Oral Talk #70
Role of KSHV in T cell Infiltration in Kaposi's Sarcoma
Microenvironment
Canrong Zhong1, Nannan Zhu1, Yuqing Li1, Yan Yuan1,2
1Sun Yat-sen University, 2University of Pennsylvania
Kaposi’s sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8 (HHV8), is an etiologic agent of Kaposi's Sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). KS is a multifocal and oligoclonal malignancy. Tumors are comprised of proliferating spindle-shaped KS cells with abnormal neoangiogenesis and abundant inflammatory infiltrate. In many early KS lesions, inflammatory cells outnumber spindle cells. Various inflammatory cytokines promote KS cell proliferation and induce spindle cell morphology. Therefore, the immune cell infiltrate and cytokine-rich microenvironment apparently plays a significant role in the progression of KS. The goal of our study is to understand the role of KSHV in establishing the unique KS microenvironment, as well as the contribution of the microenvironment to KS pathogenesis. Recently we reported that KS might originate from KSHV-infected oral mesenchymal stem cells (Li et al., 2018 Cancer Res. 78: 230-245). KSHV infection of oral MSCs resulted in expression of a large spectrum of cytokines and chemokines, including IL-6, VEGF, CCL5, CCL8, and CXCL10, reminiscent of KS lesion. Using a Transwell assay with PBMC, we found that KSHV infection of oral MSCs dramatically enhances T cells chemotaxis, which can be blocked by anti-CCL5 or anti-CCL8 antibodies. Co-cultivation of KSHV-infected MSCs with naïve T cells promoted T cell differentiation to Treg and Th17. Furthermore, we compared KSHV-infected MSCs co-cultivated with and without T cells for tumor phenotypes in mice. Results show that after being exposed to T cells, KSHV-infected MSCs exhibit increased blood vessel formation on the Matrigel plug and invasion ability. Taken together, we established an MSC model to study KS microenvironment which demonstrates how KS precursor cells recruit T cells to inflamed sites and how T cells promote the progression of KS, thus shedding light on the formation of KS microenvironment and its role in KS progression.
Microenvironment
Canrong Zhong1, Nannan Zhu1, Yuqing Li1, Yan Yuan1,2
1Sun Yat-sen University, 2University of Pennsylvania
Kaposi’s sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8 (HHV8), is an etiologic agent of Kaposi's Sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). KS is a multifocal and oligoclonal malignancy. Tumors are comprised of proliferating spindle-shaped KS cells with abnormal neoangiogenesis and abundant inflammatory infiltrate. In many early KS lesions, inflammatory cells outnumber spindle cells. Various inflammatory cytokines promote KS cell proliferation and induce spindle cell morphology. Therefore, the immune cell infiltrate and cytokine-rich microenvironment apparently plays a significant role in the progression of KS. The goal of our study is to understand the role of KSHV in establishing the unique KS microenvironment, as well as the contribution of the microenvironment to KS pathogenesis. Recently we reported that KS might originate from KSHV-infected oral mesenchymal stem cells (Li et al., 2018 Cancer Res. 78: 230-245). KSHV infection of oral MSCs resulted in expression of a large spectrum of cytokines and chemokines, including IL-6, VEGF, CCL5, CCL8, and CXCL10, reminiscent of KS lesion. Using a Transwell assay with PBMC, we found that KSHV infection of oral MSCs dramatically enhances T cells chemotaxis, which can be blocked by anti-CCL5 or anti-CCL8 antibodies. Co-cultivation of KSHV-infected MSCs with naïve T cells promoted T cell differentiation to Treg and Th17. Furthermore, we compared KSHV-infected MSCs co-cultivated with and without T cells for tumor phenotypes in mice. Results show that after being exposed to T cells, KSHV-infected MSCs exhibit increased blood vessel formation on the Matrigel plug and invasion ability. Taken together, we established an MSC model to study KS microenvironment which demonstrates how KS precursor cells recruit T cells to inflamed sites and how T cells promote the progression of KS, thus shedding light on the formation of KS microenvironment and its role in KS progression.
MSCs: Nestin+, CD31-, CD29+, CD34+, CD133+
- neural crest cell contributions to the cranial facial development, neural crest cells migrate to form the craniofacial mesenchyme that differentiates into various cranial ganglia and craniofacial cartilages and bones
- SJ Gao (kidney capsule implantation of KSHV-infected MSCs, PLLSC)
Evidence 2: KSHV infection leads to MEndT
Evidence 3: gene expression profiling reveals linagedistanc elf MSC to KS
Evidence 4: cytokines network in KSHV-MSCs: CCL8, bFGF, VEGF-A, VEGF-D, IL6, IGF1, TGFb, ANGPT2 (both by cytokines array in IHC)
Chemokine provided by KSHV-infected MSCs induces T cell chemotaxis (this is also true in KS lesion -> no B cell infiltration)
KSHV-PDLSCs promoter T cell differentiation to Th17 (and down-regulates Treg a little bit)
Activated T cell conditioned media (TCM) promotes MEndT of KSHV-infected PDLSCs Matrigel plug assay
KSHV-PDLSCs become more aggressive after co-cultivating with
Oral Talk #71
Epstein-Barr Virus LMP1 Hijacks the Host ESCRT Machinery
for Exosomal Secretion
Dingani Nkosi1, Mujeeb Cheerathodi1, Mark A Rider1, David G Meckes1
1Florida State University College of Medicine, Department of Biomedical Sciences
Exosomes are endosomal-derived extracellular vesicles (EVs) that are important mediators of cell to cell communications in both normal and pathological conditions. Latent membrane protein 1 (LMP1), one of the major viral oncogenes expressed in most Epstein-Barr virus (EBV)-associated cancers is secreted from infected cells in EVs. LMP1-modified EVs have the ability to influence the tumor microenvironment. Despite the significance of LMP1- modified EVs in EBV malignancies, very little is known about how this viral protein hijacks the host EV pathways for secretion. ESCRT-dependent and -independent cellular machineries have shown to play a major role in biogenesis of EVs and cargo sorting. Using the BioID method, we identified LMP1 proximal interacting proteins that are known to play roles in exosome formation and protein trafficking including CD63, syntenin-1, Alix, Tsg101, Hrs, charged multivesicluar body proteins (CHMPs), and sorting nexins (Rider and Cheerathodi et al., Virology, 2018). To test the contributions of the identified ESCRT genes in LMP1 exosomal trafficking, cell lines expressing inducible short hairpin RNAs (shRNAs) were generated. Cell-derived EVs were collected from LMP1 expressing cells and levels of specific cargo were evaluated. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following knock-down of syntenin-1, Alix, Hrs and Tsg101. Furthermore, the levels of intracellular and exosomal CD63, a tetraspanin protein important for LMP1 EV incorporation and regulation of LMP1- mediated signaling, was reduced following knock-down of the individual genes. Importantly, the trafficking of LMP1 to lipid rafts, plasma membrane microdomains thought to be involved in signal transduction, EV biogenesis and cargo sorting remained intact despite syntenin-1 and Alix knock-down. These results suggest that the cellular ESCRT controls the incorporation of LMP1 into the host EV pathway for secretion and may also influence LMP1-mediated signaling due to alterations in the levels of intra- and extra-cellular CD63.
for Exosomal Secretion
Dingani Nkosi1, Mujeeb Cheerathodi1, Mark A Rider1, David G Meckes1
1Florida State University College of Medicine, Department of Biomedical Sciences
Exosomes are endosomal-derived extracellular vesicles (EVs) that are important mediators of cell to cell communications in both normal and pathological conditions. Latent membrane protein 1 (LMP1), one of the major viral oncogenes expressed in most Epstein-Barr virus (EBV)-associated cancers is secreted from infected cells in EVs. LMP1-modified EVs have the ability to influence the tumor microenvironment. Despite the significance of LMP1- modified EVs in EBV malignancies, very little is known about how this viral protein hijacks the host EV pathways for secretion. ESCRT-dependent and -independent cellular machineries have shown to play a major role in biogenesis of EVs and cargo sorting. Using the BioID method, we identified LMP1 proximal interacting proteins that are known to play roles in exosome formation and protein trafficking including CD63, syntenin-1, Alix, Tsg101, Hrs, charged multivesicluar body proteins (CHMPs), and sorting nexins (Rider and Cheerathodi et al., Virology, 2018). To test the contributions of the identified ESCRT genes in LMP1 exosomal trafficking, cell lines expressing inducible short hairpin RNAs (shRNAs) were generated. Cell-derived EVs were collected from LMP1 expressing cells and levels of specific cargo were evaluated. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following knock-down of syntenin-1, Alix, Hrs and Tsg101. Furthermore, the levels of intracellular and exosomal CD63, a tetraspanin protein important for LMP1 EV incorporation and regulation of LMP1- mediated signaling, was reduced following knock-down of the individual genes. Importantly, the trafficking of LMP1 to lipid rafts, plasma membrane microdomains thought to be involved in signal transduction, EV biogenesis and cargo sorting remained intact despite syntenin-1 and Alix knock-down. These results suggest that the cellular ESCRT controls the incorporation of LMP1 into the host EV pathway for secretion and may also influence LMP1-mediated signaling due to alterations in the levels of intra- and extra-cellular CD63.
Extracellular vesicles (EVs)
- 40-120nm
- Diverted membrane enclosed particles
- Classified according to sub-cellular origin and size
- Fold I’m almost all nobody fluids
- Transfer proteins, mRNAs, miRNAs, and lipid
- Virus hijacks ENV host pathway
LMP1
- Main EBV oncogene
- Mimics CD40
EBV LMP1-modified EVs
- EBV-infected cancer cells secrete EVs containing LMP1
- Expression of LMP1 in cell causes enhancement IOC vehicle production
- LMP1 expression increases the levels of EGFR and PI3K in EVs
The LMP1 interactive com trains proteins important for exosome trafficking and biogenesis
- Cellular compartment 73% of origins identified were in vesiciepked
- TRAF2, STAT3, TDSG101, Syndin?, ITGB1, and HSC70
Hypothesis: LMP1 utilizes the ESCRT pathway to facilitate EV production
LMP1 expression increase mRNA and protein levels of EV biogenesis components
- Alix, HRD, HSC70, TSG101, Syntenin-1, CD9, CD81
- Knockdown of ESRT components reduces LMP1-mediated enhancement of vesicles
- ESRT II complex is dispensable in LMP1 exosome sorting
- SNAP not required
- VPS4A not required
- LMP1 EV secretion requires upstream, but not downstream ESCRT components
- The N-terminus and TM1 are sufficient to guide LMP1 EV sorting
Oral Talk #72
vIRF-1 Regulatesm Mitochondrial Content by Promoting
Mitophagy During HHV-8 Replication
Mai Tram Vo1, Young Bong Choi1
1Johns Hopkins School of Medicine
Human herpesvirus 8 (HHV-8) is known as an infectious agent that is causally related with AIDS-associated human malignancies including Kaposi sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) contributes to HHV-8 pathogenesis potentially by inhibiting antiviral responses such as innate immune signaling and apoptosis. We demonstrated previously that vIRF-1 localizes in part to mitochondria and inhibits associated antiviral responses, thereby promoting virus replication. However, the exact function of mitochondria-localized vIRF-1 is not fully understood. Here, we provide evidence of involvement of vIRF-1 in mitochondria quality control via mitophagy during virus replication. Firstly, we observed that cellular mitochondrial content, assessed by Cellometer image- based cytometry of mitochondria-encoded protein MTCO2, was reduced in lytic HHV-8- positive PEL cells, but this effect was reversed by vIRF-1 depletion and autophagy inhibitors, suggesting that vIRF-1 is likely to be involved in HHV-8 replication-induced mitophagy. Secondly, we found that mitophagy-related proteins including LC3-II, a lipidated form of LC3, and a mitophagy receptor, NIX, translocated to mitochondria upon virus replication. Importantly, their localization to mitochondria was significantly impaired in vIRF- 1-depleted PEL cells undergoing lytic replication, suggesting that vIRF-1 plays an essential role recruitment of the mitophagy machinery to mitochondria. Furthermore, we found that vIRF-1 could bind directly to NIX via a short linear motif in vIRF-1 containing the Asn8 and Phe10 residues. Co-transfection of vIRF-1 and NIX, but neither NIX nor vIRF-1 alone, resulted in a decrease in mitochondrial content of transfected HeLa cells, indicative of functional interaction between NIX and vIRF-1 to promote mitophagy. We also found that vIRF-1 interacts with the members of autophagy related protein 8 (ATG8) family upon mitochondria damage. Taken together, our results indicate that vIRF-1 is involved directly in mitochondrial quality control via interactions with key mitophagy proteins during HHV8 replication.
Mitophagy During HHV-8 Replication
Mai Tram Vo1, Young Bong Choi1
1Johns Hopkins School of Medicine
Human herpesvirus 8 (HHV-8) is known as an infectious agent that is causally related with AIDS-associated human malignancies including Kaposi sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) contributes to HHV-8 pathogenesis potentially by inhibiting antiviral responses such as innate immune signaling and apoptosis. We demonstrated previously that vIRF-1 localizes in part to mitochondria and inhibits associated antiviral responses, thereby promoting virus replication. However, the exact function of mitochondria-localized vIRF-1 is not fully understood. Here, we provide evidence of involvement of vIRF-1 in mitochondria quality control via mitophagy during virus replication. Firstly, we observed that cellular mitochondrial content, assessed by Cellometer image- based cytometry of mitochondria-encoded protein MTCO2, was reduced in lytic HHV-8- positive PEL cells, but this effect was reversed by vIRF-1 depletion and autophagy inhibitors, suggesting that vIRF-1 is likely to be involved in HHV-8 replication-induced mitophagy. Secondly, we found that mitophagy-related proteins including LC3-II, a lipidated form of LC3, and a mitophagy receptor, NIX, translocated to mitochondria upon virus replication. Importantly, their localization to mitochondria was significantly impaired in vIRF- 1-depleted PEL cells undergoing lytic replication, suggesting that vIRF-1 plays an essential role recruitment of the mitophagy machinery to mitochondria. Furthermore, we found that vIRF-1 could bind directly to NIX via a short linear motif in vIRF-1 containing the Asn8 and Phe10 residues. Co-transfection of vIRF-1 and NIX, but neither NIX nor vIRF-1 alone, resulted in a decrease in mitochondrial content of transfected HeLa cells, indicative of functional interaction between NIX and vIRF-1 to promote mitophagy. We also found that vIRF-1 interacts with the members of autophagy related protein 8 (ATG8) family upon mitochondria damage. Taken together, our results indicate that vIRF-1 is involved directly in mitochondrial quality control via interactions with key mitophagy proteins during HHV8 replication.
Inhibitory interaction of vIRF1 with cellular proteins
- IFN responses
- TGFb?
VIRF1 localize on mitochondria (2 previous publications)
Mitochondrial content decreased during HHV8 lytic replication
iBCBL1 (TRex BCBL1)
- MITCO2, MAVs, vIRF1, DAPY.
- VIRF1/mtDNA/DAPI
Autophagy inhibitors rescued mitochondrial content during HHV8 lytic replication
Mitochondrial content is accumulated in vIRF1 depleted lytic cells
Mitochondrial-contains autophagosomes decreased in vIRF1-depleted lytic Ellis
Mitophagy, vIRF1 promotes mitophagy to eliminate the mictochondrial damage during HHV8 lytic replication
VIRF1 is essential for NOX targeting to and modification son mitochondria
NIX oligomers are reduced during vIRF expression
NIX(BNKIP3L)
Function al interaction of vIRF1 and NIX for mitochondrial clearance
Detection f in trace lunar interaction of vIRF1 and NIX using NanoBiT system (luminescence) or by using PLA system
VIRF binding short linear motif containing ASN and PHE10 and interaction is required for NPC interaction
LC3 targets to mitochondria in a vIRF dependent way
VIRF1 binds indirectly to LC3 upon mitochondrial
Oral Talk #73
Epstein-Barr Virus Activates F-Box Protein FBXO2 to Limit
Viral Infectivity by Targeting Glycoprotein B for Degradation
Hao-Jiong Zhang1, Jinxiu Tian1, Xue-Kang Qi1, Musheng Zeng1, Yi-Xin Zeng1, Lin Feng1
1Sun Yat-sen University Cancer Center
Epstein-Barr virus (EBV) is a human cancer-related virus closely associated with lymphoid and epithelial malignancies, and EBV glycoprotein B (gB) plays an essential role in viral entry into both B cells and epithelial cells by promoting cell-cell fusion. EBV gB is exclusively modified with high-mannose-linked N-glycans and primarily localizes to the endoplasmic reticulum (ER) with low levels on the plasma membrane (PM). However, the mechanism through which gB is regulated within host cells is largely unknown. Here, we report the identification of F-box only protein 2 (FBXO2), an SCF ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans and attenuates EBV infectivity by targeting N-glycosylated gB for degradation. gB possesses seven N-glycosylation sites, and FBXO2 directly binds to these high-mannose moieties through its sugar-binding domain. The interaction promotes the degradation of glycosylated gB via the ubiquitin- proteasome pathway. Depletion of FBXO2 not only stabilizes gB but also promotes its transport from the ER to the PM, resulting in enhanced membrane fusion and viral entry. FBXO2 is expressed in epithelial cells but not B cells, and EBV infection up-regulates FBXO2 levels. In summary, our findings highlight the significance of high-mannose modification of gB and reveal a novel anti-herpes mechanism involving glycoprotein homeostasis regulation.
Viral Infectivity by Targeting Glycoprotein B for Degradation
Hao-Jiong Zhang1, Jinxiu Tian1, Xue-Kang Qi1, Musheng Zeng1, Yi-Xin Zeng1, Lin Feng1
1Sun Yat-sen University Cancer Center
Epstein-Barr virus (EBV) is a human cancer-related virus closely associated with lymphoid and epithelial malignancies, and EBV glycoprotein B (gB) plays an essential role in viral entry into both B cells and epithelial cells by promoting cell-cell fusion. EBV gB is exclusively modified with high-mannose-linked N-glycans and primarily localizes to the endoplasmic reticulum (ER) with low levels on the plasma membrane (PM). However, the mechanism through which gB is regulated within host cells is largely unknown. Here, we report the identification of F-box only protein 2 (FBXO2), an SCF ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans and attenuates EBV infectivity by targeting N-glycosylated gB for degradation. gB possesses seven N-glycosylation sites, and FBXO2 directly binds to these high-mannose moieties through its sugar-binding domain. The interaction promotes the degradation of glycosylated gB via the ubiquitin- proteasome pathway. Depletion of FBXO2 not only stabilizes gB but also promotes its transport from the ER to the PM, resulting in enhanced membrane fusion and viral entry. FBXO2 is expressed in epithelial cells but not B cells, and EBV infection up-regulates FBXO2 levels. In summary, our findings highlight the significance of high-mannose modification of gB and reveal a novel anti-herpes mechanism involving glycoprotein homeostasis regulation.
Oral Talk #74
Cell-Mediated Immune Responses to Persistent KSHV Infection
in Mice with Reconstituted Human Immune System
Components
Nicole Caduff1, Donal McHugh1, Patrick Rämer1, Christian Münz1
1Viral Immunobiology, Institute of Experimental Immunology
Cancers attributable to EBV and KSHV infection are more frequent in immunosuppressed individuals, indicating a crucial role of the immune system in controlling the respective malignancies. In contrast to EBV, the contribution of cellular immunosurveillance against KSHV is less well understood due to the lack of suitable in vitro and in vivo models for studying KSHV biology. In this project, we investigated the cellular immune control of KSHV in an in vivo model of persistent KSHV infection, recently developed in our laboratory, and based on EBV co-infection of NOD-scid γc-/- mice reconstituted with human immune system components (huNSG). In this model, EBV enables persistent KSHV infection beyond four weeks and both viruses seem to collaborate for increased PEL-like lymphomagenesis supported by lytic EBV activity. Interestingly, we observed significantly increased frequencies of unconventional CD56- CD16+ Natural Killer (NK) cells in KSHV co-infected mice compared to those solely infected with EBV, which positively correlated with the KSHV but not EBV burden in co-infected animals. This aberrant NK cell subset is associated with impaired effector functions and accordingly, KSHV loads and tumor frequencies were not affected by NK cell depletion prior to the double-infection of huNSG mice. Furthermore, we documented a significant expansion of central and effector memory T cells in KSHV co-infected compared to EBV-only infected mice, both in co-infection studies using EBV wildtype and lytically-compromised BZLF1-KO- EBV whose viral burden is not affected by KSHV co-infection. Hence, KSHV-specific T cells are likely expanding in our model. Future analysis will lead to a better understanding of how KSHV infections are controlled, which is pivotal for the development of immunotherapies. It will extend our knowledge on how these two prevalent herpesviruses shape the human immune cell landscape and further validate the potential and limitations of our newly developed in vivo co-infection model of KSHV persistence.
in Mice with Reconstituted Human Immune System
Components
Nicole Caduff1, Donal McHugh1, Patrick Rämer1, Christian Münz1
1Viral Immunobiology, Institute of Experimental Immunology
Cancers attributable to EBV and KSHV infection are more frequent in immunosuppressed individuals, indicating a crucial role of the immune system in controlling the respective malignancies. In contrast to EBV, the contribution of cellular immunosurveillance against KSHV is less well understood due to the lack of suitable in vitro and in vivo models for studying KSHV biology. In this project, we investigated the cellular immune control of KSHV in an in vivo model of persistent KSHV infection, recently developed in our laboratory, and based on EBV co-infection of NOD-scid γc-/- mice reconstituted with human immune system components (huNSG). In this model, EBV enables persistent KSHV infection beyond four weeks and both viruses seem to collaborate for increased PEL-like lymphomagenesis supported by lytic EBV activity. Interestingly, we observed significantly increased frequencies of unconventional CD56- CD16+ Natural Killer (NK) cells in KSHV co-infected mice compared to those solely infected with EBV, which positively correlated with the KSHV but not EBV burden in co-infected animals. This aberrant NK cell subset is associated with impaired effector functions and accordingly, KSHV loads and tumor frequencies were not affected by NK cell depletion prior to the double-infection of huNSG mice. Furthermore, we documented a significant expansion of central and effector memory T cells in KSHV co-infected compared to EBV-only infected mice, both in co-infection studies using EBV wildtype and lytically-compromised BZLF1-KO- EBV whose viral burden is not affected by KSHV co-infection. Hence, KSHV-specific T cells are likely expanding in our model. Future analysis will lead to a better understanding of how KSHV infections are controlled, which is pivotal for the development of immunotherapies. It will extend our knowledge on how these two prevalent herpesviruses shape the human immune cell landscape and further validate the potential and limitations of our newly developed in vivo co-infection model of KSHV persistence.
KSHV persists more frequently in the presence of EBV in huNSG mice
EBV+KSHV confection (LANA and LMP +_
PEL like dusk iunfected B cells
EBV lytic gene expression leads to enhanced tumorigeneis in EBV nad KSHV dual infected huNSF mice
Do huNSG mice moist cellular immune responses to KSHV?
- EBV infection is restricted to NK and T cells
- NK cells mainly control EBV lyrically replicating cel;s (Cell Reports 201, Chijioke et al
- EBV lytic replication
KSHV co-infection results in a redistribution of NK cell subsets
Frequency of CD56neg CD17 po NL cells correlates with KSHV but not EBV burden
NK cells seem not to control KSHV in huNSG mice
KSHBV_EBVzko
CD56negCD16pos NK cells ar associated with impaired effector functions
NK cells seem not to control KSHV during infection of huNSG mice
The cells expand significantly more in KSHV co-infected compared to EBV single-infected mice
- Central and effector memory CD8+ T cells expand in KSHV/EBVzko dual infection
- T cell depletion may lead to a higher KSHV burden and increased tumor formation (KSHV + EBV wt)
Outlook
- +T cell repertoire
- + Antigen specificuies
- + Effector functions
- + Analyze T an NK cell compartmentation of
Already published? check on
- "EBV infection on mice with reconstituted human immune system" in pdf 4759)
- Pdf 5272
- pdf 5273
Front Immunol. 2018 Feb 12;9:238. doi: 10.3389/fimmu.2018.00238. eCollection 2018.
Human γ-Herpesvirus Infection, Tumorigenesis, and Immune Control in Mice with Reconstituted Human Immune System Components.
Münz C1.
Abstract
The human γ-herpesviruses Epstein-Barr virus (EBV or HHV4) and Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) are each associated with around 2% of all tumors in humans worldwide. However, investigations into their infection, oncogenesis, and immune responses that protect from the associated tumors have been hampered by the exclusive tropism of these pathogens for humans. Mice with reconstituted human immune system components (HIS mice) provide the unique opportunity to study persistent infection, virus associated lymphoma formation, and cell-mediated immune control of EBV and KSHV. Moreover, since these pathogens are unique stimuli for cytotoxic human lymphocyte responses, they also allow us to characterize long-lasting cell-mediated immune control and the requirements for its initiation, which would also be desirable to achieve during antitumor vaccination in general. Thus, human γ-herpesvirus infection of HIS mice provides unique insights into the biology of these important human pathogens and human cell-mediated immune responses that are considered to be the main protective entity against tumors.(PDF 4759, 5272, 5273)
Oral Talk #75
Epstein-Barr Virus Infection Promotes Epithelial Cell Growth by
Attenuating Differentiation-Dependent Exit from Cell Cycle
Mark R. Eichelberg1, René Welch1, Ahmed Ali1, Kathleen Makielski1, Joseph T. Guidry2, Makoto Ohashi1, Shannon Kenney1, Sϋndϋz Keleş1, Rona Scott2, Eric Johannsen1
1University of Wisconsin-Madison, 2Louisiana State University
Latent infection of B lymphocytes and epithelial cells by Epstein-Barr virus (EBV) is causally linked to their transformation into lymphomas and carcinomas, respectively. Although carcinomas account for almost 90% of EBV associated cancers, progress had been limited due to lack of a physiologic in vitro model of EBV epithelial infection. Recently, EBV infection of normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using NOK and NOK-EBV cells we studied changes in gene expression due to EBV infection and differentiation. Although we observed that the effect of latent EBV infection in undifferentiated cells was subtle, the ability of NOK-EBV to differentiate was impaired. Keratinocytes undergoing differentiation exit the cell cycle and shut down most metabolic processes. However, RNA-seq data indicated that differentiated NOK-EBV maintained some expression of genes associated with these processes. Conversely, genes associated with differentiation were not as strongly induced in NOK-EBV. A possible mechanism for the inhibition of differentiation is expression of EBV lytic genes, since these genes are only expressed in differentiating NOK-EBV. To test this, we constructed an EBV mutant defective for lytic cycle entry by deletion of the R and Z immediate early genes (EBVΔRZ). RNA-seq analysis of NOK-EBVΔRZ suggests that lytic genes are at least partly responsible for the attenuated differentiation. In addition, we have grown NOKs, NOK-EBV, and NOK-EBVΔRZ in organotypic raft cultures. Consistent with our RNA-seq results, EBV partially disrupts the differentiation of NOK-EBV rafts, resulting in significant morphological changes and a reduction in differentiation-associated proteins in the suprabasal layers. Raft cultures of NOK-EBVΔRZ show at least partial restoration of differentiation capacity. Our results help define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers.
Attenuating Differentiation-Dependent Exit from Cell Cycle
Mark R. Eichelberg1, René Welch1, Ahmed Ali1, Kathleen Makielski1, Joseph T. Guidry2, Makoto Ohashi1, Shannon Kenney1, Sϋndϋz Keleş1, Rona Scott2, Eric Johannsen1
1University of Wisconsin-Madison, 2Louisiana State University
Latent infection of B lymphocytes and epithelial cells by Epstein-Barr virus (EBV) is causally linked to their transformation into lymphomas and carcinomas, respectively. Although carcinomas account for almost 90% of EBV associated cancers, progress had been limited due to lack of a physiologic in vitro model of EBV epithelial infection. Recently, EBV infection of normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using NOK and NOK-EBV cells we studied changes in gene expression due to EBV infection and differentiation. Although we observed that the effect of latent EBV infection in undifferentiated cells was subtle, the ability of NOK-EBV to differentiate was impaired. Keratinocytes undergoing differentiation exit the cell cycle and shut down most metabolic processes. However, RNA-seq data indicated that differentiated NOK-EBV maintained some expression of genes associated with these processes. Conversely, genes associated with differentiation were not as strongly induced in NOK-EBV. A possible mechanism for the inhibition of differentiation is expression of EBV lytic genes, since these genes are only expressed in differentiating NOK-EBV. To test this, we constructed an EBV mutant defective for lytic cycle entry by deletion of the R and Z immediate early genes (EBVΔRZ). RNA-seq analysis of NOK-EBVΔRZ suggests that lytic genes are at least partly responsible for the attenuated differentiation. In addition, we have grown NOKs, NOK-EBV, and NOK-EBVΔRZ in organotypic raft cultures. Consistent with our RNA-seq results, EBV partially disrupts the differentiation of NOK-EBV rafts, resulting in significant morphological changes and a reduction in differentiation-associated proteins in the suprabasal layers. Raft cultures of NOK-EBVΔRZ show at least partial restoration of differentiation capacity. Our results help define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers.
EBV establishes latency in B cells
hTert NOKs (Munger lab, Harvard)
NOK differentiation (methylcellulose MC)
IVL, Zta, Rta, EAD
Raft culture collagen base, cells at air interface
RNA-seq analysis NOK and NOK-EBV
- Low LMP1 and LMP2A
- High EBERs, BARTs, GFP/new
EBV has subtle effect on NOKs 45 up 88 down
However, in MC-induced differentiation many genes are altered, including ….
Pathways down regulated by the presence of EBV
EBV partially inhibits differentiation (NOK, versus NOK-EBV)
KRT10 is quickly unregulated during differentiation in the absence of EBV
MCM7
Basal NOK-EBV are invasive
EBVdeltaRZ
- This virus still strongly inhibits KRT10, ITGB1, IVL expression
Therefore,
- Latent EBV has subtle effect in ballad keratinocytesonce or more latent genes attenuated differentiation
- EBERs, LMP1, LMP2A, BNLF2a/b possible mechanisms?
Oral Talk #76
INF-γ Responses in Peripheral Blood Lymphocytes of Patients
with KSHV-associated Diseases Treated with Pomalidomide
and Liposomal Doxorubicin
Romin Roshan1, Kathryn Lurain2, Nazzarena Labo1, Ramya Ramaswami2, Thomas Uldrick2, Priscila Goncalves2, Anna Widell2, Vickie Marshall1, Robert Yarchoan2, Denise Whitby1
1Viral Oncology Section, AIDS and Cancer Viral Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, 2HIV and AIDS Mailgnancy Branch, Center for Cancer Research, National Cancer Institute
Background: KSHV-specific T-cell responses are poorly understood, including how they vary among patients with different KSHV-associated diseases or are affected by treatment. We evaluated factors associated with cell-mediated IFN-ƴ responses to the entire KSHV proteome among patients with Kaposi sarcoma +/- other KSHV-associated diseases treated with pomalidomide and liposomal doxorubicin (LD).
Methods: 12 HIV+ male patients were studied: 7 with KS alone; and 5 with KS and primary effusion lymphoma (PEL), KSHV-associated multicentric Castleman disease (KSHV-MCD) or KSHV inflammatory cytokine syndrome (KICS) at baseline and after treatment with pomalidomide and LD. PBMCs were tested using a peptide-based ELISPOT assay measuring IFN-ƴ secretion in response to 82 KSHV-encoded proteins, an SIV peptide, and a peptide pool including EBV, CMV, and influenza. Breadth and intensity of T-cell responses were compared using nonparametric univariate analyses, while response levels to individual antigens were evaluated with nested random effect models.
Results: Median age was 43 (inter-quartile range [IQR] 41-56); median CD4+ cell count was 273 (IQR 90-408), HIV VL was undetectable in all patients but 3 who had KICS, PEL or KSHV-MCD (range 28-3318 copies/mL). While positive control responses were robust (median 2327 antigens; IQR 444-3213), patients generally responded to few KSHV antigens (median 4 antigens; IQR 2-11) with a moderate mean intensity (median 163 SFU/million PBMC, IQR 147-264). Baseline responses were not different among diseases. At a second timepoint, after a median of 161 days of treatment (IQR 102-172), CD4+ counts did not change significantly and the number and intensity of responses to KSHV antigens remained essentially stable in breadth (median 4.5, IQR 2.5-12) and mean intensity (median 195 SFU/million PBMC; IQR 160-466).
Conclusions: As previously observed, HIV+ patients had sparse, modest and heterogeneous IFN-ƴ response to KSHV antigens. Treatment with pomalidomide and LD did not result in changes in response levels or breadth.
with KSHV-associated Diseases Treated with Pomalidomide
and Liposomal Doxorubicin
Romin Roshan1, Kathryn Lurain2, Nazzarena Labo1, Ramya Ramaswami2, Thomas Uldrick2, Priscila Goncalves2, Anna Widell2, Vickie Marshall1, Robert Yarchoan2, Denise Whitby1
1Viral Oncology Section, AIDS and Cancer Viral Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, 2HIV and AIDS Mailgnancy Branch, Center for Cancer Research, National Cancer Institute
Background: KSHV-specific T-cell responses are poorly understood, including how they vary among patients with different KSHV-associated diseases or are affected by treatment. We evaluated factors associated with cell-mediated IFN-ƴ responses to the entire KSHV proteome among patients with Kaposi sarcoma +/- other KSHV-associated diseases treated with pomalidomide and liposomal doxorubicin (LD).
Methods: 12 HIV+ male patients were studied: 7 with KS alone; and 5 with KS and primary effusion lymphoma (PEL), KSHV-associated multicentric Castleman disease (KSHV-MCD) or KSHV inflammatory cytokine syndrome (KICS) at baseline and after treatment with pomalidomide and LD. PBMCs were tested using a peptide-based ELISPOT assay measuring IFN-ƴ secretion in response to 82 KSHV-encoded proteins, an SIV peptide, and a peptide pool including EBV, CMV, and influenza. Breadth and intensity of T-cell responses were compared using nonparametric univariate analyses, while response levels to individual antigens were evaluated with nested random effect models.
Results: Median age was 43 (inter-quartile range [IQR] 41-56); median CD4+ cell count was 273 (IQR 90-408), HIV VL was undetectable in all patients but 3 who had KICS, PEL or KSHV-MCD (range 28-3318 copies/mL). While positive control responses were robust (median 2327 antigens; IQR 444-3213), patients generally responded to few KSHV antigens (median 4 antigens; IQR 2-11) with a moderate mean intensity (median 163 SFU/million PBMC, IQR 147-264). Baseline responses were not different among diseases. At a second timepoint, after a median of 161 days of treatment (IQR 102-172), CD4+ counts did not change significantly and the number and intensity of responses to KSHV antigens remained essentially stable in breadth (median 4.5, IQR 2.5-12) and mean intensity (median 195 SFU/million PBMC; IQR 160-466).
Conclusions: As previously observed, HIV+ patients had sparse, modest and heterogeneous IFN-ƴ response to KSHV antigens. Treatment with pomalidomide and LD did not result in changes in response levels or breadth.
Association of KSHV with immunosuppressive suggests cellular immunity is a restriction
KSHV whole proteome ELISPOT assay
- 12 mer peptide pool ~7500 overlapping peptides 82 IRGFs in 84 pools
T cell responses to KSHV ORFs are variable
Treatment: pomalidomide (3rd generation immunomodulatory drug) angiogenesis inhibitor, T cell activation , NK cell activation and increase tumor surface marker down-regulated by virus + liposomal doxoreuicin
Antigen responses did not change over time
There no statistically (NO IMMUNODOMINANT ANTIGEN IDENTIFIED)
Some patients have good CD4 response
Oral Talk #77
Epstein-Barr Virus-Infected Tonsillar Marginal Zone B Cells in
vivo as a Precursor for Immunosuppression-Related B-Cell
Lymphoma
Charles Torgbor1, Ann M. Moormann2, David A.Thorley-Lawson1
1Tufts University School of Medicine, 2University of Massachusetts Medical School
The germinal center model (GCM) of Epstein-Barr virus (EBV) persistence in humans proposes that, EBV infects and drives naïve B cells through the GC to establish a quiescent lifelong persistent infection in memory B cells. EBV causes several cancers and the normal in vivo counterparts of major EBV-associated cancers have been identified in the GCM. Yet, the normal in vivo counterparts of immunosuppression-related/ immunoblastic B-cell lymphoma (IL) which occurs in immunosuppressed transplant and HIV patients, and resembles lymphoblastoid cell lines (LCLs) remains elusive. We performed a comparative analysis of human tonsillar B cells to identify the closest normal in vivo counterparts of ILs/ LCLs. Here we show that EBV-infected tonsillar marginal zone (MZ) B cells which are GC-independent, are the closest in vivo correlates of ILs/ LCLs. Like LCLs, EBV-infected MZ B express the EBV growth program which includes high expression of the oncogenic LMP1, and the DNA mutating enzyme activation-induced cytidine deaminase (AID), in the absence of the transcription factor BCL6. Furthermore, these EBV-infected MZ B cells are proliferating extensively and are Ki67+. AID expression, rapid proliferation and LMP1 expression put these cells at high risk for tumor development. Of therapeutic importance, the high expression of the oncogenic LMP1 could prove the Achilles heel of EBV-infected MZ B cells, to be targeted. Consequently, we now propose that there are two pathways of EBV persistence in vivo: 1. the established GC model and 2. the alternate GC-independent MZ model.
Lymphoma
Charles Torgbor1, Ann M. Moormann2, David A.Thorley-Lawson1
1Tufts University School of Medicine, 2University of Massachusetts Medical School
The germinal center model (GCM) of Epstein-Barr virus (EBV) persistence in humans proposes that, EBV infects and drives naïve B cells through the GC to establish a quiescent lifelong persistent infection in memory B cells. EBV causes several cancers and the normal in vivo counterparts of major EBV-associated cancers have been identified in the GCM. Yet, the normal in vivo counterparts of immunosuppression-related/ immunoblastic B-cell lymphoma (IL) which occurs in immunosuppressed transplant and HIV patients, and resembles lymphoblastoid cell lines (LCLs) remains elusive. We performed a comparative analysis of human tonsillar B cells to identify the closest normal in vivo counterparts of ILs/ LCLs. Here we show that EBV-infected tonsillar marginal zone (MZ) B cells which are GC-independent, are the closest in vivo correlates of ILs/ LCLs. Like LCLs, EBV-infected MZ B express the EBV growth program which includes high expression of the oncogenic LMP1, and the DNA mutating enzyme activation-induced cytidine deaminase (AID), in the absence of the transcription factor BCL6. Furthermore, these EBV-infected MZ B cells are proliferating extensively and are Ki67+. AID expression, rapid proliferation and LMP1 expression put these cells at high risk for tumor development. Of therapeutic importance, the high expression of the oncogenic LMP1 could prove the Achilles heel of EBV-infected MZ B cells, to be targeted. Consequently, we now propose that there are two pathways of EBV persistence in vivo: 1. the established GC model and 2. the alternate GC-independent MZ model.
GCM: germinal center model
Search for immunologic lymphoma (IL-like B cells in human tonsils
EBV exploits normal B cell biology/differentiation route 2015 Curr top microbial Thorley-Lawson
EBV infected LCL give rise to lethal IL phenotype in immunodeficiency mice
Cancer Cell 2014, Xang et al?
LCL: IgD+, CD27+, AID+,, CD10- Bcl6-
The only cell type detected in vivo expression the EBV growth is IgD+
None of the observed in vivo are LCL type
Phenotypically LCL resemble tonsil marginal zone (MZ) B cells
MZ (spleen, tonsils,lymph nodes)
EBV gene expression in marginal zone B cells
LCLs express the potent mutagenic enzyme AIF
EBV infected cells are in the AID+MZ cells and BCL6-
PROLIFERATIN OPROFILE OF MZ B CELLS AND NAIVE B CELLS
EBV+ M B CELLS HAV EUNDERGONE MULTIPLE CELL DIVISION
While naive B cell divide very infrequently
Extended edition proliferation and e
