosinophil differentiation factor) is a mitogen and differentiation factor for preactivated murine B lymphocytes.

Recently we described a murine T-cell hybrid that produces activities that promote the differentiation of eosinophils (eosinophil differentiation factor) and cause proliferation of the BCL1 B-cell lymphoma (B-cell growth factor II activity). Both activities appear to be associated with the same molecule, which has therefore been termed interleukin 4. The hybrid does not produce any other known lymphokines. We now find that purified interleukin 4 has no effects on small resting B cells but induces naturally occurring large B cells (which have presumably been preactivated in vivo) to synthesize DNA and to secrete IgM and low levels of IgG. B cells activated by anti-Ig antibodies apparently only become responsive to the factor once they have reached late G1 stage. All bioactivities of interleukin 4 are associated with a protein of Mr 44,000 (by NaDodSO4/PAGE). Therefore these results demonstrate that this lymphokine alone is sufficient to induce clonal expansion and maturation of activated B cells.     

Interleukin 2 promotes growth and cytolytic activity in human T3+4-8- thymocytes.

Human thymocytes bearing T3 but neither T4 nor T8 antigens (T3+4-8- cells) were obtained after negative selection of thymocytes, either fresh or cultured in medium containing recombinant interleukin 2 (IL-2), by treatment with Na1/34, OKT4A and B9.4 monoclonal antibodies (which recognize T6, T4, and T8 antigens, respectively) and complement. Quantitative flow cytometry showed a 98% pure population of T3+4-8- lymphocytes, which included proliferating cells. The growth and maturation requirements of these thymocytes were characterized and related to the T3-receptor complex and IL-2 pathways, thought to be used by mature lymphocytes. The results show that addition of recombinant IL-2 promotes, in a dose-dependent way, proliferation and acquisition of effector functions by cultured T3+4-8- thymocytes, the growth being inhibitable by monoclonal antibody 33B73 (anti-Tac). Furthermore, cytolytic activity of T3+4-8- cells induced by recombinant IL-2 is specifically blocked by monoclonal antibody OKT3, showing that it operates via the T3-receptor complex and does not require either T4 or T8 molecules. The finding of in vitro responsiveness to recombinant IL-2 in T3+4-8- thymocytes suggests a role of IL-2 in the growth and maturation of cells committed to the T-cell lineage, during intrathymic differentiation, prior to expression of T4 and T8 molecules.     

B-cell growth factor: distinction from T-cell growth factor and B-cell maturation factor.

A T-cell hybridoma was derived by somatic cell hybridization between concanavalin A-activated BALB/c spleen cells and the AKR thymoma BW 5147. Media conditioned by hybridoma cells, even at high dilutions (1:1,000) support the growth of lipopolysaccharide-stimulated B-cell blasts but not that of T-cell growth factor (TCGF)-reactive T-cells. This activity, herein designated B-cell growth factor (BCGF), has a Mr of approximately equal to 20,000 and it can readily be separated from TCGF (Mr approximately equal to 30,000) by gel filtration. BCGF is constitutively produced by the hybridoma cells, it is removed from conditioned media by incubation with target cells at +4 degrees C, and it is equally effective on B-cell blasts carrying different major histocompatibility complex and Ig haplotypes. BCGF shows no T-cell replacing factor (TRF) activity, and it is poor in supporting the development of Ig-secreting plaque-forming cells in B-cell blast cultures. Terminal maturation, however, can be induced in BCGF-dependent blasts by addition of conditioned media from normal helper T cell cultures, suggesting that two distinct factors are involved in the helper cell-dependent growth and maturation of B lymphocytes.     

Changes in surface antigens of immature thymocytes under the influence of T-cell growth factor and thymic factors.

Peanut agglutinin-positive thymocytes, peanut agglutinin-negative thymocytes, cortisone-resistant thymocytes, and unfractionated thymocytes were prepared from congeneic C57BL/6 Tla mice. By using surface iodination and immunoprecipitation of solubilized antigen with specific antisera (e.g., anti-H-2D, anti-TL, anti-Qa2/3, and anti-gp70), the released specific antigens were electrophoresed on polyacrylamide gels, and their radioactivity was measured. The relative percentages of surface antigens H-2D, TL, Qa2/3, and gp70 were 3.2%, 47.5%, 2.5%, and 46.8%, respectively, for peanut agglutinin-positive thymocytes; 31.8%, 4.4%, 32.7%, and 31.1%, respectively, for cortisone-resistant thymocytes; 13.2%, 28.7%, 12.3%, and 45.8%, respectively, for peanut agglutinin-negative thymocytes; and 7.7%, 27.1%, 4.3%, and 60.9%, respectively, for unfractionated thymocytes. After incubation with thymosin fraction V or T-cell growth factor (interleukin II) for 20 hr, the changes in surface antigens of peanut agglutinin-positive thymocytes closely correlated with their normal maturation (i.e., H-2D increases and TL decreases). Thymic factors (e.g., thymosin alpha 1, thymopoietin pentapeptide, facteur thymic serique) had only small or no effects on surface antigens of peanut agglutinin-positive thymocytes. The results suggest that peptides yet to be identified in thymosin fraction V may play an important role in intrathymic evolution and that T-cell growth factor is possibly a peripheral signal derived from activated T cells that modulates T-cell receptors and may be a critical regulator of intrathymic cellular development.     

Interleukin-7 is a critical growth factor in early human T-cell development

Highly purified human CD34+ fetal liver stem cells differentiate to mature T cells when seeded in vitro into isolated fetal thymic lobes of severe combined immunodeficient (SCID) mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of interleukin-7 (IL-7) and of the alpha chain of the IL-7 receptor (IL-7R alpha) in early human T-cell development. We report that addition of either the monoclonal antibody (MoAb) M25, which neutralizes both human and mouse IL-7, or the MoAb M21, which recognizes and blocks exclusively the human high-affinity alpha-chain of the IL-7R, results in a profound reduction in human thymic cellularity. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3-CD1+ progenitor cells and subsequently toward CD4+CD8+ thymocytes. Our results reveal a critical role for IL-7 during early human thymocyte development, and may explain the absence or highly reduced levels of T cells in patients with X-linked SCID. The molecular defect in these patients has been shown to be a mutation in the gamma chain of the IL-2R. Although this gamma chain is not only present in the IL-2R, but also forms an essential part of other cytokine receptors, including IL-4, IL-7, IL-9, IL-13, and IL-15, the T- cell defect in these patients can be explained by the fact that IL-7 is not able to transduce its signal by the molecular defect of the common gamma (gamma c) chain and that IL-7 is indispensable for T-cell development.     

Interleukin 6, a possible autocrine growth and differentiation factor for the human megakaryocytic cell line, CMK

CMK is a human cell line derived from a megakaryoblastic leukaemia. It has characteristics of the megakaryocytic lineage, such as the presence of platelet peroxidase, membrane glycoproteins (GP)Ib and GPIIb/IIIa, alpha-granules, and demarcation membranes. The cell line proliferates autonomously in serum-containing medium. Here we report that the cell line expresses the gene for IL-6 and releases small quantities of the cytokine into the medium. Addition of exogenous IL-6 to cultures seeded into medium was found to promote growth of the cells. Conversely, addition of a neutralizing anti-IL-6 antibody inhibited cell growth. These data support the notion that autocrine IL-6 is one of the factors accounting for autonomous growth of the cell line.     

Interleukin 1 expression is inducible by nerve growth factor in PC12 pheochromocytoma cells.

Expression of the cytokine interleukin 1 alpha (IL-1 alpha) was demonstrated in the rat PC12 pheochromocytoma cell line by (i) immunohistochemistry using rabbit polyclonal antisera raised against the recombinant murine IL-1 alpha, (ii) an ELISA, and (iii) a specific cell conversion bioassay based on the use of LBRM33-1A5 cells. IL-1 alpha mRNA was demonstrated in the PC12 cells, by PCR amplification. Constitutive expression of IL-1 alpha in PC12 cells was demonstrated in all experiments, although the cellular levels of IL-1 alpha-like immunoreactivity varied. The expression of IL-1 alpha, as studied at the mRNA level, was inducible by mouse nerve growth factor (7S NGF), and the gene product level was inducible in a dose- and time-dependent fashion by 7S NGF. The maximum induction corresponds to a 600% increase in IL-1 alpha-like immunoreactivity above the expression level found in noninduced cells and occurred after a 3-day incubation of the cells with NGF at 0.75 micrograms/ml of culture medium. The significance of the ability of NGF to induce IL-1 expression lies in the fact that IL-1 itself also acts as a growth factor that promotes glial proliferation and, even more importantly, IL-1 itself induces the expression of NGF at peripheral nerve injury [Lindholm, D., Heumann, R., Meyer, M. & Thoenen, H. (1987) Nature (London) 330, 658-659].     

Matrix metalloproteinase-9 and vascular endothelial growth factor: a possible link in adult T-cell leukaemia cell invasion

Plasma from a total of 57 patients with adult T-cell leukaemia (ATL) (acute ATL, 39 patients; lymphoma ATL, one patient; chronic ATL, 15 patients; smouldering ATL, two patients) and 20 healthy controls was analysed for the presence of type IV gelatinase activity with clinical features. A significant elevation of plasma matrix metalloproteinase-9 (MMP-9) was observed in some ATL patients, particularly in the patients with malignant cell infiltration. MMP-9 was found to be secreted into the conditioned medium from all ATL cell lines examined. Moreover, the corresponding mRNA was detectable both in all ATL cell lines examined and in the majority of primary acute ATL cells, indicating that ATL cells are capable of synthesizing and secreting MMP-9. We previously demonstrated that a high incidence of ATL cell infiltration was closely related to a high plasma level of vascular endothelial growth factor (VEGF) produced by ATL cells themselves. This present study showed that the presence of increased plasma MMP-9 was closely associated with elevated plasma VEGF in ATL patients. Furthermore, we showed that both increased plasma MMP-9 and VEGF were significantly related to high ATL cell infiltration. All these findings strongly suggest that MMP-9 and VEGF act co-operatively in the process of ATL cell invasion.     

Interleukin 1 can act as a B-cell growth and differentiation factor.

Splenic B lymphocytes specifically reactive to the hapten fluorescein (FLU) were prepared from nonimmune adult mice by affinity fractionation on hapten-gelatin. These FLU-specific B cells were cultured as single cells or in small numbers in 10-microliter wells either in the absence of any feeder, filler, or accessory cell or in the presence of 3T3 fibroblasts acting as filler cells. A selected batch of a "T-cell-independent" antigen, FLU-Ficoll, which induces growth and differentiation only in the presence of lymphokines or cytokines acting as B-cell growth and differentiation factors (BGDF), was used as the antigenic stimulus. It was found that murine interleukin 1 prepared by recombinant DNA technology was an effective, although weak, BGDF when acting with antigen on B cells cultured either under filler cell-free conditions or in the presence of 3T3 cells. When the murine interleukin 1 was used in combination with recombinant human interleukin 2, itself a weak but effective BGDF in the system, an additive effect was observed. The results challenge the notion that interleukin 1 is exclusively or even primarily an activating cytokine. This system, in which pure factors are able to act with specific antigen on single hapten-specific B cells, will prove helpful for the further dissection of the respective roles of the various factors that can act on B cells.     

Interleukin 7 requirement for survival of T-cell acute lymphoblastic leukemia and human thymocytes on bone marrow stroma

We explored the role of interleukin-7 (IL-7) in the bone marrow (BM) stroma-mediated survival of primary T-cell acute lymphoblastic leukemia (T-ALL) cells and normal thymocytes. We presented evidence that IL-7 has a major role in the enhanced survival mediated by BM stroma both in T-ALL cells and thymocytes.     

Immature thymocytes are protected from deletion early in ontogeny.

Self/non-self discrimination occurs within the thymus, where T cells undergo positive selection to produce a repertoire that recognizes foreign antigen in the context of self major histocompatibility complex proteins and negative selection to eliminate from the repertoire those T cells with self-reactive specificities. We have previously shown that two subpopulations of immature antigen receptor-bearing thymocytes exist, one that is susceptible to negative selection induced by ligation of the T-cell receptor for antigen and a second that is resistant to T-cell receptor-mediated negative selection. In the current work, we show that all antigen receptor-bearing thymocytes in early fetal thymuses are resistant to negative selection and that thymocytes susceptible to deletion do not develop until later gestational ages. Thus, deletion is a relatively late event in T-cell ontogeny. In addition, these data suggest that a thymocyte population can be isolated early in ontogeny that is capable of transducing selective signals through the antigen receptor, which do not lead to deletion but may result in positive selection.