9 10
1Gastrointestinal Unit and Liver Center, Massachusetts General Hospital; Harvard Medical School; 11
Boston, MA, 02114; USA. 12
2Inserm, U1110; Institut de Recherche sur les Maladies Virales et Hépatiques; Strasbourg, 67000; 13
France. 14
3Université de Strasbourg; Strasbourg, 67081; France. 15
4Laboratory of Viral Hepatitis, Oswaldo Cruz Institute; FIOCRUZ, Rio de Janeiro, 21040; Brazil. 16
5Institute for Virology, University Hospital Düsseldorf; Heinrich Heine University; Düsseldorf, 40225; 17
Germany. 18
6Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, 02115; USA. 19
7Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and 20
Harvard University; Boston, MA, 02139; USA. 21
8Division of Infectious Disease; Massachusetts General Hospital; Boston, MA, 02114, USA; 22
9Institut Hospitalo-Universitaire, Pôle Hépato-digestif; Nouvel Hôpital Civil; Strasbourg, 67000; 1 France. 2 10Lead Contact 3 4 Correspondence to: 5
Georg M. Lauer, Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, 6
55 Fruit Street, Boston, MA 02114 7
glauer@mgh.harvard.edu 8
9 10
Keywords
1
Hepatitis C Virus, CD8 T cells, CD4 T cell help, T cell dysfunction, metabolism, nucleosome, adaptive 2
immunity, viral escape, network analysis, transcriptional regulation 3
4
Highlights
5
Transcriptional differences in T cells are associated with outcome and viral escape 6
T cells from different outcomes share a core of co-regulated T cell identity genes 7
Metabolic, nucleosome, and immune genes are dysregulated early in chronic infection 8
T cell dysregulation correlates with sex, age, and availability of CD4 T cell help 9
10
Summary
11
Distinct molecular pathways govern the differentiation of CD8 effector T cells into memory or 12
exhausted T cells in acute and chronic viral infection and cancer. Utilizing an integrative network and 13
differential expression-based systems approach, we investigated early transcriptional events that direct 14
the early CD8 T cell response in dichotomous outcomes of human hepatitis C virus infection. We 15
identify the core of a common T cell identity, but also highly exclusive regulatory interaction networks 16
highlighting dysregulation of crucial metabolic functions early on during persistent infection, together 17
with changes in expression of genes related to nucleosomal regulation of transcription, T cell 18
differentiation, and the inflammatory response, with associations to subject age, sex and levels of 19
available CD4 T cell help. These data identify critical pathways of T cell regulation and provide a 20
framework for investigating immune responses in human subjects. 21
Introduction
1
Over the last two decades we have gained important insights into the defining features of T cell 2
responses in the context of controlled and chronic viral infections. These advances, most notably the 3
differentiation between effective T cell memory and functionally exhausted T cells (Wherry and 4
Kurachi, 2015) have largely been driven by work performed in animal models such as lymphocytic 5
choriomeningitis virus (LCMV) infection in mice, and have had a major impact on our understanding 6
of T cell immunology beyond the scope of viral infections (Pardoll, 2012). The key concepts from 7
these studies have been successfully translated in many human infections, including those with human 8
immunodeficiency virus (HIV), hepatitis B virus (HBV), and HCV (Kahan et al., 2015). Nevertheless, 9
our understanding of how different molecular mechanisms integrate into a concerted program leading 10
to an effective or failing T cell response remains incomplete. This is especially true in the context of 11
human infections, where genetic diversity and variability, differences in age and comorbidities, as well 12
as environmental factors influence both the immune response and the outcome of infection 13
significantly (Brodin and Davis, 2016). Recent advances in sensitivity and resolution of available 14
molecular and immunological assays now allows for comprehensive investigation of fundamental 15
research questions directly in the human immune system. 16
Hepatitis C virus infection presents an exceptional opportunity to study regulation of immune 17
responses, as it is unique among human infections in that it is spontaneously and fully resolved about 18
20% of subjects within 6-9 months of infection, even after exposure a viral inoculum that will result 19
in persistent infection in other subjects (Major et al., 2004). Data from humans as well as from the 20
chimpanzee model of infection have demonstrated that the presence of adaptive immune responses 21
by both CD4 and CD8 T cells, while indispensable to resolution of infection (Grakoui, 2003; Shoukry 22
et al., 2003), is not sufficient, as they are also detected during the early phase in subjects developing 23
chronic infection (Cox et al., 2005; Schulze zur Wiesch et al., 2012). In addition to T cells, neutralizing 1
antibodies have been shown to contribute to control of the virus during the acute phase of infection, 2
where their induction is correlated with viral clearance (Osburn et al., 2014; Pestka et al., 2007). A 3
strong host component to viral control, evident also in associations between HLA expression or sex 4
with infection outcome (Bakr et al., 2006; Kuniholm et al., 2010), separates HCV from LCMV, the 5
classic mouse model of acute and chronic viral infection in inbred mouse strains, where different 6
courses of viral replication are only observed when animals are infected with different strains of the 7
virus. The analysis of the early anti-HCV T cell response in humans with different clinical outcomes 8
can therefore add additional insights and lend a unique perspective into the complexity of immune 9
response regulation in genetically diverse hosts. 10
Here, we define the molecular components of a successful CD8 T cell response to HCV infection and 11
contrast it with transcriptional events that govern HCV-specific CD8 T cells in persisting HCV 12
infection. We isolated HCV-specific HLA-multimer positive CD8 T cells from 43 subjects with 13
different infection outcomes at multiple time points during the initial 36 weeks of infection and 14
analyzed their transcriptional profiles by microarray. 15
Taking advantage of two important features of HCV infection, its dichotomous outcome, with chronic 16
viremia or complete clearance of the virus, and the occurrence of HCV viral sequence variation leading 17
to viral escape from the CD8 T cell response, we were able not only directly compare T cell responses 18
that control viremia to those that do not, but also differentiate between changes in T cell phenotype 19
and function mediated by prolonged T cell receptor (TCR) stimulation versus those mostly induced 20
by a chronic inflammatory environment. 21
Our results reveal a core T cell identity as well as unique outcome associated traits indicating early 22
dysregulation of HCV-specific CD8 T cell responses. Specifically, we identify major differences in 23
regulation of metabolic processes during early HCV infection together with differential expression 24
patterns of genes involved in regulation of apoptosis, T cell differentiation and the response to 1
inflammation. 2
By identifying a core of partially conserved transcriptional programs related to T cell immune function, 3
T cell metabolism and histone regulation, and contrasting them with programs that differentiate T 4
cells in three discrete immunological states, we gain important insights into critical diversions in the 5
regulation of the primary CD8 T cell response to viral infection and establish a reference framework 6
for more detailed mechanistic studies into T cell mediated viral control and T cell failure. 7
Results
1