Prof Paul Klenerman
|Technology Exchange:||Cell sorting|
|Scientific Themes:||Immunology & Infectious Disease|
|Keywords:||Hepatitis C virus, HIV, T cells, virus, liver and flow cytometry|
Infectious diseases such as HIV, hepatitis B and C, affect hundreds of millions of people worldwide. Our group works on the immune response to these infections, focusing on both the host and the pathogen. As there is currently no vaccine to prevent hepatitis C infection, many of our projects have addressed aspects of chronic hepatitis C virus (HCV) infection and vaccine responses. In addition, our group also looks at a range of viruses and bacteria. Overall our main contributions to date have been to define mechanisms of viral persistence, including: T cell escape, antagonism, original antigen sin, integration of non-retroviral RNA viruses; to define the key features of successful immune responses against HCV, leading to trials of a T cell vaccine; and to define the distinctive CD161+ T cell population, which dominates in the human liver.
We are currently working on three main strands of research:
1. CD161++/MAIT cell biology. These lymphocytes, which are abundant in human blood and highly enriched in the liver, have the capacity to respond to both bacterial and inflammatory signals. We are trying to understand their in vivo role in host defence and immunopathology, through the analysis of patients and in vitro studies of function and activation.
2. HCV immune defence. In collaboration with the groups of Ellie Barnes and Adrian Hill, we are involved in vaccine studies using adenoviral vectors to track vaccine-induced cells and analyse their capacity to recognise viral variants. Upcoming studies include an analysis of host responses in relation to novel drug therapies and the use of new viral sequencing approaches to define the impact of host immunity.
3. Memory inflation. Some persistent virus infections induce a striking host response, which we have termed memory "inflation"; this includes the generation of very large functional T cell populations, which can increase with time. While this was first noted in cytomegalovirus infections, we have found a similar profile of cells after adenoviral vector vaccination. Our group is working to define the qualities of these induced cells, and the critical factors that drive memory inflation.
|Dr. Georg Lauer||Gastroenterology||Harvard||United States|
|Robert Thimme||University Hospital Freiburg||Germany|
|Mala Maini||UCL||United Kingdom|
|Prof Jonathan Flint||Wellcome Trust Centre for Human Genetics||University of Oxford||United Kingdom|
|Prof Ellie (Eleanor) Barnes||Experimental Medicine Division||University of Oxford||United Kingdom|
|Prof Chris A O'Callaghan||Centre for Cellular and Molecular Physiology||University of Oxford||United Kingdom|
|Dr Nick Haining||Harvard||United States|
|Ray T Chung||Harvard||United States|
|John Wherry||U Penn||United States|
|Ramon Arens||University of Leiden||Netherlands|
|Andrea Cox||Johns Hopkins||United States|
|Gordon Freeman||Harvard||United States|
|Prof Constantino Lopez-Macias||IMSS||Mexico|
The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage. Hide abstract
A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8(+) and CD4(+) HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine. Hide abstract
BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma. Hide abstract
Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1. MAIT cells are activated by a broad range of bacteria through detection of riboflavin metabolites bound by MR1, but their direct cytolytic capacity upon recognition of cognate target cells remains unclear. We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK. Bacterial activation of MAIT cells rapidly induced GrB and perforin, licensing these cells to kill their cognate target cells. Using a novel flow cytometry-based killing assay, we show that licensed MAIT cells, but not ex vivo MAIT cells from the same donors, can efficiently kill Escherichia coli-exposed B-cell lines in an MR1- and degranulation-dependent manner. Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation. The tightly regulated cytolytic capacity of MAIT cells may have an important role in the control of intracellular bacterial infections, such as Mycobacterium tuberculosis. Hide abstract
During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity. Hide abstract
Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV. Hide abstract
HIV can be partially contained by host immunity and understanding the basis of this may inform vaccine design. The importance of B-cell function in long-term control is poorly understood. One method of investigating this is in vivo cellular depletion. In this study, we take advantage of a unique opportunity to investigate the role of B cells in an HIV-infected patient. The HIV-1(+) patient studied here was not taking antiretroviral drugs and was treated for pre-existing low-grade lymphoplasmacytoid lymphoma by depletion of CD20+ B cells using rituximab. We demonstrate that B-cell depletion results in a decline in autologous neutralizing antibody (NAb) responses and a 1.7 log(10) rise in HIV-1 plasma viral load (pVL). The recovery of NAbs results in a decline in pVL. The HIV-1 sequences diversify and NAb-resistant mutants are subsequently selected. These data suggest that B-cell function can contribute to the long-term control of pVL, and that NAbs may be more important in controlling chronic HIV-1 infection than previously suspected. Hide abstract
UNLABELLED: Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels. CONCLUSION: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus-host interaction which may be highly relevant to pathogenesis in vivo. Hide abstract
CD8(+) T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor gamma-t, P = 6 x 10(-9); RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 x 10(-7); IL-18 receptor, P = 4 x 10(-6)), and chemokine receptors (e.g., CCR6, P = 3 x 10(-8); CXCR6, P = 3 x 10(-7); CCR2, P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man. Hide abstract
UNLABELLED: Hepatitis C virus (HCV) causes chronic infection accompanied by a high risk of liver failure and hepatocellular carcinoma. CD8+ T cell responses are important in the control of viremia. However, the T cell response in chronic infection is weak both in absolute numbers and in the range of epitopes targeted. In order to explore the biology of this response further, we analyzed expression of a panel of natural killer cell markers in HCV compared with other virus-specific T cell populations as defined by major histocompatibility complex class I tetramers. We found that CD161 was significantly expressed on HCV-specific cells (median 16.8%) but not on CD8+ T cells specific for human immunodeficiency virus (3.3%), cytomegalovirus (3.4%), or influenza (3.4%). Expression was seen in acute, chronic, and resolved disease and was greatest on intrahepatic HCV-specific T cells (median 57.6%; P < 0.05). Expression of CD161 was also found on hepatitis B virus-specific CD8+ T cells. In general, CD161+CD8+ T cells were found to be CCR7- "effector memory" T cells that could produce proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) but contained scanty amounts of cytolytic molecules (granzyme B and perforin) and proliferated poorly in vitro. Expression of CD161 on CD8+ T cells was tightly linked to that of CXCR6, a chemokine with a major role in liver homing. CONCLUSION: We propose that expression of CD161 indicates a unique pattern of T cell differentiation that might help elucidate the mechanisms of HCV immunity and pathogenesis. Hide abstract
Blood donors are routinely screened for hepatitis C virus infection. Some individuals have weak or restricted virus-specific antibody responses, and are classed as indeterminate. Such donors are almost always negative for viral RNA in blood. We postulated that previous transient virus exposure might account for some of these cases. With sensitive ex-vivo analyses of T-cell responses, we identified virus-specific responses in 15 of 30 indeterminate blood donors tested, compared with none in controls (p=0.0013). Additionally, these responses were typically focused on core-derived peptides. These findings suggest previous exposure to the virus in many indeterminate blood donors. Hide abstract
Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-gamma, a "stunned" phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection. Hide abstract
Infection of adult mice with lymphocytic choriomeningitis virus (LCMV), a non-cytopathic segmented RNA virus, leads initially to generalized infection, followed by clearance and subsequent life-long immunity. Indirect evidence has suggested that viral antigens may persist in lymphoid tissues during the phase of immunological memory, but viral genomic RNA has not been detected in previous studies. During a search for persistent virus in the spleen, we identified LCMV-specific sequences present as DNA by polymerase chain reaction (PCR) in mice over 200 days after infection. In vivo and in vitro studies revealed that reverse transcription of viral RNA into complementary DNA occurred after acute infection of cells of its natural hosts, mouse and hamster, but not of other species and could be inhibited in vitro by azidothymidine (AZT), indicating that this was mediated by endogenous reverse transcriptase activity. These findings reveal a surprising and new pathway of interaction between exogenous RNA viruses and endogenous retroviral, and perhaps other host components, that results in the persistence of virally determined DNA. We speculate that the latter may function in vivo as a form of DNA vaccine. Hide abstract
Most asymptomatic individuals infected with HIV-1 have a cytotoxic T lymphocyte (CTL) response to the virus Gag proteins which can be demonstrated in vitro. Epitopes have been mapped in p17 Gag and p24 Gag restricted by HLA-B8 (p17-3 and p24-13) and -B27 (p24-14). Viruses isolated from patients who make CTL responses to these peptides vary within the genetic sequences encoding these epitopes and some mutations lead to reduction in killing activity in vitro. This was attributed to either failure of the variant epitope to bind major histocompatibility complex class I or failure of T-cell receptors to bind the presented peptide. But peptide variants of class I-restricted epitopes cause 'antagonism', that is, the presence of a variant epitope (in the form of peptide) inhibits normal lysis of targets presenting the original epitope. This mirrors similar findings in class II-restricted systems. Here we report that naturally occurring variant forms of p17-3, p24-13 and p24-14 may cause antagonism of CTL lines derived from the same individuals. The effect is present if the epitopes are derived from synthetic peptides and when they are processed from full-length proteins expressed by either recombinant vaccinia constructs or replicating HIV. Hide abstract
Activation and function of human CD161++/MAIT cells and microbial defence
CD161++/MAIT cells make up 1 in 6 circulating CD8+ T cells and up to half of CD8+ T cells in the liver during chronic inflammation, such as hepatitis C. These unique cells sit at the bridge between innate and adaptive immunity, capable of secreting a broad range of cytokines, and readily induced to kill infected cells. They can be activated in two ways – one by innate signals such as the cytokines IL-12 and IL-18, and also through their TCR, which binds the conserved MHC Class I like molecule ...
Defining RORγt-dependent transcriptional networks in human Th17 and MAIT cells
This project aims to understand how subsets of human white cells are controlled and how we can intervene to affect their function using novel small molecules. We are focusing on cytokine IL-17 producing cells including Th17 and a recently identified group of T lymphocytes called Mucosal Associated Invariant T cells (MAIT cells) which are both an important subset of human T cells and disease-related.
Translating functions of CRTH2 into therapeutic pathways to treat allergic disorders
Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is selectively expressed in certain pro-inflammatory immune cells including eosinophils, basophils, TH2 cells, and group 2 innate lymphoid cells. The aims of this project are: to understand the roles of CRTH2 in these cells and allergic diseasesto investigate the mechanisms mediating these rolesto develop CRTH2 as a novel therapeutic strategy to control allergic diseases
Defining the immune control of Hepatitis B virus (HBV) infection through HBV genomic and T cell mapping technologies; implications for immunotherapy.
HBV infects millions of people globally and is the leading cause of liver cirrhosis and hepatocellular cancer. Drug therapies are available to suppress viral replication, but in most cases therapy must be continued indefinitely. This means they are not used in resource poor setting where the burden of disease is highest.Over recent years our laboratory has built up considerable expertise in the fine mapping and functional analysis of T cell immune responses to pathogens. Our observations have ...