Optimal CD8+ T-cell memory formation following subcutaneous cytomegalovirus infection requires virus replication but not early dendritic cell responses

Cytomegalovirus (CMV) induction of large frequencies of highly functional memory T-cells has attracted much interest in the utility of CMV-based vaccine vectors, with exciting preclinical data obtained in models of infectious diseases and cancer. However, pathogenesis of human CMV (HCMV) remains a concern. Attenuated CMV-based vectors, such as replication- or spread-deficient viruses potentially offer an alternative to fully replicating vectors. However, it is not well-understood how CMV attenuation impacts vector immunogenicity, in particularly when administered via relevant routes of immunization such as the skin. Herein we used the murine cytomegalovirus (MCMV) model to investigate the impact of vector attenuation on T-cell memory formation following subcutaneous administration. We found that the spread deficient virus (ΔgL-MCMV) was impaired in its ability to induce memory CD8+ T-cells reactive to some (M38, IE1) but not all (IE3) viral antigens. Impaired memory T-cell development was associated with a preferential and pronounced loss of polyfunctional (IFN-γ+ TNF-α+) T-cells and also reduced accumulation of TCF1+ T-cells, and was not rescued by increasing the dose of replication-defective MCMV. Finally, whilst vector attenuation reduced dendritic cell (DC) recruitment to skin-draining lymph nodes, systematic depletion of multiple DC subsets during acute subcutaneous MCMV infection had a negligible impact on T-cell memory formation, implying that attenuated responses induced by replication-deficient vectors were likely not a consequence of impaired initial DC activation. Thus, overall, these data imply that the choice of antigen and/or cloning strategy of exogenous antigen in combination with the route of immunization may influence the ability of attenuated CMV vectors to induce robust functional T-cell memory

Interferon lambda is required for interferon gamma-expressing NK cell responses but does not afford antiviral protection during acute and persistent murine cytomegalovirus infection

Interferon lambda (IFNλ) is a group of cytokines that belong to the IL-10 family. They exhibit antiviral activities against certain viruses during infection of the liver and mucosal tissues. Here we report that IFNλ restricts in vitro replication of the β-herpesvirus murine cytomegalovirus (mCMV). However, IFNλR1-deficient (Ifnλr1-/-) mice were not preferentially susceptible to mCMV infection in vivo during acute infection after systemic or mucosal challenge, or during virus persistence in the mucosa. Instead, our studies revealed that IFNλ influences NK cell responses during mCMV infection. Ifnλr1-/- mice exhibited defective development of conventional interferon-gamma (IFNγ)-expressing NK cells in the spleen during mCMV infection whereas accumulation of granzyme B-expressing NK cells was unaltered. In vitro, development of splenic IFNγ+ NK cells following stimulation with IL-12 or, to a lesser extent, IL-18 was abrogated by IFNλR1-deficiency. Thus, IFNλ regulates NK cell responses during mCMV infection and restricts virus replication in vitro but is redundant in the control of acute and persistent mCMV replication within mucosal and non-mucosal tissues

IL-33 augments virus-specific memory T Cell inflation and potentiates the efficacy of an attenuated cytomegalovirus-based vaccinea

Candidate vaccines designed to generate T cell–based immunity are typically vectored by nonpersistent viruses, which largely fail to elicit durable effector memory T cell responses. This limitation can be overcome using recombinant strains of CMV. Proof-of-principle studies have demonstrated the potential benefits of this approach, most notably in the SIV model, but safety concerns require the development of nonreplicating alternatives with comparable immunogenicity. In this study, we show that IL-33 promotes the accumulation and recall kinetics of circulating and tissue-resident memory T cells in mice infected with murine CMV. Using a replication-deficient murine CMV vector, we further show that exogenous IL-33 boosts vaccine-induced memory T cell responses, which protect against subsequent heterologous viral challenge. These data suggest that IL-33 could serve as a useful adjuvant to improve the efficacy of vaccines based on attenuated derivatives of CMV

Cytomegalovirus-Specific IL-10-Producing CD4+ T Cells Are Governed by Type-I IFN-Induced IL-27 and Promote Virus Persistence

CD4+ T cells support host defence against herpesviruses and other viral pathogens. We identified that CD4+ T cells from systemic and mucosal tissues of hosts infected with the β-herpesviridae human cytomegalovirus (HCMV) or murine cytomegalovirus (MCMV) express the regulatory cytokine interleukin (IL)-10. IL-10+CD4+ T cells co-expressed TH1-associated transcription factors and chemokine receptors. Mice lacking T cell-derived IL-10 elicited enhanced antiviral T cell responses and restricted MCMV persistence in salivary glands and secretion in saliva. Thus, IL-10+CD4+ T cells suppress antiviral immune responses against CMV. Expansion of this T-cell population in the periphery was promoted by IL-27 whereas mucosal IL-10+ T cell responses were ICOS-dependent. Infected Il27rα-deficient mice with reduced peripheral IL-10+CD4+ T cell accumulation displayed robust T cell responses and restricted MCMV persistence and shedding. Temporal inhibition experiments revealed that IL-27R signaling during initial infection was required for the suppression of T cell immunity and control of virus shedding during MCMV persistence. IL-27 production was promoted by type-I IFN, suggesting that β-herpesviridae exploit the immune-regulatory properties of this antiviral pathway to establish chronicity. Further, our data reveal that cytokine signaling events during initial infection profoundly influence virus chronicity

ADCY5 couples glucose to insulin secretion in human islets

Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic β-cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism toward ATP at concentrations of the sugar >8 mmol/L. However, calcium transient generation and functional connectivity between individual human β-cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in β-cell ADCY5 expression and impaired glucose signaling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action

IFNλ can restrict mCMV replication <i>in vitro</i>.

<p>(A) <i>Ifnλr1</i> expression by 3T3 and BNLCL2 cells was determined by qPCR. (B) 3T3 (top) and BNLCL2 (bottom) cells were incubated with/without 50U/ml IFNα and/or IFNβ, or 50ng/ml IFNλ2 (IL-28A) for 24hrs and infected with mCMV at multiplicities of infection (MOI), as stated in the figure. After 4 days, infectious virions in supernatant were quantified by plaque assay. Statistical significance of PFU in IFNλ2-treated versus control cells is shown. Virus load in spleen (C), liver (D) and salivary glands (E) of WT and <i>Ifnλr1</i>^<i>-/-</i> mice was assessed 4 (D&E) and 33 (E) days p.i. (F) IFNλ2/3 protein in spleen (left), liver (middle) and salivary glands (right) was measured at day 0 and 2 days p.i (spleen and liver) or 0 and 26 days p.i (salivary glands). Results are shown as mean + SEM of 3–7 mice/group. (G) WT and <i>Ifnλr1</i>^<i>-/-</i> mice were infected (i.n) with mCMV in a volume of 25μl or 50μl and after 4 days, lung infectious viral load was quantified by plaque assay. Statistical significance was assessed using 1-way ANOVA (B) or Mann Whitney-U (C-E, G) or students T-Test (F) and is depicted where appropriate. Panel G represents merged data from two experiments whereas all other data represent at least two biological replicates performed separately.</p

IFNλ promotes IFNγ⁺ NK cell responses.

<p>(A) Neutrophil numbers in the spleens and livers of mCMV-infected WT and <i>Ifnλr1</i>^<i>-/-</i> mice was assessed 4 days p.i. IFNγ⁺ (B&D) and granzyme B⁺ (C&E) NK cell accumulations in spleens (B-C) and livers (E&F) were quantified day 4 pi. Individual mice + mean is shown from 2 independent experiments and represent 4 (liver) or 5 (spleen) experiments in all. (D) Representative bivariant FACS plots of IFNγ versus granzyme B expression by live NK1.1⁺CD3^- cells 4 days post-infection. Plots show concatenated samples from 4 (WT) and 6 (<i>Ifnλr1</i>^<i>-/-</i>) mice. Results represent 3 experiments. (G) IFNγ protein in the serum was measured 4 days pi. Mean + SEM of 3–4 mice/group is shown. (H&I) IL-12 (H) and IL-18 (I) protein was measured in naïve spleen tissue or after 2 days of mCMV infection. Mean + SEM of 3 (naïve) or 7 (infected) mice is shown. (J) IFNγ expression by NK cells stimulated with IL-12, IL-18 or IL-12/IL-18 +/- IL-28A was detected by flow cytometry. Data represent the mean +/- SD for 3 mice per group. All experiments were performed at least twice.</p

Interferon lambda is required for interferon gamma-expressing NK cell responses but does not afford antiviral protection during acute and persistent murine cytomegalovirus infection

<div><p>Interferon lambda (IFNλ) is a group of cytokines that belong to the IL-10 family. They exhibit antiviral activities against certain viruses during infection of the liver and mucosal tissues. Here we report that IFNλ restricts <i>in vitro</i> replication of the β-herpesvirus murine cytomegalovirus (mCMV). However, IFNλR1-deficient (<i>Ifnλr1</i>^-/-) mice were not preferentially susceptible to mCMV infection <i>in vivo</i> during acute infection after systemic or mucosal challenge, or during virus persistence in the mucosa. Instead, our studies revealed that IFNλ influences NK cell responses during mCMV infection. <i>Ifnλr1</i>^-/- mice exhibited defective development of conventional interferon-gamma (IFNγ)-expressing NK cells in the spleen during mCMV infection whereas accumulation of granzyme B-expressing NK cells was unaltered. <i>In vitro</i>, development of splenic IFNγ⁺ NK cells following stimulation with IL-12 or, to a lesser extent, IL-18 was abrogated by IFNλR1-deficiency. Thus, IFNλ regulates NK cell responses during mCMV infection and restricts virus replication <i>in vitro</i> but is redundant in the control of acute and persistent mCMV replication within mucosal and non-mucosal tissues.</p></div