Antiviral CD8 Research Articles

CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection

Tissue-resident memory CD8 T (TRM) cells defend against microbial reinfections at mucosal barriers; determinants driving durable TRM cell responses in non-mucosal tissues, which often harbor opportunistic persistent pathogens, are unknown. JC polyomavirus (JCPyV) is a ubiquitous constituent of the human virome. With altered immunological status, JCPyV can cause the oft-fatal brain demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV is a human-only pathogen. Using the mouse polyomavirus (MuPyV) encephalitis model, we demonstrate that CD4 T cells regulate development of functional antiviral brain-resident CD8 T cells (bTRM) and renders their maintenance refractory to systemic CD8 T cell depletion. Acquired CD4 T cell deficiency, modeled by delaying systemic CD4 T cell depletion until MuPyV-specific CD8 T cells have infiltrated the brain, impacted the stability of CD8 bTRM, impaired their effector response to reinfection, and rendered their maintenance dependent on circulating CD8 T cells. This dependence of CD8 bTRM differentiation on CD4 T cells was found to extend to encephalitis caused by vesicular stomatitis virus. Together, these findings reveal an intimate association between CD4 T cells and homeostasis of functional bTRM to CNS viral infection.

Harnessing the Induction of CD8+ T-Cell Responses Through Metabolic Regulation by Pathogen-Recognition-Receptor Triggering in Antigen Presenting Cells.

Cytotoxic CD8+ T-cells are key players of the immune responses against viruses. During the priming of a CD8+ T-cell response, the activation of a naïve T-cell by a professional antigen presenting cell (APC) involves the induction of various intracellular and metabolic pathways. The modulation of these pathways at the level of APCs or T-cells offers great potential to enhance the induction of robust effector cells and the generation of long-lived memory cells. On the one hand, signaling through pathogen recognition receptors (PRRs) expressed by APCs can greatly influence T-cell priming, and the potential of several PRR ligands as adjuvants are being studied. On the other hand, the engagement of several metabolic processes, at play in APCs and T-cells upon stimulation, implies that modulating cellular metabolism can impact on priming efficacy. Here, we review recent efforts to understand the interplay between PRR mediated signaling and metabolic pathway modulation in this context, through three examples: interplay between TLR4 and fatty acid metabolism, between TLR9 and IDO, and between STING and autophagy. These initial works highlight the potential for harnessing the induction of antiviral CD8+ T-cell responses using synergistic modulation of metabolic and PRR pathways.

Correction: Cross-Presentation of Soluble and Cell-Associated Antigen by Murine Hepatocytes Is Enhanced by Collectrin Expression.

Cross-presentation is a modular series of intracellular events dictating the internalization and subsequent MHC class I (MHC I) display of extracellular Ags. This process has been defined in dendritic cells and plays a fundamental role in the induction of CD8+ T cell immunity during viral, intracellular bacterial, and antitumor responses. Herein, acute viral infection of murine liver with adenovirus, a model for intrahepatic cross-presentation, confirms hepatocytes directly contribute to cross-presentation of Ags and priming the pool of naive CD8+ T cells within the liver microenvironment. Processing of soluble and cell-associated Ags into peptide displayed by MHC I is however defective in hepatocytes lacking collectrin, an intracellular chaperone protein that localizes within the endoplasmic reticulum-Golgi intermediate compartment. Loss of hepatic collectrin expression leads to the diminished cross-priming and expansion of cytolytic antiviral CD8+ T cells. This study demonstrates that collectrin positively regulates processing of engulfed Ags into MHC I:peptide complexes within hepatocytes. Collectrin-mediated cross-presentation supports intrahepatic adaptive antiviral immune responses and may lead to insights into the nature of how the liver acts as a primary site of CD8+ T cell activation.

Bacterial coinfection restrains antiviral CD8 T-cell response via LPS-induced inhibitory NK cells

Infection of specific pathogen-free mice with lymphocytic choriomeningitis virus (LCMV) is a widely used model to study antiviral T-cell immunity. Infections in the real world, however, are often accompanied by coinfections with unrelated pathogens. Here we show that in mice, systemic coinfection with E. coli suppresses the LCMV-specific cytotoxic T-lymphocyte (CTL) response and virus elimination in a NK cell- and TLR2/4-dependent manner. Soluble TLR4 ligand LPS also induces NK cell-mediated negative CTL regulation during LCMV infection. NK cells in LPS-treated mice suppress clonal expansion of LCMV-specific CTLs by a NKG2D- or NCR1-independent but perforin-dependent mechanism. These results suggest a TLR4-mediated immunoregulatory role of NK cells during viral-bacterial coinfections.

Combination anti-PD-1 and antiretroviral therapy provides therapeutic benefit against SIV.

Therapeutic strategies that augment antiviral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The coinhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of antiviral CD8+ T cells and B cells. Here, we tested the immunological and virological effects of PD-1 blockade combined with antiretroviral therapy (ART) in rhesus macaques. Administration of anti-PD-1 antibody 10 days prior to ART initiation rapidly enhanced antiviral CD8+ T cell function and diminished IFN-stimulated genes. This resulted in faster viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. PD-1 blockade during ART resulted in lower levels of cell-associated replication-competent virus. Following ART interruption, PD-1 antibody-treated animals showed markedly higher expansion of proliferating CXCR5+perforin+granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in better control of viremia. Our results show that PD-1 blockade can be administered safely with ART to augment antiviral CD8+ T cell function and reduce the viral reservoir, leading to improved control of viral rebound after ART interruption.

IFNγ - link between infections and cardiometabolic risk?

Viral infections induce muscle insulin resistance in mice through the stimulation of IFNγ. This results in increased insulin levels, which promote antiviral CD8+ T cell activation. In obese mice, insulin resistance triggered by IFNγ also leads to deterioration of glycaemic control. These findings highlight the intricate link between the immune system and metabolic regulation.

The Transcription Factor Runx2 Is Required for Long-Term Persistence of Antiviral CD8+ Memory T Cells.

During acute lymphocytic choriomeningitis virus infection, pathogen-specific CD8+ cytotoxic T lymphocytes undergo clonal expansion leading to viral clearance. Following this, the majority of pathogen-specific CD8+ T cells undergo apoptosis, leaving a small number of memory CD8+ T cells that persist long-term and provide rapid protection upon secondary infection. Whereas much is known about the cytokines and transcription factors that regulate the early effector phase of the antiviral CD8+ T cell response, the factors regulating memory T cell homeostasis and survival are not well understood. In this article, we show that the Runt-related transcription factor Runx2 is important for long-term memory CD8+ T cell persistence following acute lymphocytic choriomeningitis virus-Armstrong infection in mice. Loss of Runx2 in T cells led to a reduction in KLRG1lo CD127hi memory precursor cell numbers with no effect on KLRG1hi CD127lo terminal effector cell populations. Runx2 expression levels were transcriptionally regulated by TCR signal strength via IRF4, TLR4/7, and selected cytokines. These data demonstrate a CD8+ T cell–intrinsic role for Runx2 in the long-term maintenance of antiviral memory CD8+ T cell populations.

A Mitochondrial Checkpoint in Autoimmune Disease

Cell-fate decisions in innate and adaptive immune cells are metabolically regulated and modified by tissue-derived signals, thereby balancing protective and pathogenic immunity. Reporting in Science, Ling etal. (2018) have identified C1q as a mitochondrial activator that suppresses exuberant anti-self and anti-viral CD8 Tcell effector responses to prevent autoimmune disease.

Human Asymptomatic Epitope Peptide/CXCL10-Based Prime/Pull Vaccine Induces Herpes Simplex Virus-Specific Gamma Interferon-Positive CD107+ CD8+ T Cells That Infiltrate the Corneas and Trigeminal Ganglia of Humanized HLA Transgenic Rabbits and Protect against Ocular Herpes Challenge.

Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea, causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in a human leukocyte antigen (HLA) transgenic rabbit model of ocular herpes (HLA Tg rabbits). Three peptide epitopes were selected, from the HSV-1 membrane glycoprotein C (UL44400-408), the DNA replication binding helicase (UL9196-204), and the tegument protein (UL25572-580), all preferentially recognized by CD8+ T cells from "naturally protected" HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8+ T cell peptide epitopes (UL44400-408, UL9196-204, and UL25572-580), which were delivered subcutaneously with CpG2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic adeno-associated virus type 8 (AAV8) vector expressing the T cell-attracting CXCL10 chemokine (pull). The frequency and function of HSV-specific CD8+ T cells induced by the prime/pull vaccine were assessed in the peripheral blood, cornea, and trigeminal ganglion (TG). Compared to the cells generated in response to peptide immunization alone, the peptide/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-γ+) CD107+ CD8+ T cells that infiltrated both the cornea and TG. CD8+ T cell mobilization into the cornea and TG of prime/pull-vaccinated rabbits was associated with a significant reduction in corneal herpesvirus infection and disease following an ocular HSV-1 (strain McKrae) challenge. These findings draw attention to the novel prime/pull vaccine strategy for mobilizing antiviral CD8+ T cells into tissues to protect against herpesvirus infection and disease.IMPORTANCE There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8+ T cells locally into the cornea and TG, the sites of acute and latent herpesvirus infections, respectively. Mobilization of antiviral CD8+ T cells into the cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpesvirus infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues.

IL-27 regulates the number, function and cytotoxic program of antiviral CD4 T cells and promotes cytomegalovirus persistence.

The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection.

CEACAM1 promotes CD8+ T cell responses and improves control of a chronic viral infection

Dysfunction of CD8+ T cells can lead to the development of chronic viral infection. Identifying mechanisms responsible for such T cell dysfunction is therefore of great importance to understand how to prevent persistent viral infection. Here we show using lymphocytic choriomeningitis virus (LCMV) infection that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is fundamental for recruiting lymphocyte-specific protein kinase (Lck) into the T cell receptor complex to form an efficient immunological synapse. CEACAM1 is essential for activation of CD8+ T cells, and the absence of CEACAM1 on virus-specific CD8+ T cells limits the antiviral CD8+ T cell response. Treatment with anti-CEACAM1 antibody stabilizes Lck in the immunological synapse, prevents CD8+ T cell exhaustion, and improves control of virus infection in vivo. Treatment of human virus-specific CD8+ T cells with anti-CEACAM1 antibody similarly enhances their proliferation. We conclude that CEACAM1 is an important regulator of virus-specific CD8+ T cell functions in mice and humans and represents a promising therapeutic target for modulating CD8+ T cells.

The Respiratory Environment Diverts the Development of Antiviral Memory CD8 T Cells.

Our understanding of memory CD8+ T cells has been largely derived from acute, systemic infection models. However, memory CD8+ T cells generated from mucosal infection exhibit unique properties and, following respiratory infection, are not maintained in the lung long term. To better understand how infection route modifies memory differentiation, we compared murine CD8+ T cell responses to a vesicular stomatitis virus (VSV) challenge generated intranasally (i.n.) or i.v. The i.n. infection resulted in greater peak expansion of VSV-specific CD8+ T cells. However, this numerical advantage was rapidly lost during the contraction phase of the immune response, resulting in memory CD8+ T cell numerical deficiencies when compared with i.v. infection. Interestingly, the antiviral CD8+ T cells generated in response to i.n. VSV exhibited a biased and sustained proportion of early effector cells (CD127loKLRG1lo) akin to the developmental program favored after i.n. influenza infection, suggesting that respiratory infection broadly favors an incomplete memory differentiation program. Correspondingly, i.n. VSV infection resulted in lower CD122 expression and eomesodermin levels by VSV-specific CD8+ T cells, further indicative of an inferior transition to bona fide memory. These results may be due to distinct (CD103+CD11b+) dendritic cell subsets in the i.n. versus i.v. T cell priming environments, which express molecules that regulate T cell signaling and the balance between tolerance and immunity. Therefore, we propose that distinct immunization routes modulate both the quality and quantity of antiviral effector and memory CD8+ T cells in response to an identical pathogen and should be considered in CD8+ T cell-based vaccine design.

O‐021: Antiviral CD4+ but not CD8+ T cell immunity is required to prevent hepatitis E virus persistence

O‐021: Antiviral CD4+ but not CD8+ T cell immunity is required to prevent hepatitis E virus persistence

Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye.

Recent outbreaks of Ebola and Zika have highlighted the possibility that viruses may cause enduring infections in tissues like the eye, including the neural retina, which have been considered immune privileged. Whether this is a peculiarity of exotic viruses remains unclear, since the impact of more common viral infections on neural compartments has not been examined, especially in immunocompetent hosts. Cytomegalovirus is a common, universally distributed pathogen, generally innocuous in healthy individuals. Whether in immunocompetent hosts cytomegalovirus can access the eye, and reside there indefinitely, was unknown. Using the well-established murine cytomegalovirus infection model, we show that systemic infection of immunocompetent hosts results in broad ocular infection, chronic inflammation and establishment of a latent viral pool in the eye. Infection leads to infiltration and accumulation of anti-viral CD8+ T cells in the eye, and to the development of tissue resident memory T cells that localize to the eye, including the retina. These findings identify the eye as an unexpected reservoir for cytomegalovirus, and suggest that common viruses may target this organ more frequently than appreciated. Notably, they also highlight that infection triggers sustained inflammatory responses in the eye, including the neural retina.

Induction of antiviral CD8+ T lymphocyte-mediated protective memory responses.

Induction of antiviral CD8+ T lymphocyte-mediated protective memory responses.

Cutting Edge: Protection by Antiviral Memory CD8 T Cells Requires Rapidly Produced Antigen in Large Amounts.

Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design.

NK cell and Th17 responses are differentially induced in murine cytomegalovirus infected renal allografts and vary according to recipient virus dose and strain.

Human cytomegalovirus (HCMV) donor positive (D+) serostatus with acute rejection is associated with renal allograft loss, but the impact of recipient positive (R+) serostatus is unclear. In an allogeneic renal transplant model, antiviral natural killer (NK) and CD8+ T cell memory responses in murine CMV (MCMV) D+/R+ transplants were compared to D-/R- and D+/R- transplants, with recipient infection varied by MCMV dose and strain. D+/R- transplants had high primary antiviral cytolytic (interferon-γ+) and cytotoxic (granzyme B+) NK responses, whereas NK memory responses were lower in D+/R+ recipients receiving a high primary MCMV dose. Despite MCMV immunity, D+/R+ recipients receiving a low MCMV dose showed primary-like high cytolytic and cytotoxic NK responses. D+/R+ transplants infected with different D/R strains had low cytolytic NK responses but high cytotoxic NK responses. NK memory also induced a novel TNF-α+ NK response among high-dose virus recipients. MCMV+ transplants had greater Th17 responses than MCMV-uninfected transplants and Th17 inhibition ameliorated graft injury. All MCMV+ recipients had similar CD8+ T cell responses. In sum, NK and Th17 responses, but not CD8+ T cells, varied according to conditions of primary recipient infection. This variability could contribute to variable graft outcomes in HCMV D+/R+ renal transplantation.

Dendritic cell expressed H-2Db is required for generating antiviral CD8+ T cell responses and blood brain barrier disruption during Theiler’s virus infection

Abstract Blood brain barrier (BBB) disruption is a feature of multiple neurological disorders for which there is an increasingly recognized role for CD8+ T cells. Theiler’s murine encephalomyelitis virus (TMEV) infection serves as an excellent model to study CD8+ T cell mediated BBB disruption because of the well-characterized immune response to the immunodominant VP2121–130 peptide presented in the context of the H-2Db class I molecule. Intravenous injection of VP2121–130 peptide seven days post TMEV infection results in severe BBB disruption. BBB disruption is dependent on CD8+ T cell interaction with the Db: VP2121–130 epitope, but the antigen presenting cell required for BBB disruption is undefined. We hypothesized that dendritic cells are responsible for presentation of the immunodominant VP2121–130 peptide. We tested this hypothesis using a novel H-2Db Cre-LoxP conditional knockout mouse bred to MHC class I deficient CD11c-Cre expressing mice. We then evaluated these animals at 7 days post infection for induction of BBB disruption, following VP2121–130 peptide administration, using gadolinium enhanced MRI, fluorescent albumin leakage into the brain, and confocal microscopy for the tight junction protein occludin. Control CD11c-Cre(−) mice generated robust Db: VP2121–130 epitope specific CD8+ T cell responses and displayed extensive BBB disruption. In contrast, CD11c-Cre(+) littermates generated no brain infiltrating antiviral CD8+ T cell responses and were protected from BBB disruption. We conclude that CD11c+ dendritic cells are required for priming of CD8+ T cell responses and are necessary for BBB disruption during neuroinflammation in this model.

Role of glucose metabolism in herpes simplex encephalitis

Abstract Herpes simplex virus-1 (HSV-1) is an important human pathogen without an effective vaccine. Following ocular infection the virus replicates in the epithelial tissues and is transported to the trigeminal ganglia (TG) where it establishes latency. Upon periodic reactivation, the virus multiplies in the TG and the replicating virus can reenter the cornea which could result in clinical consequences in many cases. Frequent recurrences can result in chronic inflammatory reactions in the corneal stroma which impairs vision and such recurrences can ultimately result in blindness. Although rare, the virus can spread to the brain and can cause serious complications such as encephalitis especially in immunocompromised individuals. The host immune responses, especially CD8 T cells play a crucial role in controlling virus replication in the TG and prevent spread to the brain. In mouse model of ocular infection, we found that blocking glucose metabolism using 2-deoxy glucose (2DG) resulted in increased virus spread to the brain. The majority of animals treated with 2DG succumbed to herpes simplex encephalitis (HSE). Our results demonstrated that 2DG treatment impacted on both the magnitude and function of the CD8 T cells in the draining lymph nodes and TG. These data show that glucose uptake is essential for HSV specific CD8 T cell responses to be appropriately induced and function and suggest that manipulation of glucose metabolism may serve to enhance anti-viral CD8 T cell responses acting to more effectively control infection.

The transcription factor T-bet is regulated by microRNA-155 in murine anti-viral CD8+ T cells via SHIP-1.

Abstract MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that play essential roles in regulating key cellular processes, and are highly conserved across species. However, the regulatory role of microRNAs in generating effective CD8+ T cell (CTL) responses to viral infection and tumors is only now being elucidated. Microarray miRNA expression profiling identified unique expression patterns of miRNAs in naïve and effector anti-viral CTL. In particular, microRNA-155 was significantly upregulated in effector CTL. Previously, we have demonstrated that miR-155 is essential to CD8+ T cell responses to viral and intracellular bacterial infections. In the present study, we show that miR-155 overexpression in CTL augments anti-viral effector CTL and skews memory CD8+ T cells towards an effector memory phenotype. MiR-155 overexpression resulted in enhanced T-bet expression, which is known to be required for effector CTL and to promote effector memory cell formation. Importantly, we show that the proliferative effect of miR-155 on CD8+ T cells is mediated by T-bet. In CTL, T-bet levels were controlled in vivo by miR-155 via SH2 (Src homology 2)-containing inositol phosphatase-1 (SHIP-1), a known direct target of miR-155. Our studies have revealed an important and unexpected link between miR-155, T-bet transcription factor and SHIP-1 in the context of CTL responses to an in vivo viral infection.