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Supplementary Figures S1-S8; Supplementary Materials and Methods.

Purpose: Diverse immune-related effects occur with the use of epidermal growth factor receptor inhibitors (EGFRI). In addition to the cutaneous inflammation induced by EGFRIs, these agents have been associated with the exacerbation of autoimmune skin disease and contact hypersensitivity, antiviral effects, and fatal alveolar damage in the setting of lung transplantation. Because EGFR ligands can modulate MHC class I (MHCI) and II (MHCII) molecule expression, we hypothesized that some of the immune-related effects of EGFRIs are due to direct effects on the expression of MHCI and/or MHCII molecules.Experimental Design: Primary human keratinocytes and a malignant keratinocyte cell line (A431) were treated with EGFRIs alone or prior to IFN-γ, a potent inducer of MHCI and MHCII molecule expression. CIITA, MHCI, and MHCII RNA expression was measured using quantitative real-time reverse transcriptase PCR, and cell surface MHCI and MHCII protein expression was measured using flow cytometry. Skin biopsies from patients were analyzed for MHCI and MHCII protein expression before and during therapy with an EGFRI using immunohistochemistry.Results: Both EGFR tyrosine kinase inhibitors and ligand-blocking antibodies (cetuximab) augmented the induction of MHCI and MHCII molecules by IFN-γ in primary and malignant human keratinocytes. Unexpectedly, the increase in MHCI protein expression did not require the presence of IFN-γ. Consistent with these in vitro findings, skin biopsies from cancer patients exhibited increased epidermal MHCI protein expression during therapy with an EGFRI as well as increases in MHCI and MHCII molecule RNA.Conclusions: These studies suggest that EGFRIs may influence immune/inflammatory responses by directly modulating MHC expression. Clin Cancer Res; 17(13); 4400–13. ©2011 AACR.

Molecular events activating the PI3K pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to antitumor immunity. Given the ability of IFNγ to regulate antigen processing and presentation and the pivotal role of MHC class I (MHCI) and II (MHCII) expression in T-cell–mediated antitumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFNγ. We found that the induction of cell surface MHCI and MHCII molecules by IFNγ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFNγ treatment and increases accessibility at genomic STAT1-binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFNγ, and likewise, the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFNγ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intratumoral regions with high phospho-AKT IHC staining had reduced MHCI IHC staining. Implications: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.

The Ebola virus (EBOV) surface glycoprotein (GP 1,2 ) mediates host cell attachment and fusion and is the primary target for host neutralizing antibodies. Expression of GP 1,2 at high levels disrupts normal cell physiology, and EBOV uses an RNA-editing mechanism to regulate expression of the GP gene. In this study, we demonstrate that high levels of GP 1,2 expression impair production and release of EBOV virus-like particles (VLPs) as well as infectivity of GP 1,2 -pseudotyped viruses. We further show that this effect is mediated through two mechanisms. First, high levels of GP 1,2 expression reduce synthesis of other proteins needed for virus assembly. Second, viruses containing high levels of GP 1,2 are intrinsically less infectious, possibly due to impaired receptor binding or endosomal processing. Importantly, proteolysis can rescue the infectivity of high-GP 1,2 -containing viruses. Taken together, our findings indicate that GP 1,2 expression levels have a profound effect on factors that contribute to virus fitness and that RNA editing may be an important mechanism employed by EBOV to regulate GP 1,2 expression in order to optimize virus production and infectivity. IMPORTANCE The Ebola virus (EBOV), as well as other members of the Filoviridae family, causes severe hemorrhagic fever that is highly lethal, with up to 90% mortality. The EBOV surface glycoprotein (GP 1,2 ) plays important roles in virus infection and pathogenesis, and its expression is tightly regulated by an RNA-editing mechanism during virus replication. Our study demonstrates that the level of GP 1,2 expression profoundly affects virus particle production and release and uncovers a new mechanism by which Ebola virus infectivity is regulated by the level of GP 1,2 expression. These findings extend our understanding of EBOV infection and replication in adaptation of host environments, which will aid the development of countermeasures against EBOV infection.

Purpose: Diverse immune-related effects occur with the use of epidermal growth factor receptor inhibitors (EGFRI). In addition to the cutaneous inflammation induced by EGFRIs, these agents have been associated with the exacerbation of autoimmune skin disease and contact hypersensitivity, antiviral effects, and fatal alveolar damage in the setting of lung transplantation. Because EGFR ligands can modulate MHC class I (MHCI) and II (MHCII) molecule expression, we hypothesized that some of the immune-related effects of EGFRIs are due to direct effects on the expression of MHCI and/or MHCII molecules. Experimental Design: Primary human keratinocytes and a malignant keratinocyte cell line (A431) were treated with EGFRIs alone or prior to IFN-γ, a potent inducer of MHCI and MHCII molecule expression. CIITA, MHCI, and MHCII RNA expression was measured using quantitative real-time reverse transcriptase PCR, and cell surface MHCI and MHCII protein expression was measured using flow cytometry. Skin biopsies from patients were analyzed for MHCI and MHCII protein expression before and during therapy with an EGFRI using immunohistochemistry. Results: Both EGFR tyrosine kinase inhibitors and ligand-blocking antibodies (cetuximab) augmented the induction of MHCI and MHCII molecules by IFN-γ in primary and malignant human keratinocytes. Unexpectedly, the increase in MHCI protein expression did not require the presence of IFN-γ. Consistent with these in vitro findings, skin biopsies from cancer patients exhibited increased epidermal MHCI protein expression during therapy with an EGFRI as well as increases in MHCI and MHCII molecule RNA. Conclusions: These studies suggest that EGFRIs may influence immune/inflammatory responses by directly modulating MHC expression. Clin Cancer Res; 17(13); 4400–13. ©2011 AACR.

Please cite this paper as: Activating transcription factor 3 (ATF3) expression is increased in erythema multiforme and is regulated by IFN-γ in human keratinocytes. Experimental Dermatology 2010; 19: e310–e313. Abstract: Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP responsive element-binding protein (CREB) family of transcription factors and is involved in the regulation of immune responses, apoptosis, DNA repair and oncogenesis. The epidermal expression of ATF3 in the setting of cutaneous inflammation has not been well characterized. To examine the expression of ATF3 in the setting of inflammatory skin disease, ATF3 protein expression was analysed by immunohistochemistry (IHC). We found diffuse epidermal ATF3 protein expression in skin biopsies of erythema multiforme (EM). Given the role of interferon (IFN)-γ in erythema multiforme, we sought to examine the impact of IFN-γ on ATF3 expression in human keratinocytes. IFN-γ induced ATF3 mRNA and protein in primary human keratinocytes and HaCaT cells. Thus, epidermal ATF3 expression can be increased in the setting of inflammatory skin diseases and is regulated by IFN-γ in human keratinocytes.

UVR is an important environmental carcinogen and a powerful modulator of the cutaneous immune system. Exposure to UVR activates many signaling pathways leading to changes in the expression of several hundred genes. While the covalent modification of histones has been shown to play a central role in regulating gene expression, the impact of UVR on histone modifications and the contribution of histone acetyltransferases (HATs) and histone deacetylases (HDACs) to the UVR-induced transcriptional response have not been completely characterized. In this report, we have examined the impact of UVR on histone H3 K9/14 acetylation. The potential role of UVR-induced histone acetylation in the UVR transcriptional response was also explored using the HAT inhibitor curcumin and HDAC inhibitor trichostatin A (TSA). We found that UVR caused an increase in histone H3 acetylation within the promoter regions of ATF3, COX2, IL-8, MKP1 and MnSOD. In most of the regions examined, histone H3 acetylation peaked 24 h after UVR and then returned to baseline levels by 72 h. The induction of ATF3, COX2 and MKP1 was blocked in the presence of curcumin at doses that decrease in vivo histone H3 acetylation but not at lower doses that do not affect acetylation levels. We also provide evidence that for ATF3, a transcriptional superinduction occurs after repeat exposures to UVR that can be recapitulated when the second UVR exposure is replaced with TSA treatment. Thus, UVR can alter histone acetylation within human keratinocytes and these changes may contribute to the UVR-transcriptional response.

Systemic use of epidermal growth factor receptor inhibitors (EGFRIs) has been shown to alter MHC expression and that of several chemokines and to enhance immune cell recruitment into human skin. We hypothesized that EGFRIs may have value as cutaneous immune response modifiers and determined the effects of topical application of an irreversible EGFRI on a well-established murine model of influenza vaccination. We found that a single topical application of an EGFRI led to increased levels of antibodies that inhibit influenza mediated hemagglutination and viral cytopathic effects. The topically applied EGFRI significantly enhanced the generation of vaccine-specific IL-4 and IFN-γ producing cells within skin-draining lymph nodes as early as one week following vaccination. The EGFRI/vaccine group showed a twelve-fold reduction in detectable pulmonary viral load four days after infection as compared to the vaccine alone control group. The reduction in the lung viral titers correlated with the survival rate, which demonstrated 100% protection in the EGFRI/vaccine immunized group but only 65% protection in the mice immunized with vaccine alone. These findings are significant because they demonstrate that inhibition of defined signaling pathways within the skin using small molecule kinase inhibitors provides a novel approach to enhance immune responses to vaccines.

To optimally integrate targeted kinase inhibitors and immunotherapies in the treatment of melanoma, it will be critical to understand how BRAFV600E mutational status and BRAFV600E inhibition influence the expression of genes that govern antitumor immune responses. Because major histocompatibility complex (MHC) molecules are critical for interactions between tumor cells and lymphocytes, we investigated the impact of BRAFV600E-selective inhibitors on the expression of MHC molecules. We found that the treatment of A375 melanoma cells with vemurafenib enhances the induction of MHC Class I and Class II molecules by interferon γ and IFNα2b. Consistent with these findings, we observed that the forced overexpression of BRAFV600E has the opposite effect and can repress the baseline expression of MHC Class I molecules in A375 cells. Further studies utilizing eight other melanoma cell lines revealed that the vemurafenib-mediated enhancement of MHC induction by IFNγ only occurs in the context of homozygous, but not heterozygous, BRAFV600E mutation. These findings suggest that BRAFV600Eactivity directly influences the expression of MHC molecules and the response to Type I and Type II IFNs. Furthermore, our data suggest that the effect of vemurafenib on the expression of immune system-relevant genes may depend on the zygosity of the BRAFV600E mutation, which is not routinely assessed in melanoma patients.