Stanley F. Wolf

Clusters of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes

DNA sequences of the X-chromosome-linked hypoxanthine phos-phoribosyltransferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) genes have revealed the presence of clusters of CpG dinucleotides1–3, raising the possibility that such clusters are involved in the control of expression of these genes, which are expressed in all tissues. Although CpG clusters are not exclusive features of the X chromosome4–6, the analysis of X-linked genes provides the means to determine whether CpG clusters are control elements; one of the two homologous X loci in female mammals is not expressed7, so that active and inactive versions of the gene can be compared. In fact, it has been shown that these CpG clusters are undermethylated when the gene is active and extensively methylated when the gene is inactive2,8. In addition to hypomethylation, chromatin hypersensitivity to endonuclease digestion is a known hallmark of regulatory sequences in eukaryotic genes9. We report here that the CpG clusters of the active hprt and g6pd genes are not only undermethylated, but also hypersensitive to MspI, DNase I and S1 nuclease, further supporting the suggestion that they are involved in the control of expression of these genes.

Type I interferons and IL-12: convergence and cross-regulation among mediators of cellular immunity

Therapeutic use of type I IFN (IFN‐α/β) has become common. Many of the diverse diseases targeted are marked by pathogenetic abnormalities in cell‐mediated immunity (CMI), these cellular immune responses either causing injury to the host, lacking sufficient vigor for virus or tumor clearance, or both. In general, therapeutic efficacy is limited. It is thus notable that the pleiotropic effects of type I IFN on CMI remain poorly understood. We characterized the effects of type I IFN on the production of IL‐12, the central immunoregulatory cytokine of the CD4+ T cell arm of CMI. We show that type I IFN are potent inhibitors of IL‐12 production by human monocytes/macrophages. The underlying mechanism involves transcriptional inhibition of the IL‐12p40 gene, marked by down‐regulation of PU.1 binding activity at the upstream Ets site of the IL‐12p40 promoter. Type I IFN have previously been shown to be able to substitute for IL‐12 in driving IFN‐γ production from T and NK cells. The ability of IFN‐α/β to suppress IL‐12 production while up‐regulating IFN‐γ production suggests a possible mechanistic basis for the difficulties of employing these cytokines in diseases involving abnormalities of CMI.

Intranasal IL-12 produces discreet pulmonary and systemic effects on allergic inflammation and airway reactivity

IL-12 modulates T cell responses between helper T cells Th2 and Th1; however, the therapeutic potential of IL-12 for allergic diseases either directly or as an adjuvant in allergen therapy has been controversial. The role of intranasal IL-12 as an adjuvant in modulating the grass pollen allergen (GAL) therapy-induced systemic immune response and lung-specific inflammation and airway reactivity was examined in this study using a mouse model of established allergic asthma. The effects of intranasal or nebulized IL-12 with or without intranasal anti-IFN-gamma antibody were examined in groups of control and allergen-sensitized or -challenged mice. T cell cytokine patterns, antibody response profiles, pulmonary inflammation and airway reactivity were examined. Intranasal IL-12 was found to be more effective in the Th2-Th1 shifting of immune response and anti-inflammatory activity in the lung compared to nebulized IL-12 at the given doses. Intranasal IL-12 significantly decreased production of IFN-gamma, eotaxin and LTC4/D4/E4 in the lung and decreased eosinophil infiltration, resulting in attenuated airway hyper-responsiveness in GAL-sensitized (GS) mice. In contrast, intranasal IL-12 significantly increased IFN-gamma production in the thoracic lymph node cultures and decreased the IL-5/IFN-gamma ratio, suggesting a Th2-Th1 shift. Also, intranasal IL-12 increased GAL-specific IgG2a antibody response, while the IgE response remained unaffected. The systemic effects of IL-12 were IFN-gamma dependent. IL-12 induces differential expression of its own receptor beta1 and beta2 subunits in the lung tissues to augment IL-12 responsiveness. Together, these results demonstrate that intranasal IL-12 is effective in shifting the systemic immune response in the direction of Th1 in IFN-gamma-dependent manner, while decreasing pulmonary inflammation and airway reactivity independent of IFN-gamma. Thus, intranasal delivery of IL-12 may provide an approach for the treatment of asthma and may be useful as an adjuvant in local nasal immunotherapy (IT) and in asthma.

Modulation of the IL-12/IFN-γ axis by IFN-α therapy for hepatitis C

Although IFN‐α forms the foundation of therapy for chronic hepatitis C, only a minority of patients has a sustained response to IFN‐α alone. The antiviral activities of IFN‐α formed the rationale for its use in viral hepatitis. However, IFN‐α and the other Type I IFNs are also pleiotropic immune regulators. Type I IFNs can promote IFN‐γ production by activating STAT4 but can also inhibit production of IL‐12, a potent activator of STAT4 and IFN‐γ production. The efficacy of IFN‐α in the treatment of hepatitis C may therefore depend in part on the balance of IFN‐γ‐inducing and IL‐12‐suppressing effects. We characterized the effects of pegylated IFN‐α therapy for hepatitis C on the capacity of patients’ PBMC to produce IL‐12 and IFN‐γ ex vivo. Cells from patients with a sustained virological response to therapy had significantly greater levels of IFN‐α‐driven IFN‐γ production prior to treatment than those from nonresponding patients. No differences in pretreatment IL‐12 productive capacity were seen between patient groups. However, therapy with IFN‐α led to suppression of inducible IL‐12 production throughout the course of therapy in both groups of patients.

Cytokines and Cancer Immunotherapy

Interleukin 12 (IL-12) and factors that access the B7/CD28/CTLA4 pathway have been shown in mice to be potent anti-cancer agents. IL-I2 is a cytokine central to the immune response (1,2). It enhances the innate immune response, inducing NK lytic activity and IFNg secretion and directs the adaptive immune response, promoting differentiation of T cell progenitors into the Thl pathway. The cytokine profile it induces promotes MHC I and I1 expression and is thus thought to promote antigen presentation as well. IL-12 also acts at the effector stage of an immune response, enhancing T cell lytic activity, IFNg, and to a lesser extent, GM-CSF and TNF expression. IL-12 has shown considerable activity in a large number of murine tumor models including nonimunogenic tumors such as B16E10 and spontaneous tumors developed in mice transgenic for proto-oncogenes (3) or treated with methylchloranthine (4). Although a detailed understanding of mechanisms responsible for IL-12s efficacy have not been achieved, features common to many models have been induction of IFNg with consequent anti-angiogenic activity and promotion of effector CD8 cells. Based on its activities and efficacy in murine models, IL-12 moved into clinical development. After surmounting initial toxicity associated with a specific dose and schedule (5 , 6), the drug has been tested extensively as an anti-cancer agent in humans. Its development path can serve to highlight some of the issues in developing immunotherapeutics for cancer. Phase IAI studies with IL-12 suggest that clinical efficacy may be achievable. Rook et a1

A CTL assay requiring only 150 μl of mouse blood

In this paper, we present a method for measuring antigen specific cytotoxic T lymphocyte (CTL) activity from individual mouse peripheral blood samples without animal sacrifice. Peripheral blood cells are stimulated in vitro with a cocktail of antigen, cytokines, costimulatory molecules and irradiated feeder cells resulting, 7 days later, in a readily detectable antigen specific signal from a well plated under limiting dilution conditions. This highly sensitive and antigen specific assay is more efficient than conventional CTL assays and thus increases the number of mice that can be tested in a single assay. Since blood samples can be assayed from an individual mouse at multiple times during the course of an in vivo study, the assay can facilitate and strengthen correlative studies on CTL responses and in vivo results.