Phase I study of every 2- or 3-week dosing of ramucirumab, a human immunoglobulin G1 monoclonal antibody targeting the vascular endothelial growth factor receptor-2 in patients with advanced solid tumors.

BACKGROUND: Ramucirumab is a fully human immunoglobulin G1 monoclonal antibody receptor antagonist designed to block the ligand-binding site of vascular endothelial growth factor receptor-2 (VEGFR-2). An initial phase I study evaluated ramucirumab administered weekly in advanced cancer patients. This phase I study of ramucirumab [administered every 2 or 3 weeks (Q2W or Q3W)] examined safety, maximum tolerated dose, pharmacokinetics, immunogenicity, antitumor activity, and pharmacodynamics. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with escalating doses of ramucirumab i.v. over 1 h. Blood was sampled for pharmacokinetics studies throughout treatment; levels of circulating vascular endothelial growth factor-A (VEGF-A) and soluble VEGF receptors (R)-1 and -2 were assessed. RESULTS: Twenty-five patients were treated with ramucirumab: 13 with 6, 8, or 10 mg/kg Q2W, and 12 with 15 or 20 mg/kg Q3W. The median treatment duration was 12 weeks (range 2-81). No dose-limiting toxicities were observed. The most frequently reported adverse events (AEs) included proteinuria and hypertension (n = 6 each), and diarrhea, fatigue and headache (n = 4 each). Treatment-related grade 3/4 AEs were: two grade 3 hypertension (10 and 20 mg/kg), one each grade 3 vomiting, fatigue (20 mg/kg), atrial flutter (15 mg/kg), and one each grade 4 duodenal ulcer hemorrhage (6 mg/kg) and grade 4 pneumothorax (20 mg/kg). Pharmacokinetic analysis revealed low clearance and half-life of ∼110-160 h. Analysis of serum biomarkers indicated considerable patient-to-patient variability, but trends toward elevated VEGF-A and a transient decline in soluble VEGFR-2. Fifteen patients (60%) had best response of stable disease, with a median duration of 13 months (range 2-18 months) in tumor types including colorectal, renal, liver, and neuroendocrine cancers. CONCLUSION: Ramucirumab was well tolerated. Study results led to recommended phase II doses of 8 mg/kg Q2W and 10 mg/kg Q3W. Prolonged stable disease was observed, suggesting ramucirumab efficacy in various solid tumors. CLINICALTRIALSGOV: NCT00786383.

"It's crucial they're treated as patients": ethical guidance and empirical evidence regarding treating doctor-patients.

Ethical guidance from the British Medical Association (BMA) about treating doctor-patients is compared and contrasted with evidence from a qualitative study of general practitioners (GPs) who have been patients. Semistructured interviews were conducted with 17 GPs who had experienced a significant illness. Their experiences were discussed and issues about both being and treating doctor-patients were revealed. Interpretative phenomenological analysis was used to evaluate the data. In this article data extracts are used to illustrate and discuss three key points that summarise the BMA ethical guidance, in order to develop a picture of how far experiences map onto guidance. The data illustrate and extend the complexities of the issues outlined by the BMA document. In particular, differences between experienced GPs and those who have recently completed their training are identified. This analysis will be useful for medical professionals both when they themselves are unwell and when they treat doctor-patients. It will also inform recommendations for professionals who educate medical students or trainees.

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.

Retinoids synergize with interleukin-2 to augment IFN-gamma and interleukin-12 production by human peripheral blood mononuclear cells.

We have demonstrated previously that cells from both the skin and peripheral blood from patients with cutaneous T cell lymphoma (CTCL) have elevated levels of protein and mRNA for Th2 cytokines, interleukin-4 (IL-4) and IL-5, and depressed levels of Thl cytokines, IL-2 and interferon-gamma (IFN-gamma). Furthermore, IL-12 in vitro can restore IFN-gamma production by these patients' cells to near normal levels. Because retinoids exert therapeutic activity in CTCL and are potent modulators of growth and differentiation of hematopoietic cells, we investigated the role of retinoids in modulating Thl cytokine production. Peripheral blood mononuclear cells (PBMC) from normal donors and patients with CTCL were cultured with medium, IL-2, 13-cis-retinoic acid, all-trans-retinoic acid, acetretin or etretinate alone, or IL-2 plus the retinoids for 24 h, and levels of IFN-gamma were determined using ELISA. IL-2 or retinoids alone could induce low but significant levels of IFN-gamma. However, when IL-2 was cultured with each retinoid, a synergistic augmentation of IFN-gamma levels (4-fold to 90-fold) was observed except in the case of etretinate. All-trans-retinoic acid (ATRA) was the most potent IFN-y inducer. Similar studies performed using PBMC from CTCL patients indicated the IFN-gamma augmentation occurred but in a blunted manner. The IFN-y-inducing effect of ATRA and 13-cis-retinoic acid could be abrogated by addition of anti-IL-12 antibodies, suggesting that IL-12 plays a role in the synergistic upregulation of IFN-gamma. Using an IL-12 p40-specific radioimmunoassay (RIA), we confirmed the presence of IL-12 in IL-2 plus retinoid-treated culture supernatants. Purified monocytes cultured with IL-2 plus ATRA did not secrete IL-12. Only when monocytes were cocultured with lymphocytes was there an increase in IL-12 production, suggesting the involvement of a paracrine feedback loop requiring both monocytes and lymphocytes. These data suggest that retinoids can induce Th1 cytokines from normal and CTCL PBMC and that this induction may be mediated through IL-12 production.

Photoactivated hypericin is an anti-proliferative agent that induces a high rate of apoptotic death of normal, transformed, and malignant T lymphocytes: implications for the treatment of cutaneous lymphoproliferative and inflammatory disorders.

Hypericin is a photodynamic compound activated by either visible (400-700 nm) or UVA (320-400 nm) light, and has been shown to inhibit the growth of a variety of neoplastic cell types. In this study, hypericin was found to inhibit proliferative responses of malignant T cells derived from the blood of patients with cutaneous T cell lymphoma. Control cells included peripheral blood mononuclear cells (PBMC) from normal volunteers or Epstein-Barr virus-transformed lymphocytes. Cells from each of these populations were incubated with serial dilutions of hypericin or 8-methoxypsoralen and then stimulated with the mitogen ConA (10 microg per ml). Cultures were prepared in the dark to minimize photoactivation of the hypericin. Proliferation was measured by [3H]thymidine labeling after 72 h. Hypericin, photoactivated with 1.1-3.3 J white light per cm2, inhibited cellular proliferation of malignant T cells with IC50 values from 0.34 to 0.53 microM, normal PBMC with IC50 values of 0.11-0.76 microM, and Epstein-Barr virus-transformed cells with IC50 values of 0.75-3.2 microM. UVA-photoactivated hypericin (0.5-2.0 J per cm2) could also inhibit proliferation with IC50 values of 0.57-1.8 microM, 0.7-4.6 microM, and 2.0-3.7 microM for malignant, normal, or Epstein-Barr virus-transformed cells, respectively. Hypericin, photoactivated with either UVA or white light, could induce near complete apoptosis (94%) in malignant cutaneous T cell lymphoma T cells, whereas lower levels of apoptosis (37-88%) were induced in normal PBMC. These data indicate that hypericin inhibits mitogen-induced proliferation of malignant T cells from patients with cutaneous T cell lymphoma, PBMC from normal individuals, as well as Epstein-Barr virus-transformed lymphocytes, and that inhibition of cell proliferation is dependent on the concentration of hypericin used and the dose of light required to photoactivate the compound. Induction of apoptosis is, in part, one mechanism by which photoactivated hypericin inhibits malignant T cell proliferation.

Use of biological response modifiers in the treatment of cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is typically a skin-infiltrating, clonal proliferative disorder of CD4+ T cells that exhibit a T-helper type 2 cytokine phenotype. Therapeutic decisions are based on the extent of disease and the observations that host-antitumor responses occur and that these responses may be blunted by the immunosuppressive cytokines produced by the malignant T cells. Biologic response modifiers, which may enhance cell-mediated immunity and antitumor responses, are active agents in the treatment of CTCL. The rationale and use of biologic response modifiers to treat CTCL are reviewed in this article.

Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP-containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme-linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)-10 and IL-1 beta protein in the LC-like cell line XS52 as well as the reverse transcriptase-polymerase chain reaction (RT-PCR) to examine levels of mRNA for IL-10, IL-1 beta, and the 40-kDa subunit (p40) of IL-12. CGRP augmented the lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) -induced release of IL-10 protein and the induced expression of IL-10 mRNA in these cells. However, it suppressed the induction of release of IL-1 beta protein and the induction of mRNA for IL-12 p40 and IL-1 beta by LPS and GM-CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS-induced expression of IL-10 was augmented by CGRP, whereas the induction of IL-1 beta was suppressed. Northern analysis demonstrated augmentation of LPS-induced IL-10 mRNA levels and inhibition of LPS-induced IL-1 beta mRNA by CGRP. CGRP inhibited the LPS-induced induction of IL-12 mRNA as assessed by RT-PCR. Up-regulation of B7-2 expression by LPS and GM-CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co-culture with neutralizing antibodies to IL-10. Furthermore, the presence of neutralizing antibodies to IL-10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP-mediated suppression of EC presentation of tumor-associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed-type hypersensitivity in S1509a-immune mice. These data suggest that suppression of antigen-presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell-mediated immunity.

Pathogenesis of cutaneous T-cell lymphoma: implications for the use of recombinant cytokines and photopheresis.

Cutaneous T-cell lymphoma (CTCL) is a clonally derived, skin invasive malignancy of CD4+ cells with the phenotype of mature helper T cells. We previously demonstrated that the leukaemic form of CTCL (Sézary), is characterized by prominent immunological defects including depressed cell-mediated immunity. We also demonstrated increased production of T-helper type 2 (Th2) cytokines (IL-4, IL-5) and deficient Th1 cytokines (IL-2 and IFN-gamma) by their peripheral blood mononuclear cells (PBMC) and detected IL-4 and IL-5 mRNA within lesional skin of patients with all stages of CTCL. A marked defect in IL-12 production has also been noted, which may also play a role in depressed cell-mediated immunity. These results suggested that the malignant CD4+ cells were Th2 cells. Thus, the immune aberrations have been attributed to the cytokine abnormalities triggered by the malignant T-cell population. Because CTCL responds to biological response modification, we focused on strategies for reversing the cytokine and immune defects by in vitro testing of novel biological response modifiers. Our results indicate that IFN-alpha potently suppresses the abnormal IL-4 and IL-5 production, that IL-12 can correct the deficient IFN-gamma production and cell-mediated cytotoxicity, and that retinoids can enhance IFN-gamma and IL-12 production. We also studied the in vitro growth characteristics of the malignant CD4+ cells and determined that IL-12 and IFN-alpha significantly suppress growth of these cells. These studies led to a phase I trial of IL-12 to treat CTCL. Also, we have determined that photopheresis produces a high clinical response rate among Sézary syndrome patients. This therapy not only augments functions of monocytes but also induces the malignant T cells to undergo a high rate of apoptosis. We discuss how these therapies might be employed in concert to produce the optimum desired anti-tumour effect.

Calcitonin gene-related peptide inhibits proliferation and antigen presentation by human peripheral blood mononuclear cells: effects on B7, interleukin 10, and interleukin 12.

CGRP is a neuropeptide that has previously been described to possess immunosuppressive activities. CGRP is released from peripheral nerves that, in the skin, are in close physical association with dendritic APC. We sought to investigate the mechanisms by which CGRP can inhibit immune responses by studying its effects on human peripheral blood mononuclear cells (PBMC). Using allogeneic monocytes as stimulator cells, CGRP could inhibit the proliferation of PBMC by 47% when CGRP was present for the duration of culture. Interestingly, when the stimulator monocytes were incubated with CGRP for 2 h prior to irradiation then washed, the observed inhibition increased to 85%, suggesting that CGRP was exerting a direct effect on the monocyte stimulator population. Finally, the recall response to tetanus toxoid (TT) by PBMC from individuals vaccinated with TT 14 d prior was inhibited by 25-50% in the presence of CGRP. Also, CGRP decreased the levels of B7.2 but not B7.1 on treated monocytes, and this inhibition could be abrogated by the addition of anti-IL-10 antibody, suggesting that the inhibition was mediated by an increase in IL-10 production. Moreover, increased IL-10 production was confirmed by ELISA. Both IL-12 p40 and IFN-gamma levels in CGRP-treated cultures were found to be decreased by approximately 30%. The decrease in IL-12 p40 levels could be reversed by addition of anti-IL-10. These data suggest that CGRP inhibits PBMC proliferation, in part, through the release of IL-10, which in turn can downregulate important co-stimulatory molecules and the cytokines IL-12 and IFN-gamma.

Treatment of cutaneous T-cell lymphoma with extracorporeal photopheresis monotherapy and in combination with recombinant interferon alfa: a 10-year experience at a single institution.

BACKGROUND: Extracorporeal photopheresis is a pheresis-based therapy that permits the direct targeting of psoralen-mediated photochemotherapy to circulating pathogenic T cells. Although photopheresis is currently used to treat cutaneous T-cell lymphoma (CTCL), limited data are available regarding overall response rates and durability of responses among patients with advanced disease. Furthermore, little is known about the effectiveness and tolerability of combined regimens employing other biologic response modifiers including interferon alfa. OBJECTIVE: Our purpose was to determine the efficacy of photopheresis among 41 patients with the clinical and laboratory diagnosis of CTCL; the majority of patients had stage III or IV disease with the presence of circulating malignant T cells. METHODS: A retrospective chart review during a 10-year period at a single university hospital was performed for all patients receiving either photopheresis monotherapy on two consecutive days every 4 weeks (one cycle) and for an additional 12 patients who also received interferon alfa 1.5 to 5 million U subcutaneously three to five times weekly. RESULTS: Thirty-one of 41 patients (76%) were treated for six or more cycles. The remaining 10 were treated with less than six cycles because of rapidly progressing disease (n = 6), death unrelated to CTCL (n = 2), or withdrawal from treatment (n = 1); one of the 10 patients had only received five cycles of treatment but is still receiving therapy. Twenty-eight of the 31 patients treated for six or more cycles received photopheresis alone. Among the 28, seven patients (25%) had a complete remission, 13 (46%) had a partial remission defined as more than 50% clearing of skin disease, and eight (29%) did not respond to treatment. The presence of Sézary cells in the peripheral blood was associated with a favorable response. Median time to treatment failure was 18 months, whereas median survival from initiation of therapy was 77 months and from the time of diagnosis exceeded 100 months. Nine of these 28 patients went on to receive combination therapy with interferon alfa and, in some cases, other agents. Among these nine patients, five had an enhanced clinical response to the combination therapy compared with treatment with photopheresis monotherapy. The combined regimen was well tolerated. CONCLUSION: These results indicate that patients with advanced CTCL can achieve a high response rate for an extended period with photopheresis and that interferon alfa combined with photopheresis is a well-tolerated regimen that appears to produce higher response rates than photopheresis alone.

The potential therapeutic role of interleukin-12 in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a lymphoproliferative disorder characterized by skin invasion of clonally derived malignant CD4+ lymphocytes that phenotypically resemble mature T-helper (Th) cells. Sezary syndrome (SzS) represents an advanced form of CTCL associated with generalized erythroderma and involvement of the peripheral blood by the malignant cell population. We have previously demonstrated aberrant cytokine production by peripheral blood mononuclear cells (PBMCs) in SzS characterized by increased IL-4 and deficient IL-2 and IFN-gamma production, as well as increased expression of mRNA for IL-4 and IL-5 within active skin lesions, indicating that the clonal T-cell population is likely derived from the T-helper type 2 (Th2) subset of helper T lymphocytes. Furthermore, a variety of immune abnormalities have been observed in association with SzS that have been attributed to the cytokine abnormalities. Because IL-12 is a potent inducer of IFN-gamma production and causes the activation of cytotoxic lymphocytes, we assessed the production of IL-12 by PBMCs from SzS patients, and whether IL-12 could alter the unfavorable cytokine balance typical of SzS and, thus, possibly lead to correction of immune defects. In this review, we present our data, which indicate that patients with SzS exhibit marked defects in monocyte production of IL-12 p70. Moreover, in vitro culture of PBMC from SzS patients with recombinant IL-12 leads to reconstitution of normal IFN-gamma production and markedly enhances cell-mediated cytotoxicity.

Reduced surface expression of transforming growth factor beta receptor type II in mitogen-activated T cells from Sézary patients.

Sézary syndrome (SzS), the leukemic form of cutaneous T-cell lymphoma, is characterized by clonal proliferation of CD4+ T cells and immune dysfunctions, raising the possibility of cytokine-related abnormalities. We previously described a decreased response to the growth-inhibitory effects of transforming growth factor type beta (TGF-beta) in SzS T cells accompanied by apparent loss of surface type II TGF-beta receptor (TGF beta RII). To specifically determine if defects exist in TGF beta RII protein expression and/or transport in SzS patients, we developed a sensitive flow cytometric method to detect TGF beta RII on the surface and intracellularly in the CD4+ T cells. Our results indicate that unlike normal CD4+ T cells, CD4+ T cells from 9 of 12 SzS patients expressed little, if any, surface TGF beta RII in response to mitogen stimulation. At the intracellular level, however, pools of TGF beta RII were comparable to those in normal CD4+ T cells. This indicates that defective trafficking of this inhibitory cytokine receptor may contribute significantly to the development of this disease.

Inhibition of interleukin 2 production and alteration of interleukin 2 mRNA processing by human T-T cell hybridoma-derived suppressor factors.

We investigated the mechanisms by which two human T-T cell hybridoma-derived suppressor factors (SFs) (designated 160 and 169) (Platsoucas et al., Hybridoma 1987;6:589; Kunicka et al., Hybridoma 1989;8:127) inhibit the proliferative response to mitogens by human peripheral blood mononuclear cells (PBMCs). Interleukin 2 (IL-2) production by human PBMCs cultured with concanavalin A or OKT3 monoclonal antibody for 12 or 36 hr in the presence of 160 or 169 SF was found to be inhibited > 80% when compared to control PBMC cultures stimulated with mitogen in the absence of SFs. This suppression of IL-2 production was not due to the SFs interfering with IL-2-induced proliferation of the IL-2-dependent murine cell clone used to determine the levels of IL-2. The proliferative responses of SF-treated PBMCs could not be restored by addition of exogenous recombinant human IL-2 (rIL-2) (1-100 U/ml). Furthermore, inhibition of the proliferative responses by the SFs could not be reversed by addition of exogenous rIL-1, rIL-2, or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on concanavalin A-activated cultures at 12- or 36-hr time points was not affected by treatment with the SFs. Both the 160 and 169 hybridoma-derived SFs were found to cause the accumulation of an mRNA of 2.8 kb that hybridized with an IL-2-specific oligonucleotide probe. This 2.8-kb transcript was in addition to the expected 1.0-kb, transiently expressed IL-2 message, and it could be superinduced in the presence of cycloheximide. These results suggest that these SFs may be influencing RNA splicing pathways. These SFs appear to be useful molecules for probing the regulatory controls of lymphocyte proliferation and may constitute important physiological regulators of the immune response. In addition, they may have clinical activity for the treatment of patients that received transplants, patients with autoimmune diseases, and others.

Richter's syndrome associated with loss of response to transforming growth factor-beta.

A patient with chronic lymphocytic leukemia developed a large cell lymphoma apparently derived from the same neoplastic B-cell clone (Richter's syndrome). At the same time, mitogen-stimulated proliferation of the patient's circulating leukemic B-cells was no longer inhibited by the regulatory cytokine transforming growth factor-beta (TGF-beta), suggesting that such loss of inhibition might be contributing to the clinical and biological progression of the disease.

Transforming growth factor beta production and responsiveness in normal human melanocytes and melanoma cells.

Previous studies have shown that some human melanoma cells express transforming growth factor beta (TGF-beta) mRNA and are growth inhibited by exogenous TGF-beta, suggesting a possible negative autocrine role for this melanoma-derived growth factor. To better understand the role of endogenous TGF-beta in the development of melanoma, we investigated patterns of TGF-beta protein production and responsiveness of human melanoma cells as compared to normal melanocytes. Both cultured melanoma cells and normal melanocytes secreted biologically inactive, latent TGF-beta protein which, upon acid treatment, became biologically active. In melanoma cells, TGF-beta production occurred constitutively, i.e., in the absence of exogenous polypeptide growth factors. By contrast, in melanocytes, TGF-beta production depended on stimulation by exogenous growth factors such as insulin-like growth factor I. Exogenous, bioactive TGF-beta 1 at picomolar concentrations inhibited tritiated thymidine uptake of normal melanocytes, whereas melanoma cells demonstrated various degrees of resistance to TGF-beta-induced inhibition of DNA synthesis. Five of six cell lines were less sensitive than any of the melanocyte lines tested, and one cell line was completely resistant to inhibitory effects of TGF-beta on DNA synthesis. In vivo selection of melanoma cells for metastatic ability in athymic mice produced a variant cell line that was resistant to TGF-beta 1-induced inhibition of DNA synthesis and proliferation. Development of TGF-beta resistance in the variant cell line was not associated with changes in TGF-beta cell surface binding. Stable transfection of melanocytes with a plasmid expressing the Simian Virus 40 large T-antigen rendered these cells resistant to growth inhibition by TGF-beta, suggesting that TGF-beta inhibits melanoma/melanocyte growth via interaction with Simian Virus 40 large T-antigen-responsive transcription elements.

Evidence that TGF-beta can inhibit human T-lymphocyte proliferation through paracrine and autocrine mechanisms.

Transforming growth factor-beta (TGF-beta) has been documented as having an inhibitory effect on the proliferation and growth of human T-lymphocytes. We examined the relative contribution of both exogenous and endogenous TGF-beta to this inhibitory action. Purified human peripheral blood T-cells were cultured with Con A (0.2 microgram/ml), washed with methyl mannopyranoside, and then cultured in rIL-2 (5 U/ml) with or without TGF-beta (80 pM). Proliferation, as measured by uptake of tritiated thymidine at 72 hr, was inhibited by added active TGF-beta. Addition of neutralizing anti-TGF-beta antibodies at the initiation of culture abrogated the antiproliferative effects of TGF-beta. A mink lung cell bioassay was used to measure endogenous TGF-beta production by the T-cells following transient acidification of the supernatants to activate latent TGF-beta. T-lymphocytes cultured with rIL-2 alone produced low levels of TGF-beta, first detectable at 72 hr. The addition of (active) TGF-beta to these cultures resulted in earlier and higher levels of endogenously produced latent TGF-beta protein. This was reflected at the mRNA level as well. The exogenously added active TGF-beta appeared to be depleted during the culture period, presumably by the activated T-cells, which exhibited elevated levels of types I, II, and III TGF-beta receptors. The increase in TGF-beta protein levels was due to endogenous TGF-beta synthesis and secretion as supported by a capture assay using 35S-labeled culture supernatants. These findings indicate that both paracrine and autocrine mechanisms are involved in the inhibitory effects of TGF-beta on the proliferation of normal human T-lymphocytes and suggest that other TGF-beta-producing cells can augment production of TGF-beta by activated T-lymphocytes.

Induction of metalloproteinase activity in human T-lymphocytes.

Matrix metalloproteinases are thought to play major roles in a wide array of normal and pathological processes. These proteinases are involved in the degradation of the extracellular matrix and are believed to facilitate the movement of cells from one site to another. In the current study, we examined the expression of the 92 kDa gelatinase activity (MMP-9) by the human T-lymphoma cell line, HSB. Proteinase activity was greatly elevated when cells were treated with TPA. This induction was initially observed at 6 h post-TPA treatment and continued to increase up to 48 h. Proteinase induction was inhibited by actinomycin D and cycloheximide, indicating that nascent RNA and protein synthesis were required. Staurosporine, an inhibitor of protein kinase C activity, suppressed the TPA-induction of gelatinase activity. Our results suggest that TPA induces the 92 kDa gelatinase activity by activating protein kinase C. TGF-beta also induced proteinase activity, although to a lesser extent than TPA. Several criteria indicate that this enzyme is a member of the family of matrix metalloproteinases: (1) this activity was inhibited by EDTA, 1,10-phenanthroline and TIMP; (2) this activity bound to a gelatin-agarose affinity resin; (3) it has a mass of approx. 92 kDa on SDS-polyacrylamide gels; (4) it cleaves gelatin and (5) the inducible proteinase cross reacts with antiserum to MMP-9.

Transforming growth factor-beta inhibits human T-cell proliferation through multiple targets.

This study investigates further the inhibitory effects of transforming growth factor-beta (TGF-beta) on human T-lymphocyte responses to mitogenic stimulation. T cells were stimulated either with mitogenic concentrations of PHA or with submitogenic concentrations of Con A followed by the addition of IL-2. DNA synthesis ([3H]thymidine incorporation) in both systems was inhibited by 60-69% in the presence of TGF-beta, with maximal reduction occurring on days 4 and 5 of culture. Cell surface expression of transferrin receptor (TfR) and IL-2 receptor-alpha (p55) were inhibited by 20-80% in the Con A/rIL-2 system and 20-45% in the PHA system in the presence of TGF-beta. In addition, mitogen-induced up-regulation of TfR and IL-2R mRNA levels were inhibited by TGF-beta. Finally, we investigated the effect of TGF-beta on the assembly of clathrin monomers into assembled coated pits and vesicles, and essential step in TfR and IL-2R alpha turnover. Stimulation of T cells using either mitogen system resulted in an increase in the level of assembled clathrin, which was almost completely inhibited by TGF-beta. These findings suggest that TGF-beta may act at several sites in mitogen-mediated proliferative pathways to contribute to the inhibition of T-cell proliferation.

Human hybridoma-derived suppressor factor 160 and transforming growth factor-beta are different molecules.

We have previously identified a suppressor factor (SF), designated 160 constitutively produced by human T-T-cell hybridomas generated by fusing Con A-activated human peripheral blood lymphocytes from a normal donor with cells of the Jurkat tumor T-cell line (Hybridoma 8:127-151, 1989). The 160 SF inhibited in vitro proliferative responses to polyclonal activators and allogeneic cells, and immunoglobulin synthesis and secretion of human and mouse lymphocytes. We investigated whether the hybridoma-derived 160 SF and transforming growth factor-beta (TGF-beta) are distinct molecules. TGF-beta has been shown to inhibit a number of lymphocyte responses. In agreement with our previous findings, the 160 SF abrogated the proliferative responses of human peripheral blood mononuclear cells (PBMC) to mitogens and allogeneic cells in mixed lymphocyte culture. In contrast, TGF-beta, added to the PBMC cultures at the same time with the mitogen or the stimulating allogeneic cells, had no effect on the proliferative response. Acid treatment of the 160 SF completely abolished the 160 SF activity. In contrast, this treatment results in activation of the latent TGF-beta form to the active form, and acidification does not affect the function of existing active TGF-beta. A polyclonal anti-TGF-beta antibody did not detect TGF-beta by Western blotting in concentrated (10x) 160 SF preparations. In addition, the 160 SF did not induce the anchorage-independent growth of NRK fibroblasts in the presence of EGF.TGF-beta at concentrations as low as 1 ng/ml, in the presence of EGF, induced the anchorage-independent growth of the anchorage-dependent indicator NRK cells.(ABSTRACT TRUNCATED AT 250 WORDS)