Abby L. Maizel

Induction of the IL-13 receptor α2-chain by IL-4 and IL-13 in human keratinocytes : involvement of STAT6, ERK and p38 MAPK pathways

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Rα and γc and only binds IL-4, whereas, in the type II receptor, IL-4Rα dimerizes with IL-13Rα1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Rα2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Rα2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Rα2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Rα2 mRNA expression, but potentiated the effects of IL-4 or IL-13 on IL-13Rα2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Rα2 mRNA even in absence of stimulation. Our findings demonstrate, for the first time, that IL-4 and IL-13 can induce IL-13Rα2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process.

Targeted T-cell Therapy in Stage IV Breast Cancer: A Phase I Clinical Trial

Purpose: This study reports a phase I immunotherapy trial in 23 women with metastatic breast cancer consisting of eight infusions of anti-CD3 × anti-HER2 bispecific antibody (HER2Bi) armed anti-CD3–activated T cells (ATC) in combination with low-dose IL-2 and granulocyte-macrophage colony-stimulating factor to determine safety, maximum tolerated dose (MTD), technical feasibility, T-cell trafficking, immune responses, time to progression, and overall survival (OS). Experimental Design: ATC were expanded from leukapheresis product using IL2 and anti-CD3 monoclonal antibody and armed with HER2Bi. In 3+3 dose escalation design, groups of 3 patients received 5, 10, 20, or 40 × 109 armed ATC (aATC) per infusion. Results: There were no dose-limiting toxicities and the MTD was not defined. It was technically feasible to grow 160 × 109 ATC from a single leukapheresis. aATC persisted in the blood for weeks and trafficked to tumors. Infusions of aATC induced anti-breast cancer responses and increases in immunokines. At 14.5 weeks after enrollment, 13 of 22 (59.1%) evaluable patients had stable disease and 9 of 22 (40.9%) had progressive disease. The median OS was 36.2 months for all patients, 57.4 months for HER2 3+ patients, and 27.4 months for HER2 0–2+ patients. Conclusions: Targeting HER2+ and HER2− tumors with aATC infusions induced antitumor responses, increases in Th1 cytokines, and IL12 serum levels that suggest that aATC infusions vaccinated patients against their own tumors. These results provide a strong rationale for conducting phase II trials. Clin Cancer Res; 21(10); 2305–14. ©2015 AACR.

Human Immunoregulatory Molecules: Interleukin 1, Interleukin 2. and B-Cell Growth Factor

Intensive investigations over the past several years have clearly indicated that a significant degree of immune regulation is accomplished by soluble mediators. Among the host of potential immune regulators that have been described to date, the interleukins have been the most fully characterized. Yet many aspects of both the biochemistry and the cell biology of these factors remain to be determined. In an attempt to place some of the areas of current investigational interest into perspective, a brief historical overview of each factor will be undertaken. This historical overview will detail our knowledge of the biochemical properties attributed to each factor when first described. This latter point will be of significance in our subsequent attempts to unravel the biological properties of each factor and how it mediates those effects that lead to a competent immune response.

Establishment of stable human T-T hybridomas

Human T-T hybridomas potentially provide an invaluable resource for a variety of immunoregulatory molecules that modulate the immune response. To date, success in this technology, using human cell populations, has been hampered by several problems associated with proliferative and functional instability of the hybrid cells. These forms of instability are the result of a multifactorial process, with 1 parameter of importance being the chromosome number of the malignant parent cell line used for fusion. The present studies describe the production of a stable human T-T hybridoma generated by fusing a near diploid (modal chromosome number of 48) aminopterin-sensitive T cell line, CEM TG E11, and lectin-stimulated human peripheral blood lymphocytes. The rapidly growing hybrid cells have been clonally selected for the production of a B cell growth factor. Hybridization was documented by the presence of HLA phenotypes reflecting the combined antigens of the fusion partners. Fusions with 4 other partners besides CEM TG E11, where the majority of the cells had modal chromosome numbers ranging from 78 to 94, were proliferatively unstable. To date, hybrid cells derived from the CEM TG E11 fusion have been doubling approximately every 48 h for greater than 12 months, and selected clones constitutively produce B cell growth factor.

Activity of a partially purified human BCGF on murine assays for B-cell stimulatory factors. I: BCGF II-like activity of human BCGF

To further characterize a human B-cell growth factor (BCGF) produced by phytohemagglutinin (PHA) P-stimulated peripheral blood T cells, a partially purified preparation of this material was tested in a number of murine assays for B-cell stimulatory factors (BSF). Human BCGF lacked murine BSF-1 activity as assessed via the induction of polyclonal proliferation of anti-IgM-stimulated murine B cells; however, this material consistently augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1. Human BCGF also induced proliferation in unstimulated murine B cells, and augmented the proliferative response of dextran sulfate activated murine B cells. Human BCGF is therefore capable of causing proliferation of unstimulated and activated murine B cells, and by these criteria closely resembles murine BCGF II. In contrast to murine BCGF II, however, human BCGF failed to stimulate proliferation or immunoglobulin (Ig) secretion by murine BCL1 B lymphoma cells. A murine analog of this human BCGF showing the same pattern of biological responses was found in concanavalin A-stimulated supernatants of the murine MB2.1 T-cell line and D9-Cl T-cell hybridoma. The active component of the human BCGF preparation was not due to contaminating PHA, interleukin 1, interleukin 2; interferon-gamma, or endotoxin. Comparison between the above human BCGF and a commonly used source of murine BCGF II, i.e., supernatant from antigen-stimulated D10.G4.1 T cells, provided information suggestive of BCGF II heterogeneity. Both human BCGF and D10.G4.1 supernatant caused proliferation of unstimulated and dextran sulfate-stimulated murine B cells; however, only the human BCGF preparation augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1, and only the D10.G4.1 supernatant stimulated BCL1 cell proliferation and immunoglobulin secretion. The data therefore indicate that the different assays for BCGF II used in this study respond to different factors, and suggest the existence of two BCGF II-like activities.

Molecular Biology of Lung Cancer

Lung cancer continues to be the major cause of cancer death in North America. Despite some improvement in survival rates, overall only 10% of patients with non-small-cell lung cancer (NSCLC) and 3% of patients with small-cell lung cancer (SCLC) are long-term survivors (1,2). Delineation of the molecular genetics of lung carcinoma has provided significant insights into the pathogenesis of lung cancer, and can potentially enhance our ability to diagnose and manage patients. Application of conventional screening methods (e.g., chest X-rays, sputum cytology) in high-risk groups has had no impact on mortality, because most tumors are still detected too late for definitive surgical therapy (3–5). The ability to detect genetic alterations in individual tumor cells using techniques such as polymerase chain reaction (PCR) offers the possibility of molecular screening for lung cancer. These techniques offer great sensitivity, and have the potential to detect very rare tumor cells in sputum and bronchoalveolar lavage (BAL) samples, which may permit the diagnosis of tumors at a stage early enough to allow surgical cure.

Monoclonal Antibody AB1 Identifies a Human B Cell Activation Antigen and Inhibits Growth Factor-Dependent Human B Cell Proliferation

In both human and animal systems, various growth factors have been shown to be involved in B cell proliferation and differentiation (reviewed in Ref. 1). B cell growth factor (BCGF), also named B cell stimulatory factor (BSF), has been intensively studied. It has also been postulated that resting B cells are required to be activated to express BSF receptors during the early stage of activation prior to becoming responsive to BSF. Although many B cell activation antigens (2–5) have been described in man, none of them have been shown to be related to the initiation process of B cell proliferation.

IDENTIFICATION OF HUMAN B CELL GROWTH FACTOR11This work was supported in part by NIH Grant CA21927, a grant from the King Faisal Foundation and an Institutional grant from the Interferon Research Fund.

ABSTRACT Isolation of a specific human B cell growth factor (BCGF) has been the focus of the present investigation. This factor has been separated from other cytokines present in lectin stimulated peripheral blood lymphocyte conditioned media by a combination of ion exchange chromatography and multiple gel filtration steps. The isolated soluble factor possesses a molecular weight of 12-13,000 daltons and is specific for stimulating S phase entry in a proportion of activated B lymphocytes.