Shawn P. Murphy

Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress.

The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has promoted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have used polyclonal antisera raised against mouse HSF1 and HSF2 to examine the biochemical, physical, and functional properties of these two factors in unstressed and heat-shocked mouse and human cells. We have identified HSF1 as the mediator of stress-induced heat shock gene transcription. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. Also, HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in the absence of physiological stress and also provide support for a model of regulation of HSF1 and HSF2 activity by a titratable negative regulatory factor.

Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies

Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class‐I (HLA‐I) loci has made it difficult to generate locus‐specific monoclonal antibodies (mAbs). The problem of defining an antibody’s reactivity against the thousands of existing HLA‐I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA‐I to characterize experimentally the reactivity of nine mAb against 96 common HLA‐I allotypes. In conjunction with donor HLA‐I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG‐3 and JAR; and the placental cell lines HTR‐8/SVneo, Swan‐71 and TEV‐1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA‐C, HLA‐G and HLA‐E, but not HLA‐A, HLA‐B or HLA‐DR molecules in normal pregnancy. Tumour‐derived JEG‐3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.

Interferon Gamma in Successful Pregnancies

Abstract Interferon gamma (IFNG) is a proinflammatory cytokine secreted in the uterus during early pregnancy. It is abundantly produced by uterine natural killer cells in maternal endometrium but also by trophoblasts in some species. In normal pregnancies of mice, IFNG plays critical roles that include initiation of endometrial vasculature remodeling, angiogenesis at implantation sites, and maintenance of the decidual (maternal) component of the placenta. In livestock and in humans, deviations in these processes are thought to contribute to serious gestational complications, such as fetal loss or preeclampsia. Interferon gamma has broader roles in activation of innate and adaptive immune responses to viruses and tumors, in part through upregulating transcription of genes involved in cell cycle regulation, apoptosis, and antigen processing/presentation. Despite this, rodent and human trophoblast cells show dampened responses to IFNG that reflect the resistance of these cells to IFNG-mediated activation of major histocompatibility complex (MHC) class II transplantation antigen expression. Lack of MHC class II antigens on trophoblasts is thought to facilitate survival of the semiallogeneic conceptus in the presence of maternal lymphocytes. This review describes the dynamic roles of IFNG in successful pregnancy and briefly summarizes data on IFNG in gestational pathologies.

Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells.

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

Type I interferons induced by radiation therapy mediate recruitment and effector function of CD8+ T cells

The need for an intact immune system for cancer radiation therapy to be effective suggests that radiation not only acts directly on the tumor but also indirectly, through the activation of host immune components. Recent studies demonstrated that endogenous type I interferons (type I IFNs) play a role in radiation-mediated anti-tumor immunity by enhancing the ability of dendritic cells to cross-prime CD8+ T cells. However, it is still unclear to what extent endogenous type I IFNs contribute to the recruitment and function of CD8+ T cells. Little is also known about the effects of type I IFNs on myeloid cells. In the current study, we demonstrate that type I and type II IFNs (IFN-γ) are both required for the increased production of CXCL10 (IP-10) chemokine by myeloid cells within the tumor after radiation treatment. Radiation-induced intratumoral IP-10 levels in turn correlate with tumor-infiltrating CD8+ T cell numbers. Moreover, type I IFNs promote potent tumor-reactive CD8+ T cells by directly affecting the phenotype, effector molecule production, and enhancing cytolytic activity. Using a unique inducible expression system to increase local levels of IFN-α exogenously, we show here that the capacity of radiation therapy to result in tumor control can be enhanced. Our preclinical approach to study the effects of local increase in IFN-α levels can be used to further optimize the combination therapy strategy in terms of dosing and scheduling, which may lead to better clinical outcome.

Absence of MHC class II antigen expression in trophoblast cells results from a lack of class II transactivator (CIITA) gene expression

Although the mechanism(s) underlying the failure of the maternal immune system to reject the semiallogeneic fetus have not been clearly defined, the absence of MHC class II antigen expression by fetal trophoblast cells very likely plays a critical role in the maintenance of normal pregnancy. However, the regulation of class II antigen expression in trophoblast cells is poorly understood. Class II transactivator (CIITA) is a transacting factor that is required for both constitutive and IFN‐γ‐inducible class II gene transcription. In this report we demonstrate that the inability of trophoblast cells to express class II antigens is due to a lack of CIITA gene expression. Trophoblast cell lines derived from human, mouse, and rat do not express CIITA, and expression is not inducible by IFN‐γ. The absence of CIITA gene expression in trophoblasts treated with IFN‐γ does not result from a defect in the IFN‐γ receptor or the JAK/STAT pathway, because the classical IFN‐γ‐inducible gene encoding the guanylate‐binding protein is expressed. Transfection of CIITA expression vectors into trophoblast cells results in activation of class II promoters, endogenous class II mRNA expression, and subsequent expression of class II antigens on the cell surface. In contrast, class I mRNA is not expressed in human trophoblast cells transfected with CIITA expression vectors. Thus, trophoblast cells contain all of the DNA binding factors necessary for class II transcription, and ectopic expression of CIITA is sufficient to activate class II, but not class I expression. The failure of trophoblast cells to express CIITA, and therefore class II antigens, provides a potential mechanism by which the fetus is protected from the maternal immune system during pregnancy. Mol. Reprod. Dev. 51:1–12, 1998.

Regulation of major histocompatibility complex class II gene expression in trophoblast cells.

Trophoblast cells are unique because they are one of the few mammalian cell types that do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to IFN-γ. The absence of MHC class II antigen expression on trophoblast cells has been postulated to be one of the essential mechanisms by which the semi-allogeneic fetus evades immune rejection reactions by the maternal immune system. Consistent with this hypothesis, trophoblast cells from the placentas of women suffering from chronic inflammation of unknown etiology and spontaneous recurrent miscarriages have been reported to aberrantly express MHC class II antigens. The lack of MHC class II antigen expression on trophoblast cells is due to silencing of expression of the class II transactivator (CIITA), a transacting factor that is essential for constitutive and IFN-γ-inducible MHC class II gene transcription. Transfection of trophoblast cells with CIITA expression vectors activates both MHC class II and class Ia antigen expression, which confers on trophoblast cells both the ability to activate helper T cells, and sensitivity to lysis by cytotoxic T lymphocytes. Collectively, these studies strongly suggest that stringent silencing of CIITA (and therefore MHC class II) gene expression in trophoblast cells is critical for the prevention of immune rejection responses against the fetus by the maternal immune system. The focus of this review is to summarize studies examining the novel mechanisms by which CIITA is silenced in trophoblast cells. The elucidation of the silencing of CIITA in trophoblast cells may shed light on how the semi-allogeneic fetus evades immune rejection by the maternal immune system during pregnancy.

IFN-γ mediates the antitumor effects of radiation therapy in a murine colon tumor.

Cancer treatments using ionizing radiation (IR) therapy are thought to act primarily through the induction of tumor cell damage at a molecular level. However, a new concept has recently emerged, suggesting that the immune system is required for effective IR therapy. Our work here has identified interferon gamma (IFN-γ) as an essential cytokine for the efficacy of IR therapy. Local IR (15 Gy) to mice bearing Colon38, a colon adenocarcinoma, decreases tumor burden in wild-type animals. Interestingly, IR therapy had no effect on tumor burden in IFNγKO mice. We further determined that intratumoral levels of IFN-γ increased 2 days following IR, which directly correlated with a decrease in tumor burden that was not a result of direct cytotoxic effects of IFN-γ on tumor cells. T cells from IR-treated tumors exhibited a far greater capacity to lyse tumor cells in a (51)Cr release assay, a process that was dependent on IFN-γ. CD8(+) T cells were the predominant producers of IFN-γ, as demonstrated by IFN-γ intracellular staining and studies in IFN-γ reporter mice. Elimination of CD8(+) T cells by antibody treatment reduced the intratumoral levels of IFN-γ by over 90%. More importantly, elimination of CD8(+) T cells completely abrogated the effects of radiation therapy. Our data suggest that IFN-γ plays a pivotal role in mediating the antitumor effects of IR therapy.

Class II transactivator (CIITA) promoter methylation does not correlate with silencing of CIITA transcription in trophoblasts

Abstract Trophoblast cells are unique because they do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to interferon-γ (IFN-γ). The absence of MHC class II antigens on trophoblasts is thought to play a critical role in preventing rejection of the fetus by the maternal immune system. The inability of trophoblasts to express MHC class II genes is primarily due to lack of the class II transactivator (CIITA), a transacting factor that is required for constitutive and IFN-γ-inducible MHC class II transcription. We, therefore, investigated the silencing of CIITA expression in trophoblasts. In transient transfection assays, transcription from the IFN-γ-responsive CIITA type IV promoter was upregulated by IFN-γ in trophoblasts, which suggests that CIITA is silenced by an epigenetic mechanism in these cells. Polymerase chain reaction analysis demonstrated that the CIITA type IV promoter is methylated in both the human choriocarcinoma cell lines JEG-3 and Jar and in 2fTGH fibrosarcoma cells, which are IFN-γ inducible for CIITA. Conversely, methylation of the CIITA type IV promoter was not observed in human primary cytotrophoblasts isolated from term placentae or in mouse or rat trophoblast cell lines. Simultaneous treatment with IFN-γ and the histone deacetylase inhibitor trichostatin A weakly activated CIITA transcription in mouse trophoblasts. Stable hybrids between human choriocarcinoma and fibrosarcoma cells and between mouse trophoblasts and fibroblasts expressed CIITA following treatment with IFN-γ. These results suggest that silencing of CIITA transcription is recessive in trophoblasts and involves an epigenetic mechanism other than promoter methylation. The fact that CIITA is expressed in the stable hybrids implies that trophoblasts may be missing a factor that regulates chromatin structure at the CIITA promoter.

DNA Alkylating Agents Alleviate Silencing of Class II Transactivator Gene Expression in L1210 Lymphoma Cells

MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II− L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II− L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.