The Involvement of STAT/SOCS Signaling in miRNA-Induced T Cell Exhaustion in Cutaneous T-Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Zhen Han ◽  
Chingyu Su ◽  
Renee J. Estephan ◽  
Xiwei Wu ◽  
Sung Hee Kil ◽  
...  
Keyword(s):  
T Cells ◽  
Data Analysis ◽  
T Cell ◽  
Chronic Inflammation ◽  
Cell Lymphoma ◽  
Immune Checkpoints ◽  
T Cell Exhaustion ◽  
Cutaneous T Cell Lymphoma ◽  
Cell Exhaustion ◽  
Qrt Pcr

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS) arises from CD4+ T cells in a background of chronic inflammation. The chronic inflammation fosters the growth of CTCL cells and may facilitate T-cells exhaustion that is characterized by deprived effector function and sustained expression of inhibitory receptors. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. At present, the roles of miRNAs or signaling pathways involved in the expression of immune checkpoints in CTCL has yet to be elucidated. Therefore, this project is aimed to elucidate how immune checkpoints are regulated by miRNAs, and how this regulation contributes to T-cell exhaustion and the development of CTCL. Methods: We first conducted miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. The sequencing data analysis was performed at the City of Hope Integrative Genomics Core. Next, we verified the expression of 3 highly upregulated miRNAs (miR-155, -21 and -130) from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Myla 2059 and HuT 78 with anti-miR-155, -21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against SOCS, PTEN, pSTAT3 and GAPDH as a loading control. Results: The data analysis revealed that miR-155 had the highest correlation with CTLA-4 (r = 0.59, P < 0.0001), PD1 (r = 0.42, P = 0.0021), PD-L1 (r = 0.42, P = 0.0025), TIM3 (r = 0.63, P < 0.0001), LAG3 (r = 0.57, P < 0.0001), and ICOS (r = 0.74, P < 0.0001) mRNA; -21 had the highest correlation with PD-L1 (r = 0.44, P = 0.0012), TIM3 (r = 0.52, P < 0.0001), and ICOS (r = 0.37, P < 0.0073) mRNA; and -130 had the highest correlation with CTLA-4 (r = 0.5, P = 0.0002), PD-L1 (r = 0.54, P < 0.0001), TIM3 (r = 0.69, P < 0.0001), LAG3 (r = 0.49, P = 0.0003), and ICOS (r = 0.68, P < 0.0001) mRNA. qRT-PCR and ISH revealed that miRs-155, -21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a dramatic increase in SOCS proteins and significant decrease in pSTAT3 expression in Myla 2059 and HuT 78 cells transfected with anti-miRs-155, -21 or -130, compared to cells transfected with Scr. Conclusions: Immune checkpoints and ligands like PD-L1 expression in CTCL are regulated by miRs-155, -21 and -130. The SOCS family of proteins, negative regulators of STAT signaling, are involved in miRs-155, -21 and -130-induced immune checkpoints expression in CTCL. Taken together, these results demonstrate the mechanisms of miRNA-induced T cell exhaustion and pave the way for the development of miRNA therapeutics in CTCL. Disclosures Rosen: Seattle Genetics: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy. Querfeld:Celgene: Research Funding; Stemline: Consultancy; Trillium: Consultancy; MiRagen: Consultancy; Kyowa Kirin: Consultancy; Bioniz: Consultancy; Helsinn: Consultancy.

scholarly journals PD-L1 Expression Is Regulated By microRNAs-21 and -130 in Cutaneous T-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4108-4108
Author(s):  
Renee Estephan ◽  
Sung Hee Kil ◽  
Steven T Rosen ◽  
Christiane Querfeld
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lymphoma ◽  
Immune Checkpoints ◽  
T Cell Exhaustion ◽  
Cutaneous T Cell Lymphoma ◽  
Advisory Committees ◽  
Cell Exhaustion ◽  
Qrt Pcr

Abstract Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS), is characterized by the accumulation of neoplastic memory T- cells in the skin in a background of chronic inflammation. This observation suggests that chronic inflammation fosters the growth of CTCL cells, and may impair successful anti-tumor responses mediated by the CD8+ tumor-infiltrating T-cells. T-cell exhaustion plays an important role in the pathogenesis of CTCL. PD-1 and other inhibitory immune checkpoints are overexpressed in exhausted T-cells (Cancer Immunol Res 6; 2018). Their binding to corresponding ligands like PD-L1 on dendritic cells (DCs), macrophages and tumor cells in the microenvironment directly reduces the functional and proliferative capabilities of T-cells by repressing T-cell receptor signaling and inducing genes that impair T-cell function. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. The miRNA profile of CTCL correlates with a dysfunctional/exhausted immunophenotype, suggesting that epigenetic regulation is involved in the regulation of immune checkpoints. Signaling pathways such as JAK-STAT are constitutively active in CTCL and may also be involved in immune checkpoint regulation. At present, the exact role of miRNAs in regulating the expression of PD-L1 in CTCL has yet to be elucidated. Therefore, the goal of this project is to understand how PD-L1 is regulated, how this regulation contributes to T-cell exhaustion, and how this regulation is altered to contribute to the development of CTCL. To this end, we set out to test the hypothesis that PD-L1 is regulated by oncogenic miRNAs in CTCL. Methods: We first conducted a high-throughput miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. Library preparation, miRNA sequencing, and correlation calculation of miRNAs with PD-L1 mRNA expression was performed by the City of Hope Integrative Genomics Core. Next, we verified the expression of 2 highly upregulated miRs-miRs-21and -130- from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Hut78 with anti-miR-21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against PD-L1, pSTAT3 and GAPDH as a loading control. Results: The miRNAseq data revealed that miRs-21 and -130 had the highest correlation with PD-L1 mRNA. qRT-PCR and ISH revealed that miRs-21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a drastic decrease in PD-L1 and pSTAT3 expression in Hut78 cells transfected with anti-miRs-21 or -130, compared to cells transfected with Scr. Conclusions: PD-L1 expression in CTCL is regulated by miRs-21 and -130. miRs-21 and -130 may be regulating PD-L1 indirectly by silencing negative regulators of STAT signaling such as the SOCS family of proteins. This would explain the decrease in pSTAT3 levels in Hut78 cells transfected with anti-miRs. Taken together, these results demonstrate the ability of miRNAs to regulate immune checkpoints and ligands like PD-L1, and pave the way for the creation of miRNA therapeutics to reverse T-cell exhaustion and slow CTCL disease progression. Disclosures Querfeld: Acelion: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The miRNA Profile of Cutaneous T Cell Lymphoma Correlates with the Dysfunctional Immunophenotype of the Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4132-4132 ◽  
Author(s):  
Christiane Querfeld ◽  
Xiwei Wu ◽  
James F Sanchez ◽  
Joycelynne M. Palmer ◽  
Azadeh Motevalli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Immune Checkpoint ◽  
Cell Lymphoma ◽  
Immune Checkpoints ◽  
T Cell Exhaustion ◽  
Cutaneous T Cell Lymphoma ◽  
Comparison Analysis ◽  
Cell Exhaustion ◽  
Insight Into

Abstract Cutaneous T cell lymphoma (CTCL) is thought to originate from clonally expanded, effector/central memory CD4+ T-cells in a background of chronic inflammation. Our recent investigations have provided evidence for T-cell exhaustion in CTCL with high levels of PD1 and other immune checkpoints (1. Gonzalez BR, Zain J, Rosen ST, Querfeld C. Current Opinion in Oncology. 2016; 28(1): 88-96. 2. Querfeld C, Curran SA, Leung S, Myskowski PL, Horwitz SM, Halpern AC, Young JW. Blood. 2014;124[21, abstract #1695). One potential mechanism by which immune checkpoint protein synthesis can be modulated is by the induction of miRNAs. A subset of miRNAs contributes to the pathogenesis and progression of mycosis fungoides/Sézary syndrome (MF/SS)), but no experimental research has evaluated the effects of miRNAs on immune checkpoints and T-cell exhaustion in CTCL. To address whether miRNA expression correlates with disease and is involved in regulating key immune checkpoint molecules linked to T cell exhaustion, we performed miRNA and gene expression profiling on 50 MF/SS patient samples using high-throughput sequencing, to achieve further insight into the molecular pathogenesis of CTCL disease. Total RNA, which includes miRNA, was extracted from CTCL (MF and SS) samples. Library preparation and high-throughput next generation small RNA sequencing were performed by the City of Hope Integrative Genomics Core. Reads were aligned to human genome assembly hg19, and miRNA expression levels were counted based on miRBase mature miRNA sequences. Differences in miRNA and gene expression between MF and SS patients were measured. Aberrantly expressed miRNAs specific for infiltrated plaques and tumor stage MF were compared. Our preliminary results have identified molecular signatures in CTCL that are linked to disease progression and key immunomodulatory molecules. Unsupervised cluster analysis of skin samples from patients using a heat map was performed (Fig 1). Patients were subdivided into 2 distinct clusters. The top cluster contains all tumor samples with few plaques, while the bottom cluster contains all patch/plaque samples. The clusters also correlated with clinical stage (top cluster = advanced stage ≥IIB; bottom cluster = early stage IA-IIA). Comparison analysis between the top and bottom cluster identified the 20 most significant up-regulated miRNAs that correlate with immune checkpoint (PD1, PD-L1, CTLA4, LAG3, TIM3, and ICOS) expression. For one, miR-146 showed the highest correlation (Table 1). The 20 most significant down-regulated miRNAs were also identified by class comparison analysis performed between top and bottom clusters and correlated with immune checkpoint expression. In conclusion, CTCL develops in a complex microenvironment. The expression of both miRNA and immune checkpoint proteins suggest patterns that provide insight into pathogenesis and potential therapeutic options. Disclosures Querfeld: Celgene: Consultancy, Research Funding; Actelion: Consultancy; Miragen: Consultancy. Zain:Seattle Genetics: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

scholarly journals BSCI-03. T CELL EXHAUSTION SIGNATURES VARY BY TUMOR TYPE AND ARE INDEPENDENT OF INTRACRANIAL LOCATION

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i1-i1
Author(s):  
Karolina Woroniecka ◽  
Pakawat Chongsathidkiet ◽  
Xiuyu Cui ◽  
Cosette Dechant ◽  
Daniel Wilkinson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Peripheral Blood Lymphocytes ◽  
Immune Checkpoint Blockade ◽  
Immune Checkpoints ◽  
Tumor Type ◽  
T Cell Exhaustion ◽  
Murine Models ◽  
Cell Exhaustion

Abstract T-cell exhaustion is a hindrance to the efficacy of immune checkpoint blockade. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating murine models of brain metastasis, including breast, lung, and melanoma cancers. Furthermore, this study demonstrates the utility of a 4-1BB agonist antibody in certain tumors resistant to PD-1 blockade alone. METHODS: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Levels of exhaustion-associated inhibitory receptors and post-stimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. Anti-PD-1 and anti-4-1BB monoclonal antibodies were utilized as a therapeutic exhaustion-countering strategy and median survival was assessed. RESULTS: Our data reveal that tumors, regardless of their intracranial or subcutaneous location, elicit unique T-cell exhaustion signatures among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Anti-PD-1 monoclonal antibody alone did not improve median survival in any tumor type tested. In tumors with high levels of 4-1BB expression, anti-4-1BB and anti-PD-1 therapy resulted in improvement in median survival. CONCLUSIONS: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. Each tumor type demonstrated a unique T cell exhaustion signature regardless of location. 4-1BB may serve as a therapeutic adjunct to anti-PD-1 monoclonal therapy in tumors which may be resistant to PD-1 blockade alone.

MiRNA regulation of T cell exhaustion in cutaneous T cell lymphoma

2021 ◽  
Author(s):  
Zhen Han ◽  
Renee J. Estephan ◽  
Xiwei Wu ◽  
Chingyu Su ◽  
Yate-Ching Yuan ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
T Cell Exhaustion ◽  
Cutaneous T Cell Lymphoma ◽  
Mirna Regulation ◽  
Cell Exhaustion

scholarly journals Immunogenomic, single-cell and spatial dissection of CD8+T cell exhaustion reveals critical determinants of cancer immunotherapy

2021 ◽  
Author(s):  
Stefan Naulaerts ◽  
Daniel M Borras ◽  
Asier Antoranz Martinez ◽  
Julie Messiaen ◽  
Yannick Van Herck ◽  
...  
Keyword(s):  
Wound Healing ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Immune Checkpoints ◽  
Effector Memory ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

Tumoural-CD8+T cells exhibit exhausted or dysfunctional states. Contrary to immunotherapy-responsive exhausted-CD8+T cells, the clinical features of dysfunctional-CD8+T cells are disputed. Hence, we conducted large-scale multi-omics and multi-dimensional mapping of CD8+T cell-states across multiple cancer patient-cohorts. This identified tumour-specific continuum of CD8+T cell-states across 6 human cancers, partly imprinted by organ-specific immuno-modulatory niches. Herein, melanoma and glioblastoma enriched prototypical exhausted (CD8+TEXT) and severely-dysfunctional (CD8+TSDF) states, respectively. Contrary to CD8+TEXT, CD8+TSDF displayed transcriptomic and epigenetic effector/cytolytic dysfunctions, and dysregulated effector/memory single-cell trajectories, culminating into maladaptive pro-death stress and cell-cycle defects. Suboptimal antigen-priming underscored CD8+TSDF, which was distinct from immune-checkpoints 'rich' CD8+TEXT, reflecting chronic antigen-stimulation. Continuum variation also existed on tumour spatial-level, with convergent (CD8+TEXT-supportive vascular regions) and divergent features (dysfunctional CD4+T::CD8+TSDF cell-to-cell interactions) between melanoma and glioblastoma. Globally, IFNG-IL2 disparities, paucity of intra-tumoural CD4+/CD8+T cells, and myeloid TGFB/wound healing responses, distinguished CD8+TSDF-landscape. Within immuno-oncology clinical-trials, anti-PD1 immunotherapy failed to 'reinvigorate' CD8+TSDF-landscape, and instead facilitated effector-dysfunction and TGFB/wound healing. However, cellular immunotherapies (dendritic cell-vaccines, adoptive T-cell therapy) ameliorated assorted CD8+TSDF-landscape disparities, highlighting a roadmap for anti-glioblastoma multimodal-immunotherapy. Collectively, our study comprehensively expands clinical-knowledge on CD8+T cell-exhaustion and suggests that tumour-specific, pre-existing CD8+TEXT/TSDF-states, determine immunotherapy-responses.

scholarly journals Diffuse Large B-Cell Lymphoma Is Infiltrated with Functional CD8+ T-Cells Lacking the Hallmarks of Exhaustion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1518-1518
Author(s):  
Adam Greenbaum ◽  
Ajay K. Gopal ◽  
Jonathan R. Fromm ◽  
A McGarry Houghton
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
Checkpoint Inhibitors ◽  
B Cell Lymphoma ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Large B Cell Lymphoma

Background. B-cell non-Hodgkin lymphomas (NHL) are hematologic malignancies that arise in the lymph node but are not cleared by the immune cells present. The failure of anti-tumor immunity may be due to immune checkpoints such as the PD-1/PD-L1 axis, which can cause T-cell exhaustion. In contrast to Hodgkin lymphoma, checkpoint blockade in NHL has showed limited efficacy. Here we demonstrate that T-cells in DLBCL do not exhibit an exhausted phenotype which may explain the poor response to immune checkpoint inhibitors. Results. In order to better understand how the tumor microenvironment may impact NHL, we performed an extensive characterization of malignant and non-malignant human lymph nodes using high dimensional flow cytometry. We compared follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and non-malignant reactive hyperplasia (RH). Using the unsupervised clustering algorithm Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP), we identified several T-cell populations that were altered in NHL compared to RH. These included follicular helper T-cells, regulatory T-cells, CD4+ PD-1+ T-cells, and CD8+ PD-1+ T-cells. Notably, DLBCL was highly enriched with CD8+ PD-1+ T-cells. Given the important role of PD-1 in regulating T-cell exhaustion, we thus hypothesized that DLBCL is infiltrated with exhausted T-cells. Consistent with this, we demonstrated that the CD8+ T-cells also expressed other exhaustion markers and could be divided into single positive for PD-1; double positive for PD-1 and TIM-3; and triple positive for PD-1, TIM-3, and CTLA-4. Given the large expansion of CD8+ PD-1+ T-cells in DLBCL, we restricted further analysis to this histology. Since CD8+ T-cells are an important part of anti-tumor immunity, we further analyzed these cells to determine if PD-1 could serve as a potential therapeutic target. We first performed in vitro stimulation with PMA/ionomycin to determine the cytokine production capacity of CD8+ cells. Compared to PD-1- cells, CD8+ PD-1+ cells retain their production of IFNg but have decreased capacity to produce IL-2. They also express lower levels of IL-7R (CD127) and lack CD45RA consistent with an effector phenotype. Additionally, as they acquire TIM-3 and CTLA-4, they make increasing amounts of granzyme B and perforin and exhibit greater degranulation capacity. Compared to PD-1- cells, PD-1+ cells also have no baseline defects in apoptosis or proliferation. Since the cohort appeared to be infiltrated with highly activated CD8+ T-cells, we next examined whether this could be suppressed by PD-1 signaling. We identified a single lymphoma in our cohort that highly expressed PD-L1. Despite this, the CD8+ T-cells retained their ability to produce cytokines. Together, these data suggesting that CD8+ T-cells in DLBCL lack many hallmarks of exhaustion. Additionally, it suggests that PD-L1 expression by a lymphoma is insufficient by itself to cause T-cell exhaustion. Conclusion. Our work may explain the failure of single-agent immune checkpoint inhibitors in the treatment of DLBCL. Accordingly, functional differences of CD8+ T-cells in DLBCL may inform different therapeutic targeting strategies. Disclosures. A.K.G. reports grants and nonfinancial support from Teva, Bristol-Myers Squibb, Merck, Takeda, TG Therapeutics, and Effector; grants, personal fees, and nonfinancial support from Seattle Genetics, Pfizer, Janssen, Gilead, Spectrum, Amgen and Incyte; personal fees from Aptevo, BRIM Bio, Seattle Genetics, and Sanofi. Disclosures Gopal: Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte.: Consultancy; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte: Honoraria; Teva, Bristol-Myers Squibb, Merck, Takeda, Seattle Genetics, Pfizer, Janssen, Takeda, and Effector: Research Funding. Fromm:Merck, Inc.: Research Funding.

scholarly journals Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion

2021 ◽  
Vol 7 (18) ◽  
pp. eabd2710
Author(s):  
Chen Zhu ◽  
Karen O. Dixon ◽  
Kathleen Newcomer ◽  
Guangxiang Gu ◽  
Sheng Xiao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adaptor Protein ◽  
Transcriptional Analysis ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Cell Dysfunction ◽  
Autoreactive T Cell ◽  
Autoimmune Conditions ◽  
T Cell Dysfunction

T cell exhaustion has been associated with poor prognosis in persistent viral infection and cancer. Conversely, in the context of autoimmunity, T cell exhaustion has been favorably correlated with long-term clinical outcome. Understanding the development of exhaustion in autoimmune settings may provide underlying principles that can be exploited to quell autoreactive T cells. Here, we demonstrate that the adaptor molecule Bat3 acts as a molecular checkpoint of T cell exhaustion, with deficiency of Bat3 promoting a profound exhaustion phenotype, suppressing autoreactive T cell–mediated neuroinflammation. Mechanistically, Bat3 acts as a critical mTORC2 inhibitor to suppress Akt function. As a result, Bat3 deficiency leads to increased Akt activity and FoxO1 phosphorylation, indirectly promoting Prdm1 expression. Transcriptional analysis of Bat3−/− T cells revealed up-regulation of dysfunction-associated genes, concomitant with down-regulation of genes associated with T cell effector function, suggesting that absence of Bat3 can trigger T cell dysfunction even under highly proinflammatory autoimmune conditions.

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