A Rare Case of Chronic Active Epstein-Barr Virus (EBV) Infection Accompanied by the Infiltration of EBV-infected CD8+ T Cells into the Muscle

Abstract INTRODUCTION: A promising rich pipeline of combination therapies targeting checkpoint molecules expressed on T cells and/or tumor cells is currently being developed to abrogate tumor-induced immunosuppression. Novel in vivo models suitable for validating these immunotherapies and predict safety issues are warranted to accelerate their translation to patients. AIM: Epstein Barr virus (EBV) is a type 1 carcinogen that is directly associated with the development of human B cell neoplasms. We modelled EBV infection and tumor progression in long-term humanized mice and investigated the activation of T cells with PD-1 expression. Further, we performed studies evaluating the effects of an anti-PD-1 antibody (pembrolizumab/ keytruda) in on EBV infections and/or tumor growth. METHODS: Humanized mice transplanted with human cord-blood CD34+ stem cells and showing long-term (15 weeks) human T cell reconstitution were infected with an oncogenic recombinant Epstein Barr Virus (EBV), encoding enhanced firefly luciferase (fLuc) and green fluorescent protein (GFP). EBV infections were monitored by optical imaging analyses and PCR. CD8+ and CD4+ T cell subtypes (PD-1+, naïve, central memory, effector memory and terminal effector) were sequentially monitored in blood by longitudinal flow cytometry analyses and in organs at experimental endpoint. Histopathological analyses were performed to characterize EBV infection (EBER+) and PD-1+ T cell-rich infiltrates in tissues and tumors. We used the model to evaluate the effects of pembrolizumab administered after EBV challenge at low dose (first dose 1.65mg/kg and then 3.30 mg/kg, every other week, n=3) or high dose (first dose 5.00 mg/kg and then 10.00 mg/kg every other week, n=3) in respect to EBV infected controls (n=2). RESULTS: EBV-fLuc was detectable one week after infection by non-invasive optical imaging in the spleen, from where it spread rapidly and systemically. Among the EBV-infected mice, 8/18 (=44%) developed macroscopically visible tumors in the spleen. For further analyses of the data, we then compared EBV-infected mice with ("EBV-Tumor") or without ("EBV") macroscopic tumors. At 6 weeks post-infection, the relative CD8+ T cell frequencies increased significantly and constantly (control Vs. EBV p=0.0021, control Vs. EBV-Tumor p=<0.0001, EBV Vs. EBV-Tumor p=0.0072). For absolute cell counts in tissues, CD8+ T cell increases were more dramatic in mice infected with EBV and developing tumors. These differences amounted to approximately tenfold relative to controls and 3-fold relative to mice not developing tumors. Mice infected with EBV showed 90-100% of the CD4+ and CD8+ T cells in lymphatic tissues expressing PD-1. Mice with EBV-tumors showed twice as many PD-1+ CD4+ and three times as many PD-1+ CD8+ T cells as infected mice without tumors. Histopathology combined with EBER in situ hybridization, showed foci of EBV infected cells in close association with PD-1+ infiltrating lymphocytes, often in perivascular regions. This model was then used to evaluate dose-dependent effects of pembrolizumab. The check-point inhibitor controlled EBV-fLUC spread for 2 weeks, but later prompted increased levels of infections. At endpoint analyses, mice receiving pembrolizumab showed larger dissemination of tumors. CONCLUSIONS: We are currently performing additional experiments in order to elucidate this mechanism of EBV rebound. This humanized mouse model contributes to risk assessment prior to clinical trials of the use of checkpoint inhibitors in patients after transplantations at high risk of EBV infections. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

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Epstein-Barr Virus-Specific CD8 T Cells Selectively Infiltrate the Brain in Multiple Sclerosis and Interact Locally with Virus-Infected Cells: Clue for a Virus-Driven Immunopathological Mechanism

Journal of Virology ◽

10.1128/jvi.00980-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 8

Author(s):

Barbara Serafini ◽

Barbara Rosicarelli ◽

Caterina Veroni ◽

Gina Adriana Mazzola ◽

Francesca Aloisi

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Cns Injury ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr ◽

The Brain

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous herpesvirus strongly associated with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). However, the mechanisms linking EBV infection to MS pathology are uncertain. Neuropathological and immunological studies suggest that a persistent EBV infection in the CNS can stimulate a CD8 T-cell response aimed at clearing the virus but inadvertently causing CNS injury. Inasmuch as in situ demonstration of EBV-specific CD8 T cells and their effector function is missing, we searched for EBV-specific CD8 T cells in MS brain tissue using the pentamer technique. Postmortem brain samples from 12 donors with progressive MS and known HLA class I genotype were analyzed. Brain sections were stained with HLA-matched pentamers coupled with immunogenic peptides from EBV-encoded proteins, control virus (cytomegalovirus and influenza A virus) proteins, and myelin basic protein. CD8 T cells recognizing proteins expressed in the latent and lytic phases of the EBV life cycle were visualized in white matter lesions and/or meninges of 11/12 MS donors. The fraction (median value) of CD8 T cells recognizing individual EBV epitopes ranged from 0.5 to 2.5% of CNS-infiltrating CD8 T cells. Cytomegalovirus-specific CD8 T cells were detected at a lower frequency (≤0.3%) in brain sections from 4/12 MS donors. CNS-infiltrating EBV-specific CD8 T cells were CD107a positive, suggesting a cytotoxic phenotype, and stuck to EBV-infected cells. Together with local EBV dysregulation, selective enrichment of EBV-specific CD8 T cells in the MS brain supports the notion that skewed immune responses toward EBV contribute to inflammation causing CNS injury. IMPORTANCE EBV establishes a lifelong and asymptomatic infection in most individuals and more rarely causes infectious mononucleosis and malignancies, like lymphomas. The virus is also strongly associated with MS, a chronic neuroinflammatory disease with unknown etiology. Infectious mononucleosis increases the risk of developing MS, and immune reactivity toward EBV is higher in persons with MS, indicating inadequate control of the virus. Previous studies have suggested that persistent EBV infection in the CNS stimulates an immunopathological response, causing bystander neural cell damage. To verify this, we need to identify the immune culprits responsible for the detrimental antiviral response in the CNS. In this study, we analyzed postmortem brains donated by persons with MS and show that CD8 cytotoxic T cells recognizing EBV enter the brain and interact locally with the virus-infected cells. This antiviral CD8 T cell-mediated immune response likely contributes to MS pathology.

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Quantitative Analysis of Epstein‐Barr Virus (EBV)–Specific CD8+T Cells in Patients with Chronic Active EBV Infection

The Journal of Infectious Diseases ◽

10.1086/423285 ◽

2004 ◽

Vol 190 (5) ◽

pp. 985-988 ◽

Cited By ~ 42

Author(s):

Naomi Sugaya ◽

Hiroshi Kimura ◽

Shinya Hara ◽

Yo Hoshino ◽

Seiji Kojima ◽

...

Keyword(s):

T Cells ◽

Quantitative Analysis ◽

Cd8 T Cells ◽

Epstein Barr Virus ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr

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Selective Induction of Th2-Attracting Chemokines CCL17 and CCL22 in Human B Cells by Latent Membrane Protein 1 of Epstein-Barr Virus

Journal of Virology ◽

10.1128/jvi.78.4.1665-1674.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1665-1674 ◽

Cited By ~ 106

Author(s):

Takashi Nakayama ◽

Kunio Hieshima ◽

Daisuke Nagakubo ◽

Emiko Sato ◽

Masahiro Nakayama ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Regulatory T Cells ◽

Epstein Barr Virus ◽

Cytotoxic T Cells ◽

Th2 Cells ◽

Th1 Cells ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr

ABSTRACT Chemokines are likely to play important roles in the pathophysiology of diseases associated with Epstein-Barr virus (EBV). Here, we have analyzed the repertoire of chemokines expressed by EBV-infected B cells. EBV infection of B cells induced expression of TARC/CCL17 and MDC/CCL22, which are known to attract Th2 cells and regulatory T cells via CCR4, and also upregulated constitutive expression of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5, which are known to attract Th1 cells and cytotoxic T cells via CCR5. Accordingly, EBV-immortalized B cells secreted these chemokines, especially CCL3, CCL4, and CCL22, in large quantities. EBV infection or stable expression of LMP1 also induced CCL17 and CCL22 in a B-cell line, BJAB. The inhibitors of the TRAF/NF-κB pathway (BAY11-7082) and the p38/ATF2 pathway (SB202190) selectively suppressed the expression of CCL17 and CCL22 in EBV-immortalized B cells and BJAB-LMP1. Consistently, transient-transfection assays using CCL22 promoter-reporter constructs demonstrated that two NF-κB sites and a single AP-1 site were involved in the activation of the CCL22 promoter by LMP1. Finally, serum CCL22 levels were significantly elevated in infectious mononucleosis. Collectively, LMP1 induces CCL17 and CCL22 in EBV-infected B cells via activation of NF-κB and probably ATF2. Production of CCL17 and CCL22, which attract Th2 and regulatory T cells, may help EBV-infected B cells evade immune surveillance by Th1 cells. However, the concomitant production of CCL3, CCL4, and CCL5 by EBV-infected B cells may eventually attract Th1 cells and cytotoxic T cells, leading to elimination of EBV-infected B cells at latency III and to selection of those with limited expression of latent genes.

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Nivolumab treatment of relapsed/refractory Epstein-Barr virus–associated hemophagocytic lymphohistiocytosis in adults

Blood ◽

10.1182/blood.2019003886 ◽

2020 ◽

Vol 135 (11) ◽

pp. 826-833 ◽

Cited By ~ 7

Author(s):

Pengpeng Liu ◽

Xiangyu Pan ◽

Chong Chen ◽

Ting Niu ◽

Xiao Shuai ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Hemophagocytic Lymphohistiocytosis ◽

Epstein Barr Virus ◽

Sequencing Analysis ◽

Hematopoietic Stem ◽

Barr Virus ◽

Life Threatening ◽

Programmed Death Protein 1 ◽

Epstein Barr

Abstract Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening hyperinflammatory syndrome triggered by EBV infection. It often becomes relapsed or refractory (r/r), given that etoposide-based regimens cannot effectively clear the virus. r/r EBV-HLH is invariably lethal in adults without allogeneic hematopoietic stem cell transplantation. Here, we performed a retrospective analysis of 7 r/r EBV-HLH patients who were treated with nivolumab on a compassionate-use basis at West China Hospital. All 7 patients tolerated the treatment and 6 responded to it. Five of them achieved and remained in clinical complete remission with a median follow-up of 16 months (range, 11.4-18.9 months). Importantly, both plasma and cellular EBV-DNAs were completely eradicated in 4 patients. Single-cell RNA-sequencing analysis showed that HLH syndrome was associated with hyperactive monocytes/macrophages and ineffective CD8 T cells with a defective activation program. Nivolumab treatment expanded programmed death protein-1–positive T cells and restored the expression of HLH-associated degranulation and costimulatory genes in CD8 T cells. Our data suggest that nivolumab, as a monotherapy, provides a potential cure for r/r EBV-HLH, most likely by restoring a defective anti-EBV response.

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Rapid reconstitution of Epstein-Barr virus–specific T lymphocytes following allogeneic stem cell transplantation

Blood ◽

10.1182/blood.v96.8.2814.h8002814_2814_2821 ◽

2000 ◽

Vol 96 (8) ◽

pp. 2814-2821 ◽

Cited By ~ 1

Author(s):

Natalie A. Marshall ◽

John Greg Howe ◽

Richard Formica ◽

Diane Krause ◽

John E. Wagner ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cd8 T Cells ◽

Epstein Barr Virus ◽

Hla Class I ◽

Barr Virus ◽

Matched Sibling ◽

Epstein Barr

Epstein-Barr virus (EBV)–specific CD8 T lymphocytes are present at remarkably high frequencies in healthy EBV+individuals and provide protection from EBV-associated lymphoproliferative diseases. Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is a commonly used therapy in which T-cell surveillance for EBV is temporarily disrupted. Herein, human leukocyte antigen (HLA) class I tetramers were used to investigate the reestablishment of the EBV-specific CD8 T-cell repertoire in patients following allo-PBSCT. CD8+ T cells specific for lytic and latent cycle–derived EBV peptides rapidly repopulate the periphery of matched sibling allo-PBSCT patients. The relative frequencies of T cells specific for different EBV peptides in transplantation recipients closely reflect those of their respective donors. Investigation of patients at monthly intervals following unmanipulated allo-PBSCT demonstrated that the frequency of EBV-specific T cells correlates with the number of EBV genome copies in the peripheral blood and that expansion of EBV-specific T-cell populations occurs even in the setting of immunosuppressive therapy. In contrast, patients undergoing T-cell–depleted or unrelated cord blood transplantation have undetectable EBV-specific T cells, even in the presence of Epstein-Barr viremia. The protective shield provided by EBV-specific CD8 T cells is rapidly established following unmanipulated matched sibling allo-PBSCT and demonstrates that HLA class I tetramers complexed with viral peptides can provide direct and rapid assessment of pathogen-specific immunity in this and other vulnerable patient populations.

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Epstein-Barr virus–specific CD8+ T cells that re-express CD45RA are apoptosis-resistant memory cells that retain replicative potential

Blood ◽

10.1182/blood-2002-01-0160 ◽

2002 ◽

Vol 100 (3) ◽

pp. 933-940 ◽

Cited By ~ 102

Author(s):

Padraic J. Dunne ◽

Jeffery M. Faint ◽

Nancy H. Gudgeon ◽

Jean M. Fletcher ◽

Fiona J. Plunkett ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Epstein Barr Virus ◽

Acute Infection ◽

Target Cells ◽

Elderly Persons ◽

Effector Cells ◽

Barr Virus ◽

Epstein Barr ◽

Acute Infectious Mononucleosis

Abstract During acute infection, latent and lytic Epstein-Barr virus (EBV) epitope-specific CD8+ T cells have a CD45RO+CD45RA− phenotype. However, after resolution of the infection, a large proportion of these cells, particularly those specific for lytic viral epitopes, re-express the CD45RA molecule. The role of CD8+ CD45RA+ T cells in ongoing immunity to EBV and other viruses is unknown. We now demonstrate that, relative to their CD45RO+ counterparts, the EBV-specific CD8+ T cells that revert to CD45RA expression after acute infectious mononucleosis are not in cell cycle, have longer telomeres, and are more resistant to apoptosis partly because of increased Bcl-2 expression. However, the EBV-specific CD8+CD45RA+ T cells have shorter telomeres than the total CD8+ CD45RA+ T-cell pool and predominantly express low levels of the CCR7 chemokine receptor, indicating that they are not naive cells. In addition, EBV-specific CD8+CD45RA+ T cells can be induced to proliferate and exhibit potent cytotoxic activity against target cells loaded with specific peptide. Our results strongly suggest, therefore, that EBV-specific CD8+ CD45RA+ T cells represent a stabilized virus-specific memory pool and not terminally differentiated effector cells. The identification of mechanisms that enable stable virus-specific CD8+ T cells to persist after acute infection may lead to the enhancement of antiviral immunity in immunocompromised and elderly persons.

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