Publications by authors named "Renata B Filler"

21 Publications

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Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

PLoS Biol 2021 03 17;19(3):e3001143. Epub 2021 Mar 17.

Department of Laboratory Medicine, Yale University, New Haven, Connecticut, United States of America.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.
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http://dx.doi.org/10.1371/journal.pbio.3001143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007021PMC
March 2021

Non-steroidal anti-inflammatory drugs dampen the cytokine and antibody response to SARS-CoV-2 infection.

J Virol 2021 Jan 13. Epub 2021 Jan 13.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). As PGs play diverse biological roles in homeostasis and inflammatory responses, inhibiting PG production with NSAIDs could affect COVID-19 pathogenesis in multiple ways, including: (1) altering susceptibility to infection by modifying expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2; (2) regulating replication of SARS-CoV-2 in host cells; and (3) modulating the immune response to SARS-CoV-2. Here, we investigate these potential roles. We demonstrate that SARS-CoV-2 infection upregulates COX-2 in diverse human cell culture and mouse systems. However, suppression of COX-2 by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on expression, viral entry, or viral replication. In contrast, in a mouse model of SARS-CoV-2 infection, NSAID treatment reduced production of pro-inflammatory cytokines and impaired the humoral immune response to SARS-CoV-2 as demonstrated by reduced neutralizing antibody titers. Our findings indicate that NSAID treatment may influence COVID-19 outcomes by dampening the inflammatory response and production of protective antibodies rather than modifying susceptibility to infection or viral replication.Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19). NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which are critical for the generation of prostaglandins - lipid molecules with diverse roles in homeostasis and inflammation. Inhibition of prostaglandin production by NSAIDs could therefore have multiple effects on COVID-19 pathogenesis. Here, we demonstrate that NSAID treatment reduced both the antibody and pro-inflammatory cytokine response to SARS-CoV-2 infection. The ability of NSAIDs to modulate the immune response to SARS-CoV-2 infection has important implications for COVID-19 pathogenesis in patients. Whether this occurs in humans and whether it is beneficial or detrimental to the host remains an important area of future investigation. This also raises the possibility that NSAIDs may alter the immune response to SARS-CoV-2 vaccination.
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http://dx.doi.org/10.1128/JVI.00014-21DOI Listing
January 2021

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

J Virol 2021 01 13;95(3). Epub 2021 Jan 13.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a conditional knockout ( ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV) In contrast, the nonpersistent MNoV strain CW3 (MNoV) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (), neutrophils (), and dendritic cells () did not affect MNoV viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV; that myelomonocytic cells are a major, but not exclusive, target cell of MNoV; and that STAT1 signaling restricts the cellular tropism of MNoV This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis. Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV infection. Mortality associated with the MNoV strain in mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism .
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http://dx.doi.org/10.1128/JVI.01652-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925115PMC
January 2021

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication.

bioRxiv 2020 Sep 25. Epub 2020 Sep 25.

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE , one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

Importance: Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NSAIDs function by inhibiting the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes are critical for the generation of prostaglandins, lipid molecules with diverse roles in maintaining homeostasis as well as regulating the inflammatory response. While COX-1/COX-2 signaling pathways have been shown to affect the replication of many viruses, their effect on SARS-CoV-2 infection remains unknown. We found that SARS-CoV-2 infection induced COX-2 expression in both human cell culture systems and mouse models. However, inhibition of COX-2 activity with NSAIDs did not affect SARS-CoV-2 entry or replication. Our findings suggest that COX-2 signaling may instead regulate the lung inflammation observed in COVID-19 patients, which is an important area for future studies.
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http://dx.doi.org/10.1101/2020.09.24.312769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523115PMC
September 2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

Single-cell longitudinal analysis of SARS-CoV-2 infection in human bronchial epithelial cells.

bioRxiv 2020 May 7. Epub 2020 May 7.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT, USA.

SARS-CoV-2, the causative agent of COVID-19, has resulted in more than 3,000,000 infections and 200,000 deaths. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as the major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon stimulated genes in both infected and bystander cells. Here, we have conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and provide a detailed characterization of genes, cell types, and cell state changes associated with the infection.
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http://dx.doi.org/10.1101/2020.05.06.081695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263511PMC
May 2020

CD300lf is the primary physiologic receptor of murine norovirus but not human norovirus.

PLoS Pathog 2020 04 6;16(4):e1008242. Epub 2020 Apr 6.

Departments of Laboratory Medicine and Immunobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV.
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http://dx.doi.org/10.1371/journal.ppat.1008242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162533PMC
April 2020

Langerhans Cells Facilitate UVB-Induced Epidermal Carcinogenesis.

J Invest Dermatol 2015 Nov 8;135(11):2824-2833. Epub 2015 Jun 8.

Department of Dermatology, Yale School of Medicine, New Haven, Connecticut, USA. Electronic address:

UVB light is considered the major environmental inducer of human keratinocyte (KC) DNA mutations, including within the tumor-suppressor gene p53, and chronic exposure is associated with cutaneous squamous cell carcinoma formation. Langerhans cells (LCs) comprise a dendritic network within the suprabasilar epidermis, yet the role of LCs in UVB-induced carcinogenesis is largely unknown. Herein we show that LC-intact epidermis develops UVB-induced tumors more readily than LC-deficient epidermis. Although levels of epidermal cyclopyrimidine dimers following acute UVB exposure are equivalent in the presence or absence of LCs, chronic UVB-induced p53 mutant clonal islands expand more readily in association with LCs, which remain largely intact and are preferentially found in proximity to the expanding mutant KC populations. The observed LC facilitation of mutant p53 clonal expansion is completely αβ and γδ T-cell independent and is associated with increased intraepidermal expression of IL-22 and the presence of group 3 innate lymphoid cells. These data demonstrate that LCs have a key role in UVB-induced cutaneous carcinogenesis and suggest that LCs locally stimulate KC proliferation and innate immune cells that provoke tumor outgrowth.
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http://dx.doi.org/10.1038/jid.2015.207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640962PMC
November 2015

Mechanisms of chemical cooperative carcinogenesis by epidermal Langerhans cells.

J Invest Dermatol 2015 May 18;135(5):1405-1414. Epub 2014 Sep 18.

Department of Dermatology, Yale School of Medicine, New Haven, Connecticut, USA. Electronic address:

Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential. The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal among which are polyaromatic hydrocarbons (PAHs) ubiquitous in the environment, tobacco smoke, and broiled meats. Langerhans cells (LCs) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). LCs rapidly internalize and accumulate DMBA as numerous membrane-independent cytoplasmic foci. Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility. LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1 independent. Thus, LCs make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens. Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation.
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http://dx.doi.org/10.1038/jid.2014.411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364923PMC
May 2015

IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis.

J Invest Dermatol 2014 Jul 31;134(7):1903-1911. Epub 2014 Jan 31.

Department of Dermatology, University of Maryland, Baltimore, Maryland, USA; Department of Microbiology/Immunology, University of Maryland, Baltimore, Maryland, USA. Electronic address:

The cytokine IL-9, derived primarily from T-helper 9 (Th9) lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also has a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch-test sites from non-atopic patients were assayed using quantitative PCR and immunohistochemistry. The cytokines IFN-γ, IL-4, IL-17A, IL-9, and PU.1, a Th9 associated transcription factor, were elevated when compared with paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+ CD3+ and PU.1+ CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. Peripheral blood mononuclear cells from nickel-allergic patients, but not nonallergic controls, show significant IL-9 production in response to nickel. Blocking studies with mAbs to HLA-DR (but not HLA-A, -B, -C) or chloroquine significantly reduced this nickel-specific IL-9 production. In addition, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9(-/-) mice shows enhanced Th1 lymphocyte immune responses, when compared with wild-type mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.
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http://dx.doi.org/10.1038/jid.2014.61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303591PMC
July 2014

Langerhans cells facilitate epithelial DNA damage and squamous cell carcinoma.

Science 2012 Jan;335(6064):104-8

Department of Dermatology, Yale University School of Medicine, New Haven, CT 06520, USA.

Polyaromatic hydrocarbons (PAHs) are prevalent, potent carcinogens, and 7,12-dimethylbenz[a]anthracene (DMBA) is a model PAH widely used to study tumorigenesis. Mice lacking Langerhans cells (LCs), a signatory epidermal dendritic cell (DC), are protected from cutaneous chemical carcinogenesis, independent of T cell immunity. Investigation of the underlying mechanism revealed that LC-deficient skin was relatively resistant to DMBA-induced DNA damage. LCs efficiently metabolized DMBA to DMBA-trans-3,4-diol, an intermediate proximal to oncogenic Hras mutation, and DMBA-treated LC-deficient skin contained significantly fewer Hras mutations. Moreover, DMBA-trans-3,4-diol application bypassed tumor resistance in LC-deficient mice. Additionally, the genotoxic impact of DMBA on human keratinocytes was significantly increased by prior incubation with human-derived LC. Thus, tissue-associated DC can enhance chemical carcinogenesis via PAH metabolism, highlighting the complex relation between immune cells and carcinogenesis.
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http://dx.doi.org/10.1126/science.1211600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753811PMC
January 2012

Characterization of the DNA copy-number genome in the blood of cutaneous T-cell lymphoma patients.

J Invest Dermatol 2012 Jan 1;132(1):188-97. Epub 2011 Sep 1.

Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous non-Hodgkin's lymphoma that may variably involve the skin, lymph nodes, and peripheral blood. Malignant burden ranges from cutaneous patches and plaques with little evidence of blood involvement to erythroderma often in association with frank leukemia, as in Sézary syndrome. Toward a better understanding of the pathogenesis of this CD4+ T-cell malignancy, we conducted a high-resolution genomic analysis combining DNA (23 samples) and mRNA (12 samples) data of peripheral blood isolates from CTCL patients across a spectrum of stages. Strikingly, even patients with limited involvement, e.g., normal CD4 counts, contained significant copy-number alterations. Defining genomic characteristics of CTCL blood involvement included gains on 8q and 17q, and deletions on 17p and chromosome 10. A consensus analysis of 108 leukemic CTCL samples demonstrated global similarities among patients with varied blood involvement, narrowing 38 of 62 loci. Toward an annotated framework for in vitro testing, we also characterized genomic alterations in five CTCL cell lines (HH, HUT78, PNO, SeAx, and Sez4), revealing intact core features of leukemic CTCL. Together, these studies produce the most comprehensive view of the leukemic CTCL genome to date, with implications for pathogenesis, molecular classification, and potential future therapeutic developments.
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http://dx.doi.org/10.1038/jid.2011.254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841973PMC
January 2012

Molecular analysis of tumor-promoting CD8+ T cells in two-stage cutaneous chemical carcinogenesis.

J Invest Dermatol 2010 Jun 19;130(6):1726-36. Epub 2009 Nov 19.

Department of Dermatology and Skin Diseases Research Center, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

T-pro are tumor-infiltrating TCRalphabeta(+)CD8(+) cells of reduced cytotoxic potential that promote experimental two-stage chemical cutaneous carcinogenesis. Toward understanding their mechanism of action, this study uses whole-genome expression analysis to compare T-pro with systemic CD8(+) T cells from multiple groups of tumor-bearing mice. T-pro show an overt T helper 17-like profile (high retinoic acid-related orphan receptor-(ROR)gammat, IL-17A, IL-17F; low T-bet and eomesodermin), regulatory potential (high FoxP3, IL-10, Tim-3), and transcripts encoding epithelial growth factors (amphiregulin, Gro-1, Gro-2). Tricolor flow cytometry subsequently confirmed the presence of TCRbeta(+) CD8(+) IL-17(+) T cells among tumor-infiltrating lymphocytes (TILs). Moreover, a time-course analysis of independent TIL isolates from papillomas versus carcinomas exposed a clear association of the "T-pro phenotype" with malignant progression. This molecular characterization of T-pro builds a foundation for elucidating the contributions of inflammation to cutaneous carcinogenesis, and may provide useful biomarkers for cancer immunotherapy in which the widely advocated use of tumor-specific CD8(+) cytolytic T cells should perhaps accommodate the cells' potential corruption toward the T-pro phenotype. The data are also likely germane to psoriasis, in which the epidermis may be infiltrated by CD8(+) IL-17-producing T cells.
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http://dx.doi.org/10.1038/jid.2009.362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920801PMC
June 2010

Acute upregulation of an NKG2D ligand promotes rapid reorganization of a local immune compartment with pleiotropic effects on carcinogenesis.

Nat Immunol 2008 Feb 6;9(2):146-54. Epub 2008 Jan 6.

Peter Gorer Department of Immunobiology, King's College London School of Medicine at Guy's Hospital, London SE1 9RT, UK.

The self-encoded ligands MICA (human) and Rae-1 (mouse) for the cytotoxic lymphocyte activating receptor NKG2D are highly expressed in carcinomas and inflammatory lesions and have been linked to immunosurveillance and graft rejection. However, whether NKG2D ligands have an intrinsic ability to acutely regulate tissue-associated immune compartments is not known. Here we show that epidermis-specific upregulation of Rae-1 induced rapid, coincident and reversible changes in the organization of tissue-resident V(gamma)5V(delta)1 TCRgammadelta+ intraepithelial T cells and Langerhans cells, swiftly followed by epithelial infiltration by unconventional alphabeta T cells. Whereas local V(gamma)5V(delta)1+ T cells limited carcinogenesis, Langerhans cells unexpectedly promoted it. These results provide unique insight into the early phases of tissue immunosurveillance and indicate that acute changes in NKG2D ligands may alone initiate a rapid, multifaceted immunosurveillance response in vivo.
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http://dx.doi.org/10.1038/ni1556DOI Listing
February 2008

Characterizing tumor-promoting T cells in chemically induced cutaneous carcinogenesis.

Proc Natl Acad Sci U S A 2007 Apr 5;104(16):6770-5. Epub 2007 Apr 5.

Department of Dermatology, Yale University School of Medicine, New Haven, CT 06520-8059, USA.

There is a longstanding but poorly understood epidemiologic link between inflammation and cancer. Consistent with this, we previously showed that alphabeta T cell deficiency can increase resistance to chemical carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene and promoted by phorbol 12-myristate 13-acetate. This provoked the hypothesis that alphabeta T cell deficiency removed T regulatory cells that limit the anti-tumor response or removed a specific tumor-promoting (T-pro) T cell population. Here we provide evidence for the latter, identifying a novel CD8(+) subset that is a candidate for T-pro cells. We demonstrate that CD8 cell-deficient mice show substantially less tumor incidence and progression to carcinoma, whereas susceptibility is restored by CD8(+) cell reconstitution. To characterize the putative T-pro cells, tumor-infiltrating lymphocytes were isolated from normal and CD4(-/-) mice, revealing an activated population of T cell receptor alphabeta(+)CD8(+)CD44(+)CD62L(-) cells expressing the inflammatory mediators IFNgamma, TNFalpha, and cyclooxygenase-2, but deficient in perforin, relative to recirculating cells of equivalent phenotype. This novel population of CD8(+) T cells has intriguing similarities with other lymphocytes that have been associated with tissue growth and invasiveness and has implications for inflammation-associated carcinogenesis, models of cancer immunosurveillance, and immunotherapeutic strategies.
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http://dx.doi.org/10.1073/pnas.0604982104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1871860PMC
April 2007

Cutaneous two-stage chemical carcinogenesis.

CSH Protoc 2007 Sep 1;2007:pdb.prot4837. Epub 2007 Sep 1.

Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA.

INTRODUCTIONThe induction of cutaneous carcinogenesis in mice by topical administration of chemicals enables the local, systemic, and environmental factors that influence tumor susceptibility, growth, and progression to be studied in the laboratory. Chemical carcinogenesis in mouse skin has been used for several decades, and continues to help in the identification of important molecular and immunological pathways involved in cutaneous malignancy. This protocol describes the two-stage application of chemicals to the skin for the initiation and promotion of cutaneous tumors. In the two-stage model of induction, a single application of the chemical initiator mutagen 7,12-dimethylbenz[a]anthracene (DMBA) is followed by repeated applications of a pro-inflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; otherwise known as phorbol 12-myristate 13-acetate [PMA]). Resulting tumors may develop in the form of benign papillomas that regress or progress to SCC, or SCC may arise without an apparent precursor lesion. Thus, two-stage chemical carcinogenesis provides an opportunity to monitor early and late events in cancer development and progression.
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http://dx.doi.org/10.1101/pdb.prot4837DOI Listing
September 2007

Environmentally responsive and reversible regulation of epidermal barrier function by gammadelta T cells.

J Invest Dermatol 2006 Apr;126(4):808-14

Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06520-8059, USA.

The intraepithelial lymphocyte (IEL) network possibly composes the largest T-cell compartment in the body, but it is poorly understood. IELs show limited T-cell receptor (TCR) diversity and have been proposed to respond to generic stress signals rather than pathogen-specific antigens. Consistent with this, skin-resident TCRgammadelta+ cells, known as dendritic epidermal T cells (DETC), downregulate cutaneous inflammation, promote wound healing, and protect against cutaneous neoplasia. These pleiotropic effects collectively suggest that DETC (and IEL more generally) may contribute to epithelial maintenance and barrier function. The present studies test this hypothesis. Using skin surface impedance analysis to measure hydration status and transepidermal water loss, we show that the epidermal barrier is defective in gammadelta T-cell deficient mice. However, this does not represent a constitutive role of gammadelta cells, but rather one that is dependent on environmental challenge, consistent with the primary role for lymphocytes being the response of the host to its environment. Likewise, the importance of the physiologic DETC-associated TCR is demonstrated by showing that Vgamma5+ fetal thymocytes reconstitute the barrier function defect in TCRdelta-/- mice, while Vgamma5-/- mice also show environmentally responsive defects in cutaneous physiology.
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http://dx.doi.org/10.1038/sj.jid.5700120DOI Listing
April 2006

The distinct contributions of murine T cell receptor (TCR)gammadelta+ and TCRalphabeta+ T cells to different stages of chemically induced skin cancer.

J Exp Med 2003 Sep;198(5):747-55

Department of Dermatology and the Yale Skin Diseases Research Core Center, Yale University, New Haven, CT 065420, USA.

Epithelial tissues in which carcinomas develop often contain systemically derived T cell receptor (TCR)alphabeta+ cells and resident intraepithelial lymphocytes that are commonly enriched in TCRgammadelta+ cells. Recent studies have demonstrated that gammadelta cells protect the host against chemically induced cutaneous malignancy, but the role of alphabeta T cells has been enigmatic, with both protective and tumor-enhancing contributions being reported in different systems. This study aims to clarify the contributions of each T cell type to the regulation of squamous cell carcinoma induced in FVB mice by a two-stage regimen of 7,12-dimethylbenz[a]anthracene initiation followed by repetitive application of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. This protocol permits one to monitor the induction of papillomas and the progression of those papillomas to carcinomas. The results show that whereas gammadelta cells are strongly protective, the nonredundant contributions of alphabeta T cells to the host's protection against papillomas are more modest. Furthermore, at both high and low doses of carcinogens, alphabeta T cells can contribute to rather than inhibit the progression of papillomas to carcinomas. As is likely to be the case in humans, this study also shows that the contribution of T cells to tumor immunosurveillance is regulated by modifier genes.
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http://dx.doi.org/10.1084/jem.20021282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194182PMC
September 2003

Anti-inflammatory effects in the skin of thymosin-beta4 splice-variants.

Immunology 2003 May;109(1):1-7

Department of Dermatology and the Yale Skin Diseases Research Core Center, Yale University, New Haven, Connecticut 06520, USA.

The intraepithelial lymphocyte (IEL) network of T-cell receptor gammadelta+ (Vgamma5+) dendritic epidermal T cells (DETC) in murine skin down-regulates cutaneous inflammation, although the mechanism is unknown. Thymosin-beta4 (Tbeta4), identified by serial analysis of gene expression as a predominant transcript in gut IEL, encodes both a ubiquitous actin-binding protein (UTbeta4) with demonstrated capacity to inhibit neutrophilic infiltration, and a splice-variant limited to lymphoid tissue (LTbeta4) with unknown bioactivity. Freshly isolated Vgamma5+ DETCs expressed both forms, while only LTbeta4 was preferentially up-regulated after cellular activation in vitro. To compare the anti-inflammatory properties of LTbeta4 and UTbeta4 in the skin in vivo, the biological activities of synthesized polypeptides were assessed using three different strategies: neutrophil infiltration by footpad lambda-carrageenan injection; irritant contact dermatitis to 12-O-tetradecanoylphorbol 13-acetate; and allergic contact dermatitis to 2,4-dinitrofluorobenzene. These studies clearly showed that the anti-inflammatory activities of LTbeta4 were broader and most often stronger than those of UTbeta4. Thus, the activation-responsive expression of the lymph-specific form of Tbeta4 may be one mechanism by which DETC, and possibly other IELs, down-regulate local inflammation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1782938PMC
http://dx.doi.org/10.1046/j.1365-2567.2003.01616.xDOI Listing
May 2003

Resident skin-specific gammadelta T cells provide local, nonredundant regulation of cutaneous inflammation.

J Exp Med 2002 Apr;195(7):855-67

Department of Dermatology and the Yale Skin Diseases Research Core Center, Yale University, New Haven, CT 06520, USA.

The function of the intraepithelial lymphocyte (IEL) network of T cell receptor (TCR) gammadelta(+) (Vgamma5(+)) dendritic epidermal T cells (DETC) was evaluated by examining several mouse strains genetically deficient in gammadelta T cells (delta(-/-) mice), and in delta(-/-) mice reconstituted with DETC or with different gammadelta cell subpopulations. NOD.delta(-/-) and FVB.delta(-/-) mice spontaneously developed localized, chronic dermatitis, whereas interestingly, the commonly used C57BL/6.delta(-/-) strain did not. Genetic analyses indicated a single autosomal recessive gene controlled the dermatitis susceptibility of NOD.delta(-/-) mice. Furthermore, allergic and irritant contact dermatitis reactions were exaggerated in FVB.delta(-/-), but not in C57BL/6.delta(-/-) mice. Neither spontaneous nor augmented irritant dermatitis was observed in FVB.beta(-/-) delta(-/-) mice lacking all T cells, indicating that alphabeta T cell-mediated inflammation is the target for gammadelta-mediated down-regulation. Reconstitution studies demonstrated that both spontaneous and augmented irritant dermatitis in FVB.delta(-/-) mice were down-regulated by Vgamma5(+) DETC, but not by epidermal T cells expressing other gammadelta TCRs. This study demonstrates that functional impairment at an epithelial interface can be specifically attributed to absence of the local TCR-gammadelta(+) IEL subset and suggests that systemic inflammatory reactions may more generally be subject to substantial regulation by local IELs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2193718PMC
http://dx.doi.org/10.1084/jem.20012000DOI Listing
April 2002