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

The antiviral restriction factor IFN-induced transmembrane protein 3 prevents cytokine-driven CMV pathogenesis

The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3–/– mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3–/– mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity

NBEAL2 is required for neutrophil and NK cell function and pathogen defense.

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity

IL-27 promotes CD4⁺IL-10⁺ development and impairs anti-MCMV T_H1 immunity and control of MCMV persistence.

<p><i>Il-27r</i>α^-/- or WT (C57BL/6) mice were infected with MCMV and spleen and salivary gland CD4⁺/IFNγ⁺ (A) responses were quantified and expressed as mean + SEM of 11 mice/group. (B) Replicating virus in salivary gland homogenates of <i>Il-27r</i>α^-/- and WT mice is shown as individual mice + median. Data is representative of 2 experiments. (C) MCMV genomes in saliva were quantified by qPCR. Data is shown as mean ± SEM from 11 mice per group from 2 replicative experiments.</p

IL-27 promotes splenic CD4⁺IL-10⁺ T cell development whereas ICOS is required for salivary gland CD4⁺IL-10⁺ T cell accumulation.

<p><i>Il-27r</i>α^-/- or WT (C57BL/6) mice were infected with MCMV and spleen and salivary gland CD4⁺/IL-10⁺ (A) responses were quantified and expressed at mean + SEM of 11 mice/group. (B) Representative bivariant FACS plots of IFNγ versus IL-10 expression by splenic CD4⁺CD3⁺ T cells. (C&D) gp130 and (E) ICOS expression by IL-10⁺ (Thy1.1⁺) and IL-10^- (Thy1.1^-) CD4⁺CD3⁺ T cells was assessed in 10-Bit mice and shown as representative FACS plots (C) and histogram overlays (E) with mean + SEM of 5–6 mice/group (D). Gating was determined using Thy1.1⁺ CD4⁺CD3⁺ cells derived from fluorescent minus one-stained samples from mice infected for 14 days. (F&G) WT (C57BL/6) mice were infected with MCMV and at d6 and d10 pi αICOS or Isotype antibody was added. At d14 pi (F) salivary glands and (G) spleen CD4⁺/IL-10⁺ responses were quantified and expressed as mean + SEM of 6 mice/group.</p

Type-I IFN induces IL-27 expression during MCMV infection.

<p>(A) Heat map of IL-27 p28 mRNA expression by WT, IFNβ^-/- and IFNαR^-/- macrophages following stimulation with WT MCMV (C3X), replication-deficient (ΔIE3) MCMV or poly(I:C). (B) Expression of IL-27 p28 by splenic leukocytes was assessed d2 pi in Cre^- mice treated/not with 2 mg anti-IFNαR-1. Mean + SEM of 8 mice/group is shown and data is representative of 2 separate experiments. (C) WT (C57BL/6) mice were infected with MCMV treated with anti-IL-27, Isotype or virus alone. At d6 pi mice were treated /not with anti-IL-10R and at d14 pi spleen CD4⁺/IFNγ⁺ (D) responses were quantified and expressed as mean ± SEM of 4–15 mice/group. (E) MCMV genomes in saliva were quantified by qPCR. Data shown as mean ± SEM from 4–15 mice/ group.</p

T cell-derived IL-10 impairs anti-MCMV T cell immunity and promotes virus persistence.

<p>CD4-Cre^-IL-10^flox/flox(Cre^-) and CD4-Cre⁺IL-10^flox/flox (Cre⁺) mice were infected with MCMV and at day 7, 14, 30 and 60 pi peptide-specific CD4⁺ IFNγ⁺ responses in the salivary glands (A) and spleen (B) were measured. Data is shown as mean ± SEM of cell numbers with mean of 8–17 mice per group. (C) Splenic (top) and pulmonary (bottom) virus-specific CD8⁺ T cells were quantified with tetramers refolded around peptides from MCMV antigens IE3, M38, m139 and M45. Total CD8⁺/CD3⁺ Tetramer⁺ cells are plotted with 4–8 mice per group representative of 2 experiments. (D) Replicating virus in salivary gland homogenates at day 7, 14 and 28 pi were measured by plaque assay. Data is shown as individual mice + median and represents 2–3 experiments. (E) Viral genomes were measured in saliva by qPCR day 7, 14, 21 and 28 pi. Data is shown as mean ± SEM from 5 mice/group from 3 replicative experiments. (F) Replicating virus in salivary gland homogenates d14 pi in <i>rag1</i>^-/- mice following transfer of WT or <i>IL-10</i>^-/- CD4⁺ T cells. Data is shown as individual mice + median and represents two separate experiments.</p

HCMV specific T-cells in peripheral blood and mucosal tissue secrete IL-10.

<p>(A) Total leukocytes from colon and peripheral blood (left) or peripheral blood CD4⁺ T-cells isolated by negative selection (right) were isolated from colorectal patients undergoing surgery (left) or HCMV sero-positive healthy donors (right) were stimulated with γ-irradiated autologous targets that were pre-pulsed with vehicle control, pp65 or gB peptide pool. γ-irradiated autologous targets pre-pulsed with vehicle control or pp65 is also shown from healthy CMV sero-positive individuals. After 16hrs, cytokine expression was analyzed using a human IFNγ/IL-10 fluorospot kit where green spots represent IFNγ⁺ cells, red spots represent IL-10⁺ cells and yellow spots indicate dual IFNγ⁺/IL-10⁺ cells. (B) Peptide-specific spot forming cell (SFC) were calculated after subtraction of SFCs in medium-stimulated wells. Data is expressed as mean + SEM of duplicate values. Individual donors are shown.</p

Virus-specific CD4⁺ T-cells produce IL-10 during MCMV infection.

<p>CD4-Cre^-IL-10^flox/flox (Cre^-) mice were infected with MCMV and at day 7, 14, 30 and 60 pi leukocytes from the salivary glands (A-C &F) and spleen (A, &D-F) were isolated. (A) Representative bivariant FACS plots of IL-10 versus IFNγ expression by viable (aqua live/dead^-) salivary gland (top) and splenic (bottom) CD4⁺CD3⁺ cells. CD4⁺/IFNγ⁺ and CD4⁺/IL-10⁺ responses were measured after 6-hour stimulation with m09, M25, m139 and m142 MHCII MCMV antigens. Data is shown as mean ± SEM percent IL-10⁺ (B&D) and percent + SEM IFNγ⁺ (C&E) CD4⁺ cells and (F) IL-10⁺, IFNγ⁺ and double positive cells over time. Data represents 8–16 mice per group and is representative of 2–3 experiments per time-point.</p

Transcription factor profiling of CD4⁺/IL-10⁺ producing T cells.

<p>10-BiT reporter mice were infected with MCMV and at day 7 and d14 pi spleen and salivary glands were isolated. (A) Representative bivariant FACS plot of viable (aqua live/dead^-) Thy1.1 expression by CD4⁺CD3⁺ cells (top) and representative histograms of T-Bet, BLIMP-1 and c-maf expression or (D) contour FACS plot of CXCR3/CCR5 expression by Thy1.1^- or Thy1.1⁺ CD4⁺CD3⁺ cells. Control = fluorescent minus one-stained Thy1.1⁺ samples from each time-point. (B&C) Intensity of transcription factor staining by CD4⁺CD3⁺ cells in the spleen (B) and salivary glands (C) is shown as mean + SEM of 5–6 mice/group and is representative of 2 separate experiments.</p