Dr Chris Willberg

Research Area: Immunology
Technology Exchange: Cell sorting, Cellular immunology, Flow cytometry and Immunohistochemistry
Scientific Themes: Immunology & Infectious Disease
Keywords: T cells, Innate, Inflammation and Flow Cytometry
Web Links:
T cell responses

T cell responses

Germinal Centres

Germinal Centres

Germinal centre Follicular Dendritic cells

Germinal centre Follicular Dendritic cells

Tingible body macrophage

Tingible body macrophage

3D flow plot

3D flow plot

I lead a number of projects within Prof. Paul Klenerman’s group, as well as run the Flow Cytometry unit within the Peter Medawar Building.

My research interests fall into three distinct areas:

Firstly, with Prof. Paul Klenerman, we are interested in the activation and regulation of mucosal associated invariant T cells (MAIT cells). MAIT cells represent the most abundant innate-like T cell population within the human body. They play a particularly important role in our fight against bacterial infections. However, they have also been associated with a number of inflammatory disease settings, including multiple sclerosis and psoriasis. Thus, these cells have the potential to play important roles in both maintaining health, but also in promoting inflammation and subsequent pathology.

Secondly, I am interested in the control and regulation of the germinal centre reaction. The germinal centre is a dynamic microanatomical structure within which B cells compete for antigen presented on follicular dendritic cells. This competition results in the selection of B cells that produce high affinity antibodies, which is crucial from the development of a robust B cell response.

Finally, in line with my role as flow cytometry manager, we are continuously looking to develop and utilise novel cytometry techniques and applications. These include multi-parametric cytotoxicity assays and proliferation assays.

Name Department Institution Country
Professor Paul Klenerman Experimental Medicine Division University of Oxford United Kingdom
Professor Anna Katharina (Katja) A Simon Experimental Medicine Division University of Oxford United Kingdom
Professor Ellie (Eleanor) Barnes Experimental Medicine Division University of Oxford United Kingdom
Emeritus Professor Helen Chapel Experimental Medicine Division University of Oxford United Kingdom
Dr Teresa Marafioti UCL United Kingdom
Turtle L, Bali T, Buxton G, Chib S, Chan S, Soni M, Hussain M, Isenman H et al. 2016. Human T cell responses to Japanese encephalitis virus in health and disease. J Exp Med, 213 (7), pp. 1331-1352. | Show Abstract | Read more

Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8(+) and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4(+) and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4(+) T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4(+) and CD8(+) T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus.

Ussher JE, van Wilgenburg B, Hannaway RF, Ruustal K, Phalora P, Kurioka A, Hansen TH, Willberg CB, Phillips RE, Klenerman P. 2016. TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells. Eur J Immunol, 46 (7), pp. 1600-1614. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1-mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1-mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1-mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B-cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B-cell lines, suggesting differential regulation in different cell types. APC activation and NF-κB signaling were critical for MR1-mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1-mediated MAIT cell activation. Overall, MR1-mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.

Leitman EM, Willberg CB, De Burgh-Thomas A, Streeck H, Goulder PJ, Matthews PC. 2016. Subdominant Gag-specific anti-HIV efficacy in an HLA-B*57-positive elite controller. AIDS, 30 (6), pp. 972-974. | Read more

Fergusson JR, Hühn MH, Swadling L, Walker LJ, Kurioka A, Llibre A, Bertoletti A, Holländer G et al. 2016. CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut. Mucosal Immunol, 9 (2), pp. 401-413. | Show Abstract | Read more

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Hurst J, Hoffmann M, Pace M, Williams JP, Thornhill J, Hamlyn E, Meyerowitz J, Willberg C et al. 2015. Immunological biomarkers predict HIV-1 viral rebound after treatment interruption. Nat Commun, 6 pp. 8495. | Show Abstract | Read more

Treatment of HIV-1 infection with antiretroviral therapy (ART) in the weeks following transmission may induce a state of 'post-treatment control' (PTC) in some patients, in whom viraemia remains undetectable when ART is stopped. Explaining PTC could help our understanding of the processes that maintain viral persistence. Here we show that immunological biomarkers can predict time to viral rebound after stopping ART by analysing data from a randomized study of primary HIV-1 infection incorporating a treatment interruption (TI) after 48 weeks of ART (the SPARTAC trial). T-cell exhaustion markers PD-1, Tim-3 and Lag-3 measured prior to ART strongly predict time to the return of viraemia. These data indicate that T-cell exhaustion markers may identify those latently infected cells with a higher proclivity to viral transcription. Our results may open new avenues for understanding the mechanisms underlying PTC, and eventually HIV-1 eradication.

Baxter AE, Russell RA, Duncan CJ, Moore MD, Willberg CB, Pablos JL, Finzi A, Kaufmann DE et al. 2014. Macrophage infection via selective capture of HIV-1-infected CD4+ T cells. Cell Host Microbe, 16 (6), pp. 711-721. | Show Abstract | Read more

Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

Fergusson JR, Smith KE, Fleming VM, Rajoriya N, Newell EW, Simmons R, Marchi E, Björkander S et al. 2014. CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages. Cell Rep, 9 (3), pp. 1075-1088. | Show Abstract | Read more

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.

Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K, Kang YH, Walker LJ, Hansen TH, Willberg CB, Klenerman P. 2015. MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets. Mucosal Immunol, 8 (2), pp. 429-440. | Show Abstract | Read more

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.

Lopez-Granados E, Stacey M, Kienzler AK, Sierro S, Willberg CB, Fox CP, Rigaud S, Long HM et al. 2014. A mutation in X-linked inhibitor of apoptosis (G466X) leads to memory inflation of Epstein-Barr virus-specific T cells. Clin Exp Immunol, 178 (3), pp. 470-482. | Show Abstract | Read more

Mutations in the X-linked inhibitor of apoptosis (XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus (EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP (G466X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4(+) CD8(+) population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon (IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAP(G466X) mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease.

Jo J, Tan AT, Ussher JE, Sandalova E, Tang XZ, Tan-Garcia A, To N, Hong M et al. 2014. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver. PLoS Pathog, 10 (6), pp. e1004210. | Show Abstract | Read more

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright) mucosal-associated invariant T (MAIT) and CD56(Bright) NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

Ussher JE, Klenerman P, Willberg CB. 2014. Mucosal-associated invariant T-cells: new players in anti-bacterial immunity. Front Immunol, 5 (OCT), pp. 450. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population involved in anti-bacterial immunity. In human beings, MAIT cells are abundant, comprising ~10% of the CD8(+) T-cell compartment in blood. They are enriched at mucosal sites and are particularly prevalent within the liver. MAIT cells are defined by the expression of a semi-invariant T-cell receptor (Vα7.2-Jα33/12/20) and are restricted by the non-polymorphic, highly evolutionarily conserved MHC class Ib molecule, MHC-related protein (MR)1. MR1 has recently been shown to present an unstable pyrimidine intermediate derived from a biosynthetic precursor of riboflavin; riboflavin biosynthesis occurs in many bacteria but not in human beings. Consistent with this, MAIT cells are responsive to riboflavin-metabolizing bacteria, including Salmonella. In mouse models, MAIT cells have been shown to play a non-redundant role in anti-bacterial immunity, including against Escherichia coli, Klebsiella pneumoniae, and Mycobacterium bovis BCG. In human beings, MAIT cells are decreased in frequency in the blood of patients with tuberculosis or pneumonia, and their frequency has been inversely correlated with the risk of subsequent systemic bacterial infection in patients in intensive care. Intriguingly, MAIT cells are also depleted from the blood early in HIV infection and fail to recover with anti-retroviral therapy, which may contribute to the susceptibility of patients infected with HIV to certain bacterial infections, including non-typhoidal Salmonella. In this review, we will discuss what is currently known about MAIT cells, the role that Salmonella has played in elucidating MAIT cell restriction and function, and the role MAIT cells might play in the control of Salmonella infection.

Newey PJ, Gorvin CM, Cleland SJ, Willberg CB, Bridge M, Azharuddin M, Drummond RS, van der Merwe PA, Klenerman P, Bountra C, Thakker RV. 2013. Mutant prolactin receptor and familial hyperprolactinemia. N Engl J Med, 369 (21), pp. 2012-2020. | Show Abstract | Read more

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

Ussher JE, Bilton M, Attwod E, Shadwell J, Richardson R, de Lara C, Mettke E, Kurioka A, Hansen TH, Klenerman P, Willberg CB. 2014. CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner. Eur J Immunol, 44 (1), pp. 195-203. | Show Abstract | Read more

CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-α chain 7.2 (Vα7.2)-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) Vα7.2(+) and CD161(++) Vα7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.

Barnes E, Folgori A, Capone S, Swadling L, Aston S, Kurioka A, Meyer J, Huddart R et al. 2012. Hepatitis Hide and Seek SCIENCE TRANSLATIONAL MEDICINE, 4 (115),

Barnes E, Folgori A, Capone S, Swadling L, Aston S, Kurioka A, Meyer J, Huddart R et al. 2012. Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man. Sci Transl Med, 4 (115), pp. 115ra1. | Show Abstract | Read more

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.

Kang YH, Seigel B, Bengsch B, Fleming VM, Billerbeck E, Simmons R, Walker L, Willberg CB et al. 2012. CD161(+)CD4(+) T cells are enriched in the liver during chronic hepatitis and associated with co-secretion of IL-22 and IFN-γ. Front Immunol, 3 (NOV), pp. 346. | Show Abstract | Read more

Hepatitis C virus infection is a major cause of chronic liver disease. CD4(+) T cells play a key role in disease outcome. However, the critical functions and associated phenotypes of intrahepatic CD4(+) T cells are not well defined. We have previously shown that CD8(+) T cells expressing the C type lectin CD161 are highly enriched in the human liver, especially during chronic hepatitis. These cells are associated with a type 17 differentiation pattern and express cytokines including IL-17A, IL-22, and IFN-γ. We therefore analyzed expression of CD161 on CD4(+) T cells in blood and liver and addressed the relevant phenotype and functional capacity of these populations. We observed marked enrichment of CD161(+)CD4(+) T cells in the liver during chronic hepatitis such that they are the dominant subtype (mean 55% of CD4(+) T cells). IL-22 and IL-17 secreting CD4(+) T cells were readily found in the livers of HCV(+) and NASH donors, although not enriched compared to blood. There was, however, specific enrichment of a novel subset of IL-22/IFN-γ dual secretors (p = 0.02) compared to blood, a result reconfirmed with direct ex vivo analyses. These data indicate the dominance of CD161(+) expressing lymphocyte populations within the hepatic infiltrate, associated with a distinct cytokine profile. Given their documented roles as antiviral and hepatoprotective cytokines respectively, the impact of co-secretion of IFN-γ and IL-22 in the liver may be particularly significant.

Sharp ER, Willberg CB, Kuebler PJ, Abadi J, Fennelly GJ, Dobroszycki J, Wiznia AA, Rosenberg MG, Nixon DF. 2012. Association of differentiation state of CD4+ T cells and disease progression in HIV-1 perinatally infected children. PLoS One, 7 (1), pp. e29154. | Show Abstract | Read more

BACKGROUND: In the USA, most HIV-1 infected children are on antiretroviral drug regimens, with many individuals surviving through adolescence and into adulthood. The course of HIV-1 infection in these children is variable, and understudied. METHODOLOGY/PRINCIPAL FINDINGS: We determined whether qualitative differences in immune cell subsets could explain a slower disease course in long term survivors with no evidence of immune suppression (LTS-NS; CD4%≥25%) compared to those with severe immune suppression (LTS-SS; CD4%≤15%). Subjects in the LTS-NS group had significantly higher frequencies of naïve (CCR7+CD45RA+) and central memory (CCR7+CD45RA-) CD4+ T cells compared to LTS-SS subjects (p = 0.0005 and <0.0001, respectively). Subjects in the rapid progressing group had significantly higher levels of CD4+ T(EMRA) (CCR7-CD45RA+) cells compared to slow progressing subjects (p<0.0001). CONCLUSIONS/SIGNIFICANCE: Rapid disease progression in vertical infection is associated with significantly higher levels of CD4+ T(EMRA) (CCR7-CD45RA+) cells.

Rajoriya N, Willberg C, Seigal B, Kang Y, Phillips-Hughes J, Collier J, Barnes E, Thimme R, Klenerman P. 2011. CD161+gamma-delta T CELLS: DEFINING THEIR ROLE IN PATIENTS WITH AND WITHOUT CHRONIC HEPATITIS C GUT, 60 (Suppl 2), pp. A43-A43. | Read more

Sharp ER, Willberg CB, Kuebler PJ, Abadi J, Fennelly GJ, Dobroszycki J, Wiznia AA, Rosenberg MG, Nixon DF. 2011. Immunodominance of HIV-1 specific CD8+ T-cell responses is related to disease progression rate in vertically infected adolescents. PLoS One, 6 (7), pp. e21135. | Show Abstract | Read more

BACKGROUND: HIV-1 vertically infected children in the USA are living into adolescence and beyond with the widespread use of antiretroviral drugs. These patients exhibit striking differences in the rate of HIV-1 disease progression which could provide insights into mechanisms of control. We hypothesized that differences in the pattern of immunodomination including breadth, magnitude and polyfunctionality of HIV-1 specific CD8+ T cell response could partially explain differences in progression rate. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we mapped, quantified, and assessed the functionality of these responses against individual HIV-1 Gag peptides in 58 HIV-1 vertically infected adolescents. Subjects were divided into two groups depending upon the rate of disease progression: adolescents with a sustained CD4%≥25 were categorized as having no immune suppression (NS), and those with CD4%≤15 categorized as having severe immune suppression (SS). We observed differences in the area of HIV-1-Gag to which the two groups made responses. In addition, subjects who expressed the HLA- B*57 or B*42 alleles were highly likely to restrict their immunodominant response through these alleles. There was a significantly higher frequency of naïve CD8+ T cells in the NS subjects (p = 0.0066) compared to the SS subjects. In contrast, there were no statistically significant differences in any other CD8+ T cell subsets. The differentiation profiles and multifunctionality of Gag-specific CD8+ T cells, regardless of immunodominance, also failed to demonstrate meaningful differences between the two groups. CONCLUSIONS/SIGNIFICANCE: Together, these data suggest that, at least in vertically infected adolescents, the region of HIV-1-Gag targeted by CD8+ T cells and the magnitude of that response relative to other responses may have more importance on the rate of disease progression than their qualitative effector functions.

Sims S, Willberg C, Klenerman P. 2010. MHC-peptide tetramers for the analysis of antigen-specific T cells. Expert Rev Vaccines, 9 (7), pp. 765-774. | Show Abstract | Read more

The development of the fluorescently labeled tetrameric MHC-peptide complex has enabled the direct visualization, quantification and phenotypic characterization of antigen-specific T cells using flow cytometry and has transformed our understanding of cellular immune responses. The combination of this technology with functional assays provides many new insights into these cells, allowing investigation into their lifecycle, manner of death and effector function. In this article, we hope to provide an overview of the techniques used in the construction of these tetramers, the problems and solutions associated with them, and the methods used in the study of antigen-specific T cells. Understanding how the antigen-specific cells develop and function in different circumstances and with different pathogens will be key to understanding natural host defense, as well as vaccine design and assessment.

Billerbeck E, Kang YH, Walker L, Lockstone H, Grafmueller S, Fleming V, Flint J, Willberg CB et al. 2010. Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties. Proc Natl Acad Sci U S A, 107 (7), pp. 3006-3011. | Show Abstract | Read more

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.

Willberg CB, Garrison KE, Jones RB, Meiklejohn DJ, Spotts G, Liegler TJ, Ostrowski MA, Karlsson AC, Hecht FM, Nixon DF. 2010. Rapid progressing allele HLA-B35 Px restricted anti-HIV-1 CD8+ T cells recognize vestigial CTL epitopes. PLoS One, 5 (4), pp. e10249. | Show Abstract | Read more

BACKGROUND: The HLA-B*35-Px allele has been associated with rapid disease progression in HIV-1 infection, in contrast to the HLA-B*35-Py allele. METHODOLOGY/PRINCIPAL FINDINGS: Immune responses to two HLA-B*35 restricted HIV-1 specific CTL epitopes and their variants were followed longitudinally during early HIV-1 infection in 16 HLA-B*35+ individuals. Subjects expressing HLA-B*35-Px alleles showed no difference in response to the consensus epitopes compared to individuals with HLA-B*35-Py alleles. Surprisingly, all the HLA-B*35-Px+ individuals responded to epitope-variants even in the absence of a consensus response. Sequencing of the viral population revealed no evidence of variant virus in any of the individuals. CONCLUSIONS/SIGNIFICANCE: This demonstrates a novel phenomenon that distinguishes individuals with the HLA-B*35-Px rapid progressing allele and those with the HLA-B*35-Py slower progressing allele.

Fox J, Willberg C, Ziprin P, Goldin R, Weber J, McClure M, Klenerman P, Fidler S. 2009. The role of the gut mucosa in protection from HIV-1 in highly exposed persistently seronegative individuals (HEPS) HIV MEDICINE, 10 pp. 5-5.

Erickson AL, Willberg CB, McMahan V, Liu A, Buchbinder SP, Grohskopf LA, Grant RM, Nixon DF. 2008. Potentially exposed but uninfected individuals produce cytotoxic and polyfunctional human immunodeficiency virus type 1-specific CD8(+) T-cell responses which can be defined to the epitope level. Clin Vaccine Immunol, 15 (11), pp. 1745-1748. | Show Abstract | Read more

We measured CD8(+) T-cell responses in 12 potentially exposed but uninfected men who have sex with men by using cytokine flow cytometry. Four of the individuals screened exhibited polyfunctional immune responses to human immunodeficiency virus type 1 Gag or Vif. The minimum cytotoxic T lymphocyte epitope was mapped in one Gag responder.

Willberg CB, McConnell JJ, Eriksson EM, Bragg LA, York VA, Liegler TJ, Hecht FM, Grant RM, Nixon DF. 2008. Immunity to HIV-1 is influenced by continued natural exposure to exogenous virus. PLoS Pathog, 4 (10), pp. e1000185. | Show Abstract | Read more

Unprotected sexual intercourse between individuals who are both infected with HIV-1 can lead to exposure to their partner's virus, and potentially to super-infection. However, the immunological consequences of continued exposure to HIV-1 by individuals already infected, has to our knowledge never been reported. We measured T cell responses in 49 HIV-1 infected individuals who were on antiretroviral therapy with suppressed viral loads. All the individuals were in a long-term sexual partnership with another HIV-1 infected individual, who was either also on HAART and suppressing their viral loads, or viremic (>9000 copies/ml). T cell responses to HIV-1 epitopes were measured directly ex-vivo by the IFN-gamma enzyme linked immuno-spot assay and by cytokine flow cytometry. Sexual exposure data was generated from questionnaires given to both individuals within each partnership. Individuals who continued to have regular sexual contact with a HIV-1 infected viremic partner had significantly higher frequencies of HIV-1-specific T cell responses, compared to individuals with aviremic partners. Strikingly, the magnitude of the HIV-1-specific T cell response correlated strongly with the level and route of exposure. Responses consisted of both CD4(+) and CD8(+) T cell subsets. Longitudinally, decreases in exposure were mirrored by a lower T cell response. However, no evidence for systemic super-infection was found in any of the individuals. Continued sexual exposure to exogenous HIV-1 was associated with increased HIV-1-specific T cell responses, in the absence of systemic super-infection, and correlated with the level and type of exposure.

Semmo N, Krashias G, Willberg C, Klenerman P. 2007. Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection. J Viral Hepat, 14 (7), pp. 492-502. | Show Abstract | Read more

CD4(+) T-cell responses are important for the outcome of hepatitis C virus (HCV) infection. However, the functional status of HCV-specific CD4(+) T cells in persistent infection is poorly understood. It is generally recognized that proliferative capacity of HCV-specific CD4(+) T cells is weak or absent in persistent infection, but whether this results from deletion of antigen-specific cells or represents maintenance of antigen-specific but poorly proliferative populations is not defined. We used a set of ex vivo assays to evaluate the functionality of HCV specific CD4(+) T cells in persistent and resolved infection. Peripheral blood mononuclear cells (PBMC) from 24 prospectively recruited HCV polymerase chain reaction (PCR) positive individuals, 12 spontaneously resolved individuals (i.e. anti-HCV+, PCR-) and 11 healthy controls were analysed for interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) secretion by enzyme linked immunospot assays (ELISpot). HCV-specific CD4(+) proliferative responses of carboxy fluorescein succinimidyl ester-labelled PBMC were assessed using a sensitive single cell flow cytometric assay. Sustained IFN-gamma ELISpot responses were observed in the PCR+ group. However, proliferation of HCV-specific CD4(+) T cells in the PCR+ group was substantially reduced on a per cell basis, in parallel to IL-2 secretion, compared with responses in the PCR- group. In PCR- individuals, a strong relationship between cytokine secretion and proliferative capacity was seen. However, in PCR+ individuals, IFN-gamma secretion far exceeded proliferative capacity. During persistent HCV infection, some CD4(+) T-cell specificities appear to be lost, as measured using a range of techniques, but others, potentially important, are maintained as IFN-gamma secretors but with low proliferative capacity even using a highly sensitive assay. Such subsets may yet play a significant role in vivo and also provide a template for modulation in immunotherapeutic interventions.

Willberg CB, Pillai SK, Sharp ER, Rosenberg MG, Agudelo JD, Barbour JD, Nixon DF. 2007. Rational peptide selection to detect human immunodeficiency virus type 1-specific T-cell responses under resource-limited conditions. Clin Vaccine Immunol, 14 (6), pp. 785-788. | Show Abstract | Read more

Understanding human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses is important for the development of vaccines and therapies. We describe a novel method for the rational selection of peptides that target stable regions of the HIV-1 genome, rich in epitopes specifically recognized by the study population. This method will be of particular use under resource/sample-limited conditions.

Willberg CB, Ward SM, Clayton RF, Naoumov NV, McCormick C, Proto S, Harris M, Patel AH, Klenerman P. 2007. Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha. PLoS One, 2 (8), pp. e791. | Show Abstract | Read more

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

Willberg CB, Nixon DF. 2007. Treatment interruption in chronic HIV-1 infection: does it deliver? Curr Opin HIV AIDS, 2 (1), pp. 26-30. | Show Abstract | Read more

PURPOSE OF REVIEW: This review sets out to overview treatment interruption in chronic HIV-1 infection: what treatment interruption promised, results from recent trials, and what the future holds. RECENT FINDINGS: Recent studies have produced mixed results; several trials have been prematurely halted, whereas others have reported more positive outcomes. One consistent finding has been the identification of the CD4 T-cell count nadir as a critical parameter in determining the outcome of treatment interruption. SUMMARY: The use of treatment interruption is still controversial, but it is becoming clear that certain individuals could benefit, and partial treatment interruption strategies warrant further investigation.

Meier UC, Owen RE, Taylor E, Worth A, Naoumov N, Willberg C, Tang K, Newton P et al. 2005. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections. J Virol, 79 (19), pp. 12365-12374. | Show Abstract | Read more

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.

Clayton RF, Rinaldi A, Kandyba EE, Edward M, Willberg C, Klenerman P, Patel AH. 2005. Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int, 25 (2), pp. 389-402. | Show Abstract | Read more

BACKGROUND: Liver cell lines closely resembling primary hepatocyte are essential for research on hepatitis viruses and hepatocyte function. Currently used cell lines are derived from hepatic tumours and have altered gene expression. AIMS: The generation and characterisation of novel human hepatocyte lines (HHLs) derived from healthy human liver, retaining the primary hepatocyte phenotype. RESULTS: Primary hepatocytes were immortalised with Moloney's mouse leukaemia virus expressing E6 and E7 proteins of human papillomavirus, and cultures propagated long-term. All HHLs contained markers of hepatocyte and biliary phenotype (cytokeratins 7, 8, 18 and 19), Cytochrome P450 and albumin. The HHLs did not express high levels of p53 or alpha-fetoprotein. When grown in a collagen sandwich culture, or at the air-liquid interface, HHLs were maintained as monolayer whereas Huh-7 and HepG2 formed thick layers. All HHLs showed increased capacity to bind recombinant hepatitis C virus-like particles in comparison with Huh-7 and HepG2. We also demonstrate that HHLs contained active gap junctions, and that the cells respond to stimulation with IFN-alpha by upregulation of major histocompatibility complex (MHC)-I and -II. CONCLUSIONS: These HHLs retain primary hepatocyte phenotype and should be useful for investigating mechanisms of entry and replication of hepatotropic viruses, and should also be valuable in the study of hepatocyte biology and pathology.

Hansasuta P, Dong T, Thananchai H, Weekes M, Willberg C, Aldemir H, Rowland-Jones S, Braud VM. 2004. Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific. Eur J Immunol, 34 (6), pp. 1673-1679. | Show Abstract | Read more

The recognition of MHC class I molecules by killer cell immunoglobulin-like receptors (KIR) is central to the control of NK cell function and can also modulate the CTL activation threshold. Among KIR receptors, KIR3DL2 is thought to interact with HLA-A3 and -A11, although direct evidence has been lacking. In this study, we show that HLA-A3 and -A11 tetramers specifically bind to KIR3DL2*001 transfectants and that this recognition is peptide-specific. Single amino acid substitutions in the nonamer peptide underline a critical role for residue 8 in recognition of KIR3DL2. However, the role of this interaction in vivo still remains to be established.

Lucas M, Vargas-Cuero AL, Lauer GM, Barnes E, Willberg CB, Semmo N, Walker BD, Phillips R, Klenerman P. 2004. Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8+ T cells. J Immunol, 172 (3), pp. 1744-1753. | Show Abstract

Recent studies using MHC class I tetramers have shown that CD8(+) T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8(+) T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8(+) T cells between these groups. In the HCV-infected cohort, CMV-specific CD8(+) T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8(+) T cell population, a novel feature that may be a hallmark of this infection.

Willberg C, Barnes E, Klenerman P. 2003. HCV immunology--death and the maiden T cell. Cell Death Differ, 10 Suppl 1 (S1), pp. S39-S47. | Show Abstract | Read more

Cellular immune responses play an important role in the control of hepatitis C virus (HCV), although in the majority of cases they ultimately fail. We examine the mechanisms by which virus-specific T cells may interact with a cell that is infected with HCV and how this interaction may explain the success and failure of the immune response. As an infected cell presenting foreign antigen, the hepatocyte will interact with a large number of lymphocytes, both by direct cell to cell contact and by indirect means through the secretion of cytokines and chemokines. These interactions may lead on the one hand to the death of infected hepatocytes or suppression of viral replication and on the other hand to the death of T lymphocytes or down regulation of their function. We suggest that activation of lymphocytes in lymphoid organs leads to generation of effector T cells (positive loop), while at the same time presentation of antigen in the liver either on hepatocytes or other specialised antigen presenting cells depresses these responses (negative loop). This model helps to explain both the specific phenotype and low frequencies of HCV specific CTL in chronic infection, through early elimination of cells before expansion and maturation can occur. The outcome of HCV infection is likely to result from the early balance between these two simultaneous loops.

García P, Llano M, de Heredia AB, Willberg CB, Caparrós E, Aparicio P, Braud VM, López-Botet M. 2002. Human T cell receptor-mediated recognition of HLA-E. Eur J Immunol, 32 (4), pp. 936-944. | Show Abstract | Read more

The HLA-E class Ib molecule presents hydrophobic peptides derived from the leader sequences of other class I molecules, constituting the ligands for CD94/NKG2 lectin-like receptors. Along the course of our studies on human CD94+ T cells, we characterized an alpha beta CD8+CD94/NKG2C+ CTL clone (K14). In cytolytic assays against the murine TAP-deficient RMA-S cells transfected with human beta2 microglobulin and HLA-E (RMA-S/HLA-E), loaded with different synthetic peptides, K14 displayed a pattern of specific recognition distinct to that observed in CD94/NKG2C+ NK clones tested in parallel. RMA-S/HLA-E cells loaded with some but not all HLA class I leader sequence peptides were efficiently recognized by K14 but not by CD94/NKG2C clones, andvice versa. Remarkably, K14 also reacted with HLA-E loaded with a peptide derived from the BZLF-1 Epstein-Barr virus protein. Anti-CD94 mAb did not prevent K14 cytotoxicity against RMA-S/HLA-E cells, whereas incubation with anti-clonotypic mAb specific for the K14 TCR markedly inhibited lysis. Soluble HLA-E tetramers refolded with different peptides (i.e. VMAPRTVLL, VMAPRTLIL, VMAPRTLFL) specifically stained K14 cells. HLA-E tetramer binding was minimally reduced by pretreatment with anti-CD94 mAb alone, but was completely prevented in combination with anti-clonotypic mAb. Altogether, the data unequivocally imply the generation of human T cells potentially recognizing through the alpha beta TCR HLA-E molecules that bind to class I- and virus-derived peptides.

Mackenzie CR, Willberg CB, Däubener W. 1998. Inhibition of group B streptococcal growth by IFN gamma-activated human glioblastoma cells. J Neuroimmunol, 89 (1-2), pp. 191-197. | Show Abstract | Read more

Group B streptococci are the most important bacteria inducing neonatal septicemia and meningitis. The aim of this study was to assess the role of IFNgamma in the induction of anti-microbial effector mechanisms in human brain tumor cells. Different human glioblastoma/astrocytoma cell lines, stimulated with IFNgamma, restricted the growth of group B streptococci. In addition, we found that TNF alpha is able to enhance the IFNgamma-mediated anti-microbial effect. In contrast to group B streptococci, other bacteria which are also capable of inducing meningitis, like E. coli and all but one of the tested Streptococcus pneumoniae strains, were not influenced by the IFNgamma treated cells. We found that the IFNgamma or the IFNgamma/TNF alpha induced activation of indoleamine 2,3-dioxygenase is responsible for the inhibition of streptococcal growth, since the addition of supplemental L-tryptophan completely blocks the IFNgamma induced bacteriostasis.

van Wilgenburg B, Scherwitzl I, Hutchinson EC, Leng T, Kurioka A, Kulicke C, de Lara C, Cole S et al. 2016. MAIT cells are activated during human viral infections. Nat Commun, 7 pp. 11653. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Llibre A, López-Macías C, Marafioti T, Mehta H, Partridge A, Kanzig C, Rivellese F, Galson JD et al. 2016. LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation. J Immunol, 196 (5), pp. 2085-2094. | Show Abstract | Read more

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans.

Ussher JE, Phalora P, Cosgrove C, Hannaway RF, Rauch A, Günthard HF, Goulder P, Phillips RE, Willberg CB, Klenerman P. 2015. Molecular Analyses Define Vα7.2-Jα33+ MAIT Cell Depletion in HIV Infection: A Case-Control Study. Medicine (Baltimore), 94 (29), pp. e1134. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an abundant antibacterial innate-like lymphocyte population. There are conflicting reports as to their fate in HIV infection. The objective of this study was to determine whether MAIT cells are truly depleted in HIV infection. In this case-control study of HIV-positive patients and healthy controls, quantitative real-time polymerase chain reaction was used to assess the abundance of messenger RNA (mRNA) and genomic DNA (gDNA) encoding the canonical MAIT cell T cell receptor (Vα7.2-Jα33). Comparison was made with flow cytometry. Significant depletion of both Vα7.2-Jα33 mRNA and gDNA was seen in HIV infection. Depletion of Vα7.2+CD161++ T cells was confirmed by flow cytometry. In HIV infection, the abundance of Vα7.2-Jα33 mRNA correlated most strongly with the frequency of Vα7.2+CD161++ cells. No increase was observed in the frequency of Vα7.2+CD161- cells among CD3+CD4- lymphocytes. MAIT cells are depleted from blood in HIV infection as confirmed by independent assays. Significant accumulation of a CD161- MAIT cell population is unlikely. Molecular approaches represent a suitable alternative to flow cytometry-based assays for tracking of MAIT cells in HIV and other settings.

Ussher JE, van Wilgenburg B, Hannaway RF, Ruustal K, Phalora P, Kurioka A, Hansen TH, Willberg CB, Phillips RE, Klenerman P. 2016. TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells. Eur J Immunol, 46 (7), pp. 1600-1614. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1-mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1-mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1-mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B-cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B-cell lines, suggesting differential regulation in different cell types. APC activation and NF-κB signaling were critical for MR1-mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1-mediated MAIT cell activation. Overall, MR1-mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.

Fergusson JR, Smith KE, Fleming VM, Rajoriya N, Newell EW, Simmons R, Marchi E, Björkander S et al. 2014. CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages. Cell Rep, 9 (3), pp. 1075-1088. | Show Abstract | Read more

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.

Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K, Kang YH, Walker LJ, Hansen TH, Willberg CB, Klenerman P. 2015. MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets. Mucosal Immunol, 8 (2), pp. 429-440. | Show Abstract | Read more

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.

Jo J, Tan AT, Ussher JE, Sandalova E, Tang XZ, Tan-Garcia A, To N, Hong M et al. 2014. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver. PLoS Pathog, 10 (6), pp. e1004210. | Show Abstract | Read more

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright) mucosal-associated invariant T (MAIT) and CD56(Bright) NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

Ussher JE, Klenerman P, Willberg CB. 2014. Mucosal-associated invariant T-cells: new players in anti-bacterial immunity. Front Immunol, 5 (OCT), pp. 450. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population involved in anti-bacterial immunity. In human beings, MAIT cells are abundant, comprising ~10% of the CD8(+) T-cell compartment in blood. They are enriched at mucosal sites and are particularly prevalent within the liver. MAIT cells are defined by the expression of a semi-invariant T-cell receptor (Vα7.2-Jα33/12/20) and are restricted by the non-polymorphic, highly evolutionarily conserved MHC class Ib molecule, MHC-related protein (MR)1. MR1 has recently been shown to present an unstable pyrimidine intermediate derived from a biosynthetic precursor of riboflavin; riboflavin biosynthesis occurs in many bacteria but not in human beings. Consistent with this, MAIT cells are responsive to riboflavin-metabolizing bacteria, including Salmonella. In mouse models, MAIT cells have been shown to play a non-redundant role in anti-bacterial immunity, including against Escherichia coli, Klebsiella pneumoniae, and Mycobacterium bovis BCG. In human beings, MAIT cells are decreased in frequency in the blood of patients with tuberculosis or pneumonia, and their frequency has been inversely correlated with the risk of subsequent systemic bacterial infection in patients in intensive care. Intriguingly, MAIT cells are also depleted from the blood early in HIV infection and fail to recover with anti-retroviral therapy, which may contribute to the susceptibility of patients infected with HIV to certain bacterial infections, including non-typhoidal Salmonella. In this review, we will discuss what is currently known about MAIT cells, the role that Salmonella has played in elucidating MAIT cell restriction and function, and the role MAIT cells might play in the control of Salmonella infection.

Ussher JE, Bilton M, Attwod E, Shadwell J, Richardson R, de Lara C, Mettke E, Kurioka A, Hansen TH, Klenerman P, Willberg CB. 2014. CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner. Eur J Immunol, 44 (1), pp. 195-203. | Show Abstract | Read more

CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-α chain 7.2 (Vα7.2)-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) Vα7.2(+) and CD161(++) Vα7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.

Barnes E, Folgori A, Capone S, Swadling L, Aston S, Kurioka A, Meyer J, Huddart R et al. 2012. Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man. Sci Transl Med, 4 (115), pp. 115ra1. | Show Abstract | Read more

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.

Sharp ER, Willberg CB, Kuebler PJ, Abadi J, Fennelly GJ, Dobroszycki J, Wiznia AA, Rosenberg MG, Nixon DF. 2011. Immunodominance of HIV-1 specific CD8+ T-cell responses is related to disease progression rate in vertically infected adolescents. PLoS One, 6 (7), pp. e21135. | Show Abstract | Read more

BACKGROUND: HIV-1 vertically infected children in the USA are living into adolescence and beyond with the widespread use of antiretroviral drugs. These patients exhibit striking differences in the rate of HIV-1 disease progression which could provide insights into mechanisms of control. We hypothesized that differences in the pattern of immunodomination including breadth, magnitude and polyfunctionality of HIV-1 specific CD8+ T cell response could partially explain differences in progression rate. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we mapped, quantified, and assessed the functionality of these responses against individual HIV-1 Gag peptides in 58 HIV-1 vertically infected adolescents. Subjects were divided into two groups depending upon the rate of disease progression: adolescents with a sustained CD4%≥25 were categorized as having no immune suppression (NS), and those with CD4%≤15 categorized as having severe immune suppression (SS). We observed differences in the area of HIV-1-Gag to which the two groups made responses. In addition, subjects who expressed the HLA- B*57 or B*42 alleles were highly likely to restrict their immunodominant response through these alleles. There was a significantly higher frequency of naïve CD8+ T cells in the NS subjects (p = 0.0066) compared to the SS subjects. In contrast, there were no statistically significant differences in any other CD8+ T cell subsets. The differentiation profiles and multifunctionality of Gag-specific CD8+ T cells, regardless of immunodominance, also failed to demonstrate meaningful differences between the two groups. CONCLUSIONS/SIGNIFICANCE: Together, these data suggest that, at least in vertically infected adolescents, the region of HIV-1-Gag targeted by CD8+ T cells and the magnitude of that response relative to other responses may have more importance on the rate of disease progression than their qualitative effector functions.

Sims S, Willberg C, Klenerman P. 2010. MHC-peptide tetramers for the analysis of antigen-specific T cells. Expert Rev Vaccines, 9 (7), pp. 765-774. | Show Abstract | Read more

The development of the fluorescently labeled tetrameric MHC-peptide complex has enabled the direct visualization, quantification and phenotypic characterization of antigen-specific T cells using flow cytometry and has transformed our understanding of cellular immune responses. The combination of this technology with functional assays provides many new insights into these cells, allowing investigation into their lifecycle, manner of death and effector function. In this article, we hope to provide an overview of the techniques used in the construction of these tetramers, the problems and solutions associated with them, and the methods used in the study of antigen-specific T cells. Understanding how the antigen-specific cells develop and function in different circumstances and with different pathogens will be key to understanding natural host defense, as well as vaccine design and assessment.

Billerbeck E, Kang YH, Walker L, Lockstone H, Grafmueller S, Fleming V, Flint J, Willberg CB et al. 2010. Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties. Proc Natl Acad Sci U S A, 107 (7), pp. 3006-3011. | Show Abstract | Read more

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.

Willberg CB, Garrison KE, Jones RB, Meiklejohn DJ, Spotts G, Liegler TJ, Ostrowski MA, Karlsson AC, Hecht FM, Nixon DF. 2010. Rapid progressing allele HLA-B35 Px restricted anti-HIV-1 CD8+ T cells recognize vestigial CTL epitopes. PLoS One, 5 (4), pp. e10249. | Show Abstract | Read more

BACKGROUND: The HLA-B*35-Px allele has been associated with rapid disease progression in HIV-1 infection, in contrast to the HLA-B*35-Py allele. METHODOLOGY/PRINCIPAL FINDINGS: Immune responses to two HLA-B*35 restricted HIV-1 specific CTL epitopes and their variants were followed longitudinally during early HIV-1 infection in 16 HLA-B*35+ individuals. Subjects expressing HLA-B*35-Px alleles showed no difference in response to the consensus epitopes compared to individuals with HLA-B*35-Py alleles. Surprisingly, all the HLA-B*35-Px+ individuals responded to epitope-variants even in the absence of a consensus response. Sequencing of the viral population revealed no evidence of variant virus in any of the individuals. CONCLUSIONS/SIGNIFICANCE: This demonstrates a novel phenomenon that distinguishes individuals with the HLA-B*35-Px rapid progressing allele and those with the HLA-B*35-Py slower progressing allele.

Fox J, Willberg C, Ziprin P, Goldin R, Weber J, McClure M, Klenerman P, Fidler S. 2009. The role of the gut mucosa in protection from HIV-1 in highly exposed persistently seronegative individuals (HEPS) HIV MEDICINE, 10 pp. 5-5.

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 ...

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