Christian B. Willberg

Novel Adenovirus-Based Vaccines Induce Broad and Sustained T Cell Responses to HCV in Man

An adenoviral HCV vaccine induces antiviral T cell responses in human volunteers. Hepatitis Hide and Seek Like venture capitalists and Wall Street bankers, patients receiving results of their blood work don’t like surprises, and more than money is at stake. Because infections caused by the hepatitis C virus (HCV) frequently are asymptomatic, patients might not know they’ve been infected: Symptoms don’t usually appear until irreversible liver scarring has occurred, which may cause cirrhosis, liver failure, or cancer. Even if infection is caught early, current therapies to combat this stealth virus have serious side effects, and there is no vaccine to prevent or treat HCV infection. Now, Barnes et al. demonstrate that vaccines developed with adenoviral vectors can induce broad and sustained immune responses to HCV in humans. Adenoviral vectors have shown promise in vaccine trials in animal models; however, preexisting immunity to common serotypes in humans has limited their use. In a phase 1 clinical trial, Barnes et al. vaccinated healthy subjects with two rare serotype adenoviral vectors that expressed an HCV protein. Both the human and the chimp adenoviral vaccinations elicited HCV-specific immune responses in the recipients that responded to multiple HCV antigens, were sustained for at least a year with boost, and elicited memory responses. And the researchers got a surprise they liked: Vaccination primed T cells to respond to multiple HCV strains at a level consistent with protective immunity. Further trials will be needed to confirm protective or therapeutic roles in HCV-infected individuals. 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.

Recognition of HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific.

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.

CD161 ++ CD8 + T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner

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.

Shared Alterations in NK Cell Frequency, Phenotype, and Function in Chronic Human Immunodeficiency Virus and Hepatitis C Virus Infections

ABSTRACT 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 CD56dim cell fraction compared to CD56bright 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 CD56dim 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.

MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets

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.

Toll-Like Receptor 8 Agonist and Bacteria Trigger Potent Activation of Innate Immune Cells in Human Liver

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 CD161Bright mucosal-associated invariant T (MAIT) and CD56Bright 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.

Pervasive Influence of Hepatitis C Virus on the Phenotype of Antiviral CD8 + T Cells

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.

Human T cell receptor-mediated recognition of HLA-E

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 α β CD8+CD94/NKG2C+ CTL clone (K14). In cytolytic assays against the murine TAP‐deficient RMA‐S cells transfected with human β2 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 α β TCR HLA‐E molecules that bind to class I‐ and virus‐derived peptides.

MAIT cells are activated during human viral infections.

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.

CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages

Summary 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.