Session 10A 11:00 AM – 12:30 PM Immunity & Immune Therapy (Shannon Hall)
Session Chairs:
Dirk Dittmer & Rajiv Khanna
A Role for EBNA2 in Mechanisms that Foster Some
Autoimmune Diseases
John B. Harley1,2,3,4,5, Xiaoting Chen1, Mario Pujato1, Daniel Miller1, Avery Maddox1, Carmy
Forney1, Albert F. Magnusen1, Arthur Lynch1, Kashish Chetal6, Masashi Yukawa7, Artem Barski4,7,8,
Nathan Salomonis4,6, Kenneth M. Kaufman1,2,4,5, Leah Kottyan1,4, Matthew T. Weirauch1,3,4,6
1Center for Autoimmune Genomics and Etiology (CAGE), Cincinnati Children's Hospital Medical
Center, 2Division of Immunobiology, Cincinnati Children's Hospital Medical Center, 3Division of
Developmental Biology, Cincinnati Children's Hospital Medical Center , 4Department of Pediatrics,
University of Cincinnati, 5US Department of Veterans Affairs Medical Center , 6Division of
Biomedical Informatics, Cincinnati Children's Hospital Medical Center, 7Division of Allergy and
Immunology, Cincinnati Children's Hospital Medical Center, 8Division of Human Genetics,
Cincinnati Children's Hospital Medical Center
Explaining the genetics of many diseases is challenging because most associations
localize to incompletely understood regulatory regions. We show transcription factors (TFs)
occupy multiple loci of individual complex genetic disorders by using novel computational
methods. Application to 213 phenotypes and 1,544 TF binding datasets identifies 2,264
relationships between hundreds of TFs and 94 phenotypes, including AR in prostate
cancer and GATA3 in breast cancer. Strikingly, nearly half of the systemic lupus
erythematosus risk loci from Europeans are occupied by the Epstein-Barr virus (EBV)
EBNA2 protein (OR=6, P
1. Strong viral associations with SLE among Filipinos. Lupus Sci Med. 2017 Jul 28;4(1):e000214. doi: 10.1136/lupus-2017-000214. eCollection 2017.
2. A genome-wide integrative genomic study localizes genetic factors influencing antibodies against Epstein-Barr virus nuclear antigen 1 (EBNA-1). PLoS Genet. 2013;9(1):e1003147.
PMID: 23326239
3. Identification of unique microRNA signature associated with lupus nephritis. PLoS One. 2010 May 11;5(5):e10344.
4. Aberrant Epstein-Barr viral infection in systemic lupus erythematosus. Autoimmun Rev. 2009 Feb;8(4):337-42.
5. Lupus-like autoantibody development in rabbits and mice after immunization with EBNA-1 fragments. J Autoimmun. 2008 Dec;31(4):362-71.
6. The curiously suspicious: a role for Epstein-Barr virus in lupus.
Harley JB, Harley IT, Guthridge JM, James JA.
Lupus. 2006;15(11):768-77. Review. PMID: 17153849
Disruption of IL-15 Signaling and NK Cells Facilitates
Oncogenesis of Gammaherpesviruses in SIV-infected Rhesus
Macaques
Yoshinori Fukazawa1,2, Helen Li1,2, Devin Fachko1,2, Minsha Manoharan1,2, Derick M. Duell1,2,
Amanda Johnson1,2, Ann Lewis2, Jeffrey D. Lifson3, Keith A. Reinmann4, Michael K. Axthelm1,2,
Rebecca L. Skalsky1,2, Scott W. Wong1,2, Louis J. Picker1,2, Afam A. Okoye1,2
1Vaccine and Gene Therapy Institute, Oregon Health & Science University, 2Oregon National
Primate Research Center, Oregon Health & Science University, 3AIDS and Cancer Virus Program,
Leidos Biomedical Research, Inc., Frederick National Laboratory, 4MassBiologics, University of
Massachusetts Medical School
Background: Lymphocryptovirus (LCV) and rhesus rhadinovirus (RRV), simian homologs of
EBV and KSHV, respectively, naturally infect rhesus macaques (RM) and both viruses are
associated with the development of lymphoproliferative disorder (LPD) and B cell
lymphoma in simian immunodeficiency virus (SIV) infected RM. To evaluate the role of NK
cells on SIV infection and LCV and RRV reactivation and lymphomagenesis, we used an
IL-15-neutalizing monoclonal antibody (mAb), which profoundly depletes NK cells.
Methods: RM received anti-IL-15 at 2-week intervals either given from -6 to 8 weeks after
SIV infection (long duration group, 8 doses; n=8), or given only prior to infection (short
duration group, 3 doses; n=8). Another group received a control mAb on the same
schedule as the long duration group (8 doses; n=8). SIV RNA in plasma and, RRV/LCV
DNA in blood were quantified by RT-PCR and qPCR, respectively. RM were euthanized
with AIDS-defining malignancies, which included LPD and B cell lymphomas.
Histopathological examination included immunostaining for cellular markers, EBV nuclear
antigen 2 (EBNA2) and RRV major capsid protein to detect EBV and RRV-infected cells,
respectively.
Results: Despite massive and prolonged NK cell depletion in anti-IL-15 treatment RM, SIV
replication kinetics and CD4+ T cell depletion were comparable between groups. However,
in the long duration, anti-IL-15-treated group, 6 of 8 RM manifested accelerated RRV
reactivation and replication as early as 6 weeks post-infection, and 5 of 8 RM presented
with confirmed cases of B lymphoma. Interestingly, most tumors had high frequencies of
EBNA2+ cells that were CD79adim and CD20dim/–. Almost half of the EBNA2+ cells were
Ki67+ and some RM showed a preferential distribution of EBNA2+ in B cell follicles.
Conclusions: These data suggest that IL-15 and/or NK cells play a key role in protection
from RRV reactivation and/or LCV-associated tumor lymphomagenesis during SIV-
associated malignancies.
Dramatically Decreased and Delayed CD8 T-Cell Responses
During Asymptomatic EBV Seroconversion
Liisa K. Selin1, Fransenio Clark1, Rabinarayan Mishra1, Anna Gil1, Ramakanth Chirravuri2, Dario
Ghersi2, Katherine Luzuriaga3
1Department of Pathology, University of Massachusetts, 2University of Nebraska, 3Program in
Molecular Medicine, University of Massachusetts Medical School
Infection with EBV is strongly associated with the syndrome of acute infectious
mononucleosis (AIM), with symptoms of mild to severe lymphadenopathy, sore throat,
splenomegaly and prolonged fatigue. The symptoms are associated with a massive
expansion of CD8+ T cells in the peripheral blood, which are mostly directed to two EBV
immunodominant epitopes: BMLF1280-288 and BRLF1109-118 in HLA-A2:01+ patients.
During AIM there is an expansion of unique cross-reactive CD8+ T cells between influenza
A (IAV)-M158 and EBV-BMLF1, which can modulate the immune response to EBV with
their frequency directly correlating with disease severity. However, little is known about
EBV-specific and crossreactive CD8 T-cell responses in the vast majority of individuals,
who asymptomatically seroconvert and become persistently infected with EBV. Here, we
hypothesized that asymptomatic donors would have decreased EBV-specific and
crossreactive CD8 T-cell responses near the time of sero-conversion as compared to AIM
patients. By tracking seronegative donors at the time of college admission every 3-4
months for their 4 years in college we have identified 20 asymptomatic sero-converters
who had surprisingly low or non-existent EBV lytic epitope-specific responses in the first
year after sero-conversion with no evidence of cross-reactive IAV-M1/EBV-BMLF1 memory
CD8 T-cell activation ex vivo. In the presence of these much lower and delayed CD8 T-cell
responses, their viral loads were equivalent to AIM patients. There was tremendous
individual variation in the kinetics of their CD8 T-cell responses. Surprisingly, EBV
seronegative young donors (14/30) grew EBV-BRLF1-specific cells in BRLF1-peptide-
stimulated cultures even years prior to sero-conversion. A few EBV seronegative young
donors grew crossreactive IAV-M1 cells in EBV-BRLF1 and/or EBV-BMLF1-stimulated
cultures like we have reported in long-term EBV middle-aged seronegative donors. These
results are consistent with CD8 T-cell activation mediating the symptomatology of AIM, and
strongly suggest that in the asymptomatic donor EBV is silently infecting the host.
MBio. 2017 Dec 5;8(6). pii: e01841-17.
Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires. Aslan N1, Watkin LB1, Gil A1, Mishra R1, Clark FG1,2, Welsh RM1, Ghersi D3, Luzuriaga K4,2, Selin LK5,2.
Abstract
Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to
massive CD8 T-cell expansion and
acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome.
We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between
influenza virus A-M158 (IAV-M1) and
EBV BMLF1280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students.
Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer+ frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer+ cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter.IMPORTANCE The pathogenic impact of immune responses that by chance cross-react to unrelated viruses has not been established in human infections. Here, we demonstrate that the severity of acute infectious mononucleosis (AIM), an Epstein-Barr virus (EBV)-induced disease prevalent in young adults but not children, is associated with increased frequencies of T cells cross-reactive to EBV and the commonly acquired influenza A virus (IAV). The T-cell receptor (TCR) repertoire and functions of these cross-reactive T cells differed between mild- and severe-AIM patients, most likely because these two groups of patients had selected different memory TCR repertoires in response to IAV infections encountered earlier. This heterologous immunity may explain variability in disease outcome and why young adults with more-developed IAV-specific memory T-cell pools have more-severe disease than children, who have less-developed memory pools. This study provides a new framework for understanding the role of heterologous immunity in human health and disease and highlights an important developing field examining the role of T-cell repertoires in the mediation of immunopathology.
PMID: 29208744
Gammaherpesvirus Infected B Cells Display Abnormal
Repertoire
Monika Zelazowska1, Qiwen Dong2, Joshua Plummer1, Yunxiang Mu1, Yi Zhong1, Bin Liu1, Laurie
Krug2, Kevin McBride1
1Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson
Cancer Center, 2Department of Molecular Genetics and Microbiology, Stony Brook University
Gammaherpesviruses (γHV) are oncogenic viruses that establish lifelong latency in B cells.
Chronic infection with human γHV, Kaposi's sarcoma-associated herpesvirus (KSHV) and
Epstein-Barr virus (EBV) is associated with the development of lymphoproliferative
diseases, lymphomas and other types of cancer. The murine equivalent of human γHV,
MHV68, is widely used as an animal model for γHV infection. Primary infection is
associated with mucosal tissue replication, expansion in germinal center (GC) B cells and
chronic persistence in host’s memory B cells. The GC cells undergo somatic hypermutation
(SHM), class switch recombination (CSR) and repertoire selection which generate long
lived antibody-secreting plasma cells and memory B cells. It is unknown how the repertoire
of infected cells compares to that of uninfected cells within the compartment of germinal
center B cells.
We isolated and comparatively analyzed the B cell repertoire of infected and non-infected
GC cells from individual mice. This was done by sequencing repertoire from both the single
cells and GC populations using NGS high-throughput sequencing. We determined the
effect of infection on diversity, selection, mutagenesis and expansion of clones within the
GC population of a mouse. Our data indicate that repertoire from MHV68 infected B cell
are distinct from noninfected GC cells and is characterized by unique clones, repertoire
bias and inappropriate expansion of certain clones. Moreover, both B cell populations
share CDR3 variants only on minimum level. Thus, our findings demonstrate that viral
infection cause a subversion of GC mutagenic and selective processes which result in a
skewed repertoire.
Defective EBV Particles Equipped with Latent Epitopes Offer
Increased Protection Against EBV Infection
Dwain van Zyl1,2,3,4, Ming-Han Tsai1,3,4,5, Anatoliy Shumilov1,3,4, Viktor Schneidt1,2,3,4, Rémy
Poirey1,3,4,Bettina Schlehe6, Herbert Fluhr6, Josef Mautner7, Henri-Jacques Delecluse1,3,4
1German Cancer Research Center, 2Faculty of Biosciences, Heidelberg University, 3Institut National
de la Santé et de la Recherche Médicale (INSERM) Unit U1074, 4German Center for Infection
Research (DZIF), 5Institute of Microbiology and Immunology, National Yang-Ming University,
6Frauenklinik, University Hospital Heidelberg, 7Children’s Hospital, Technische Universität
München, & Helmholtz Zentrum München
The Epstein-Barr virus (EBV) infects approximately 95% of the adult population, is the
primary cause of infectious mononucleosis and has been implicated in the development of
several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that
comprises virus lytic replication and several latency programs. Considering EBV infection
holistically, we rationalised that a prophylactic EBV vaccine should prime the immune
system against lytic and latent proteins. To this end, we have engineered DNA-free EBV
virus-like particles and light particles (VLPs/LPs) to contain latent proteins. This was
achieved by fusing latent antigens to the major tegument protein of EBV, enabling their
incorporation into VLPs/LPs. In vitro and ex vivo analysis confirmed that the modified
VLPs/LPs were capable of stimulating CD4+ T cells specific for lytic and latent proteins.
Moreover, T cells stimulated ex vivo with modified VLPs/LPs were shown to be cytolytic
and capable of restricting EBV-infected cells. Vaccination of humanised mice with
VLPs/LPs containing EBNA1 afforded significantly higher protection against wild-type EBV.
This suggests that increasing the spectrum of EBV antigens included in a vaccine will
enhance its ability to confer sterile immunity.
Leukemia. 2018 Jun 20.
Antibodies conjugated with viral antigens elicit a cytotoxic T cell response against primary CLL ex vivo.
Schneidt V1,2,3,4, Ilecka M1,3,4, Dreger P5,
van Zyl DG1,2,3,4, Fink S1,3,4,6, Mautner J7,8,9, Delecluse HJ10,11,12,13.
Abstract
Chronic lymphocytic leukemia (CLL) is the most frequent B cell malignancy in Caucasian adults. The therapeutic armamentarium against this incurable disease has recently seen a tremendous expansion with the introduction of specific pathway inhibitors and innovative immunotherapy. However, none of these approaches is curative and devoid of side effects.
We have used B-cell-specific antibodies conjugated with antigens (AgAbs) of the Epstein-Barr virus (EBV) to efficiently expand memory CD4+ cytotoxic T lymphocytes (CTLs) that recognized viral epitopes in 12 treatment-naive patients with CLL.
The AgAbs carried fragments from the EBNA3C EBV protein that is recognized by the large majority of the population. All CLL cells pulsed with EBNA3C-AgAbs elicited EBV-specific T cell responses, although the intensity varied across the patient collective. Interestingly, a large proportion of the EBV-specific CD4+ T cells expressed granzyme B (GrB), perforin, and CD107a, and killed CLL cells loaded with EBV antigens with high efficiency in the large majority of patients. The encouraging results from this preclinical ex vivo study suggest that AgAbs have the potential to redirect immune responses toward CLL cells in a high percentage of patients in vivo and warrant the inception of clinical trials.
PMID: 29925906
Candidate Public T-Cell Responses to Kaposi Sarcoma
Identified through T-Cell Receptor Sequencing of Tumor
Infiltrating Lymphocytes
Warren Phipps1,2, Andrea Towlerton1, David Coffey1,2, Nixon Niyonzima3, Edus H Warren1,2
1Fred Hutchinson Cancer Research Center, 2University of Washington, 3Uganda Cancer Institute
BACKGROUND: Kaposi sarcoma (KS) development is strongly associated with immune
dysfunction in the context of HIV infection, but little is known about T-lymphocyte
responses against KS tumor cells or human herpesvirus-8 (HHV-8), the viral cause of KS.
By comparing the composition and dynamics of the T-cell repertoire of tumor-infiltrating
lymphocytes (TIL) in KS tumor samples from patients with and without favorable response
to therapy, we aim to identify TIL characteristics associated with tumor regression.
METHODS: High-throughput sequencing of the T-cell receptor β chain (TRB) locus was
performed of TIL from 1-2 pre-treatment and 1-2 post-treatment KS tumors and in
corresponding normal skin samples, which were obtained from HIV-infected adults with KS
receiving care at the Uganda Cancer Institute in Kampala, Uganda. We compared the TRB
repertoire observed in serially collected tumors and in the corresponding normal skin
samples to identify TRB sequences carried in candidate tumor-reactive T cells.
RESULTS: TRB sequencing has been performed to date on 182 KS tumors and 46
corresponding normal skin samples obtained from 65 HIV-infected adults with KS. TRB
repertoire analysis has identified 31 unique TRB CDR3 sequences that were (i) observed
in 10 or more different biopsies, and (ii) not observed in a large control population of
healthy adults. Unsupervised hierarchical cluster analysis of the tumors containing these
31 sequences revealed that all of sequences were detected in multiple biopsies from
individual patients and in different patients sharing class I MHC-alleles. These
observations suggest that these sequences may be markers for public HHV-8-specific T
cell responses.
CONCLUSIONS: Our data support the existence of public T-cell responses in KS TIL,
which could have significant therapeutic value. Improved understanding of how cellular
immune responses are associated with KS tumor regression may ultimately enhance KS
staging approaches and guide use of increasingly targeted and effective immune-based
therapies.
