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• W4384385597 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1. Editor's evaluation This study analyzes the development and functional relevance of IL-10-producing regulatory T cells in a mouse model of cytomegalovirus infection. The results indicate that IL-10-producing CD4+ T cells express genes associated with chronically activated TH1-like cells, undergo clonal expansion, and inhibit antiviral T cell responses via the secretion of arginase, an enzyme that breaks down an amino acid required for T cell activation and proliferation. These findings reveal a novel and important immunoregulatory mechanism that facilitates viral persistence. https://doi.org/10.7554/eLife.79165.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Immune dysregulation occurs during many persistent viral infections. High levels of ongoing viral replication, which characterize human immunodeficiency virus (HIV), hepatitis B virus (HBV), and, in mice, lymphocytic choriomeningitis virus (LCMV), typically lead to T cell exhaustion, defined by impaired effector functions, the expression of inhibitory cytokines and receptors (Wherry, 2011), and substantial alterations in cellular gene expression (Doering et al., 2012). Moreover, inducible and naturally occurring FoxP3+ regulatory T cells accumulate during many chronic viral infections, presumably to limit excessive immune activation (Veiga-Parga et al., 2013), and T helper (TH)1-like cells that express the immunosuppressive cytokine interleukin (IL)-10 can be induced by LCMV (Parish et al., 2014), HIV (Graziosi et al., 1994), and human/murine cytomegalovirus (HCMV/MCMV) (Clement et al., 2016; Jones et al., 2010; Mason et al., 2013). Evidence from parasitic infections suggests that IL-10-producing TH1-like cells protect against immune pathology, akin to classical FoxP3+ regulatory T cells (Anderson et al., 2007; Jankovic et al., 2007). However, experimental deletion of IL-10 production in T cells has been shown to promote the clearance of LCMV without any obvious collateral effects (Clement et al., 2016; Parish et al., 2014; Richter et al., 2013), suggesting a potential therapeutic role for similar manipulations in humans, albeit with the possibility of an attendant risk to the development of CD8+ T cell memory (Laidlaw et al., 2015). The mechanisms that induce IL-10 expression in T cells require further clarification, despite proposed roles for costimulatory receptors, cytokines, transcription factors, and signals delivered via the T cell receptor (TCR) (Saraiva and O’Garra, 2010). For example, chronic antigen exposure and the transcription factor Blimp-1 appear to be important for the development of IL-10-producing CD4+ T cells in mice infected with LCMV (Parish et al., 2014), and the inhibitory receptor TIGIT is known to act upstream of IL-10 (Schorer et al., 2020). However, it is clear that viral persistence can be facilitated by IL-10, exemplified in the context of MCMV infection by ongoing replication in the salivary glands (SGs) (Humphreys et al., 2007; Mandaric et al., 2012). Interferon (IFN)γ-expressing CD4+ T cells have been shown to limit viral replication in the SGs of mice infected with MCMV (Jonjić et al., 1989; Lucin et al., 1992; Walton et al., 2011). Nonetheless, CD4+ T cells also represent an important source of IL-10 (Clement et al., 2016; Humphreys et al., 2007), the production of which is promoted by IL-27 during acute infection with MCMV. In contrast, less is known about the mucosal IL-10-producing CD4+ T cells that appear during chronic infection with MCMV, which are phenotypically distinct from type 1 regulatory T (Tr1) cells, specifically lacking concurrent expression of CD49d and LAG-3, and develop independently of IL-27 (Clement et al., 2016). These cells express high levels of various transcription factors, such as c-Maf and T-bet (Clement et al., 2016), and often coexpress other molecules with putative inhibitory functions, such as PD-1, TIM-3, and IL-21 (Apetoh et al., 2010; Awasthi et al., 2007; Chihara et al., 2018; Pot et al., 2009; Zhu et al., 2015). However, the functional relevance of these characteristics has remained obscure, along with the ontogeny of IL-10-producing CD4+ T cells during chronic infection with MCMV. To address these issues, we performed a comprehensive functional, phenotypic, and transcriptomic analysis of IL-10-producing CD4+ T cells isolated from the SGs of mice infected with MCMV. Our data revealed that these cells were clonally expanded and highly differentiated TH1-like cells with gene expression signatures that indicated a key developmental role for T-bet. In addition, we identified an inhibitory effect attributable to arginase-1 (Arg1), which was upregulated among IL-10-producing CD4+ T cells during viral chronicity and facilitated the site-specific persistence of MCMV. Results IL-10-producing CD4+ T cells display a TH1-like profile To better understand the development and functionality of inhibitory CD4+ T cells that develop during viral chronicity, we infected MCMV IL-10 reporter (10BiT) mice with MCMV. These mice express Thy1.1 under the Il10 promoter (Maynard et al., 2007). Unlike mucosal sites in the respiratory tract (Zhang et al., 2019), ongoing viral replication in this model occurs primarily in the SGs, facilitated by the induction of CD4+ T cells that produce IL-10 (Humphreys et al., 2007), which peak on day 14 post-infection (p.i.) (Clement et al., 2016). At this time point, we found that approximately 10–30% of CD4+ T cells in the SGs were Thy1.1+, of which ~95% displayed an effector memory phenotype (CD44hi CD62Llo) (Figure 1—figure supplement 1A). IL-10-producing CD4+ T cells were also induced by polyclonal stimulation and universally expressed Thy1.1 (Figure 1—figure supplement 1B). We then compared the transcriptional profiles of endogenously generated IL-10+ and IL-10− CD4+ T cells, isolated via fluorescence-activated cell sorting (FACS) as Thy1.1+ (IL-10+) and Thy1.1− (IL-10−) CD44hi CD62Llo CD4+ T cells (Figure 1—figure supplement 1A). Principal component analysis (PCA) of the RNA-seq data revealed that Thy1.1+ CD4+ T cells were transcriptionally distinct from Thy1.1− CD4+ T cells (Figure 1—figure supplement 1C). As expected, Il10 was highly upregulated in Thy1.1+ CD4+ T cells (Figure 1A), and chromatin was more open in the Il10 promoter region compared with Thy1.1− CD4+ T cells (Figure 1B). Genes associated with localization and cell migration (Ccl7, Cxcl2, Cxcl12, Ccl5, Cxcl14, Ccl28, Ccl12, Ccr1, and Ccr5), cell signaling (Ceacam1, Havcr2, Tigit, Lag3, Cd40, Cd36, and Itgb4), regulation of cellular processes (Prdm1, Gata2, Yes1, Card10, and Il33), and metabolism (Elovl7, Galnt3, Car13, Aldh1l1, and Ildrl), including glycolysis and the tricarboxylic acid cycle (Fbp2 and Sdhc), oxidative phosphorylation (Osgin1), and the mitochondrial respiratory chain (Mt-Nd1, Ndufs6, Ndufb8, Uqcrfs1, and Uqcr11), were also upregulated in Thy1.1+ CD4+ T cells, alongside genes associated with activation (Fgl2, Cxcr2, and Nfil3) and antiviral effector functions (Gzmb, Prf1, Gzmk, and Lyz2) (Figure 1A, C, D and Figure 1—figure supplement 1D). These latter gene profiles suggested the potential for cytolytic activity, but we found no evidence of a concomitant increase in the expression levels of granzyme B protein among Thy1.1+ CD4+ T cells (data not shown), which also lacked gene signatures classically associated with cytotoxic CD4+ T cells, such as the upregulation of Klrc1 and Crtam (https://doi.org/10.5281/zenodo.7243956). Figure 1 with 1 supplement see all Download asset Open asset Interleukin (IL)-10-producing CD4+ T cells display a TH1-like profile. 10BiT mice were infected with 3 × 104 pfu of murine cytomegalovirus (MCMV). Leukocytes were isolated from the salivary glands (SGs) on day 14 p.i. and sorted as CD4+ CD44+ CD62L− CD90/90.1+ (Thy1.1+) or CD90/90.1− (Thy1.1−) populations via fluorescence-activated cell sorting (FACS). (A) Volcano plot highlighting differentially upregulated genes in Thy1.1+ CD4+ T cells (red) versus Thy1.1− CD4+ T cells (blue). (B) ATAC-seq profiles showing accessible chromatin regions in the Il10 gene for Thy1.1+ CD4+ T cells (red) and Thy1.1− CD4+ T cells (blue). Data are shown as normalized values accounting for the total number of reads per lane. The black box indicates a major difference in chromatin accessibility. Black arrows indicate binding motifs for Tbx21. (C) Gene ontology analysis of data from (A) indicating the top six modules that were upregulated (red) or downregulated (blue) in Thy1.1+ CD4+ T cells. (D) Heatmap comparing data from (A) (left column) with published data from T-bet+ versus T-bet-knockout CD4+ T cells (middle column, GSE38808) and TH1 versus naive CD4+ T cells (right column, E-MTAB-2582). Displayed genes were selected according to relevant pathways identified via gene ontology analysis and tabulated against respective functions (all p < 0.05). (E) Venn diagram showing the overlap between genes enriched in Thy1.1+ CD4+ T cells (A, D) and genes enriched in TH1-like CD4+ T cells (E-MTAB-2582). Data in (A–E) are shown as pooled analyses from a minimum of n = 5 mice per group representing three independent experiments. (F) Representative histograms (top) and summary bar graphs (bottom) showing the expression of IL-7R and TCF1/7 among Thy1.1+ CD4+ T cells (red) and Thy1.1− CD4+ T cells (blue). The fluorescence-minus-one control is shown in sky blue (top). Bottom: data are shown as mean ± standard error of the mean (SEM; n = 10 mice per group representing two independent experiments). ****p < 0.0001 (Mann–Whitney U test). (G) Representative flow cytometry plots showing the expression of CD39 and TIM-3 among Thy1.1+ CD4+ T cells (red) and Thy1.1− CD4+ T cells (blue). Data are shown as pooled analyses from a minimum of n = 10 mice per group representing two independent experiments. Figure 1—source data 1 Interleukin (IL)-10-producing CD4+ T cells display a TH1-like profile. Source data Figure 1C: gene ontology analysis indicating the top six modules that were upregulated (red) or downregulated (blue) in Thy1.1+ CD4+ T cells isolated from the salivary glands (SGs) on day 14 p.i. (10BiT mice). Source data Figure 1F: percent expression of IL-7R and TCF1/7 among Thy1.1+ CD4+ T cells and Thy1.1− CD4+ T cells isolated from the SGs on day 14 p.i. (10BiT mice). https://cdn.elifesciences.org/articles/79165/elife-79165-fig1-data1-v1.xlsx Download elife-79165-fig1-data1-v1.xlsx Thy1.1+ CD4+ T cells are known to express the TH1-associated chemokine receptors CXCR3 and CCR5 (Clement et al., 2016). We found that MCMV-induced Thy1.1+ CD4+ T cells shared many transcripts with CD4+ TH1 cells generated in vitro (Stubbington et al., 2015; Figure 1E), encompassing genes associated with numerous cellular and immunological processes (https://doi.org/10.5281/zenodo.7447477), and further expressed IFNγ in response to polyclonal stimulation at a population frequency of ~25% (Figure 1—figure supplement 1E). These data suggested that Thy1.1+ CD4+ T cells were commonly derived from antigen-specific TH1 cells, especially given that IFNγ detection via flow cytometry likely underestimates the composite frequency of CD4+ T cells that specifically recognize MCMV (Jeitziner et al., 2013). However, genes associated with the induction of IFNγ, including Il18r1 and il18rap, were actually downregulated in Thy1.1+ CD4+ T cells (Figure 1A, C, D), and in two of three replicates, a similar pattern was observed for Ifng (https://doi.org/10.5281/zenodo.7243956). Comparable findings were reported previously in functional studies of IFNγ expression at the protein level among IL-10-producing CD4+ T cells specific for HCMV or MCMV (Clement et al., 2016; Mason et al., 2013). Other genes that were downregulated in Thy1.1+ CD4+ T cells included Il7 and Tcf1/7 (Figure 1A, D), which extended to the protein level (Figure 1F). The relative underexpression of these cell survival-associated factors coincided temporally with the rapid contraction of virus-specific IL-10-producing CD4+ T cells that typically occurs during the early stages of viral chronicity (Clement et al., 2016). In contrast, Thy1.1+ and Thy1.1− CD4+ T cells expressed similar levels of transcripts encoding DR5 (https://doi.org/10.5281/zenodo.7243956), which engages natural killer (NK) cell-expressed TRAIL and induces CD4+ T cell death in the SGs (Schuster et al., 2014). IL-10 production among CD4+ T cells has been associated with the expression of inhibitory molecules and markers of exhaustion (Saraiva and O’Garra, 2010). Counterintuitively, we found that MCMV-induced Thy1.1+ CD4+ T cells downregulated the exhaustion-associated transcription factor Tox1 but nonetheless expressed a module of inhibitory genes, including Lag3, Fgl2, Havcr2, and Entpd1 (Figure 1D). These inhibitory molecules were also expressed at the protein level, alongside PD-1 (Figure 1G and Figure 1—figure supplement 1F). In addition, differential bystander activation seemed unlikely, because Thy1.1+ CD4+ T cells expressed LAG-3 and PD-1 more commonly than Thy1.1− CD4+ T cells after preselection based on the induction of IFNγ (Figure 1—figure supplement 1E). Collectively, these data showed that IL-10-producing CD4+ T cells exhibited a highly differentiated TH1-like profile, characterized by the upregulation of various inhibitory molecules and the downmodulation of IFNγ expression lacking concordance with known signatures of exhaustion, during chronic infection with MCMV. IL-10-producing CD4+ T cells exhibit prominent clonal structures IL-10-producing CD4+ T cells recognize a broad range of viral antigens during chronic infection with MCMV (Clement et al., 2016). To characterize these interactions in more detail and evaluate the clonal relationship between IL-10+ (Thy1.1+) and IL-10− (Thy1.1−) CD4+ T cells, we used a next-generation approach to sequence the corresponding TCRs. The repertoires of Thy1.1+ CD4+ T cells were less diverse and incorporated more prominent clonal expansions compared with the repertoires of Thy1.1− CD4+ T cells (Figure 2A, B and Figure 2—figure supplement 1A). Several features also indicated that these expansions represented antigen-focused responses confined largely to Thy1.1+ CD4+ T cells (Figure 2C–G). First, the number of nucleotide variants that encoded each complementarity-determining region (CDR)3α and CDR3β amino acid sequence, an indicator of antigen-specific convergence (Logunova et al., 2020), was higher overall among Thy1.1+ CD4+ T cells versus Thy1.1− CD4+ T cells (Figure 2C). Second, there were some differences in Trbv gene use that distinguished Thy1.1+ CD4+ T cells from Thy1.1− CD4+ T cells, albeit with a general preference for Trbv3, Trbv5, and Trbv31 (Figure 2D). Third, clusters of homologous TCRβ variants, identified using the statistical model ALICE (Pogorelyy et al., 2019), were detected predominantly among Thy1.1+ CD4+ T cells (Figure 2E–G). Importantly, this latter model accounts for generation probabilities, reliably separating immunologically relevant and irrelevant public TCRs. Figure 2 with 1 supplement see all Download asset Open asset Interleukin (IL)-10-producing CD4+ T cells exhibit prominent clonal structures. 10BiT mice were infected with 3 × 104 pfu of murine cytomegalovirus (MCMV). Leukocytes were isolated from the salivary glands (SGs) on day 14 p.i. and sorted as CD4+ CD44+ CD62L− CD90/90.1+ (Thy1.1+) or CD90/90.1− (Thy1.1−) populations via fluorescence-activated cell sorting (FACS). (A) Clonality and (B) diversity metrics calculated for the T cell receptor (TCR)α (top) and TCRβ repertoires (bottom) derived from Thy1.1+ CD4+ T cells and Thy1.1− CD4+ T cells. (C) TCR convergence measured as the average number of nucleotide sequences encoding amino acid-identical complementarity-determining region (CDR)3α (top) and CDR3β loops (bottom) across the 2000 most prevalent clonotypes. (A–C) p values were calculated using a paired t-test with Benjamini–Hochberg correction. ns, not significant. (D) Hierarchical clustering of Trbv gene use weighted by clonotype frequency. (E–G) Cluster analysis of the 500 most prevalent TCRβ clonotypes using the tcrgrapher pipeline. (E) The cumulative frequency of tcrgrapher hits per sample is shown in gray. The frequency of each cluster comprising at least two tcrgrapher hits was calculated for each sample and averaged across all six repertoires. The five most prevalent clusters are shown in color. (F) Amino acid logos for each of the five most prevalent clusters. (G) Visual representation of clusters comprising at least two tcrgrapher hits. Nodes represent unique amino acid sequences. Edges connect sequences with a single amino acid mismatch. Amino acid sequences present only in Thy1.1+ CD4+ T cells are shown in red, amino acid sequences present only in Thy1.1− CD4+ T cells are shown in blue, and amino acid sequences present in both Thy1.1+ CD4+ T cells and Thy1.1− CD4+ T cells are shown in gray. Data are shown as pooled analyses from n = 4 mice per group representing three independent experiments (groups 1–3). It should be noted that none of these differences were absolute. For example, the clusters of TCRβ variants identified among Thy1.1+ CD4+ T cells also occurred at lower cumulative frequencies among Thy1.1− CD4+ T cells (Figure 2E–G), and the TCRα and TCRβ repertoires overlapped considerably between Thy1.1+ CD4+ T cells and Thy1.1− CD4+ T cells (Figure 2—figure supplement 1B). Moreover, there were no prominent differences in the physicochemical properties of amino acids in the central parts of the CDR3α and CDR3β loops, which generally differ among functionally discrete subsets of CD4+ T cells (Kasatskaya et al., 2020), to indicate an ontogenetic divergence between Thy1.1+ CD4+ T cells and Thy1.1− CD4+ T cells (Figure 2—figure supplement 1C). Collectively, these data revealed the presence of common molecular signatures that predominated among Thy1.1+ CD4+ T cells, consistent with the notion of an antigen-driven process of differentiation leading to the production of IL-10. IL-10-producing CD4+ T cells are enriched for expression of Arg1 Our analysis of inhibitory gene expression revealed one particularly intriguing feature, namely that Thy1.1+ CD4+ T cells significantly upregulated Arg1 (Figure 1D). Arg1 promotes the catalytic breakdown of L-arginine (Munder, 2009) and has been shown to inhibit the proliferation of T cells (Czystowska-Kuzmicz et al., 2019; Rodriguez et al., 2004; Rodriguez et al., 2002). A previous study also reported that T cells could express Arg1 (Washburn et al., 2019), although the functional relevance of this observation has remained obscure. To address this knowledge gap, we first confirmed expression at the protein level via Western blotting (Figure 3A) in experiments incorporating control mice lacking the ability to express Arg1 in the CD4+ lineage (Cd4Cre/+Arg1flox/flox). We then revealed the open chromatin structure around Arg1 in Thy1.1+ CD4+ T cells (Figure 3B) and further probed the expression of Arg1 versus Thy1.1 among CD4+ T cells via flow cytometry (Figure 3C). Our data showed that Arg1 was expressed by CD4+ T cells almost exclusively in the SGs (Figure 3A, C). Of note, Arg1 expression was also detected among CD8+ T cells but not among NK T cells via flow cytometry, although intracellular discrimination was subtle (Figure 3—figure supplement 1A). Depletion experiments nonetheless revealed that only CD4+ T cells contributed significantly to Arg1 protein concentrations in SG homogenates during chronic infection with MCMV (Figure 3—figure supplement 1B). Figure 3 with 1 supplement see all Download asset Open asset Interleukin (IL)-10-producing CD4+ T cells are enriched for expression of arginase-1 (Arg1). (A) Expression of Arg1 among leukocytes isolated via magnetic separation from the salivary glands (SGs) and spleens of Cd4+/+Arg1flox/flox or Cd4Cre/+Arg1flox/flox mice on day 14 p.i. detected by Western blot. (B, C) 10BiT mice were infected with 3 × 104 pfu of murine cytomegalovirus (MCMV). (B) Leukocytes were isolated from the SGs on day 14 p.i. and sorted as CD4+ CD44+ CD62L− CD90/90.1+ (Thy1.1+) or CD90/90.1− (Thy1.1−) populations via fluorescence-activated cell sorting (FACS). ATAC-seq profiles show accessible chromatin regions in the Arg1 gene for Thy1.1+ CD4+ T cells (red) and Thy1.1− CD4+ T cells (blue). Data are shown as normalized values accounting for the total number of reads per lane. The black box indicates a major difference in chromatin accessibility. Black arrows indicate binding motifs for Tbx21. Data are shown as pooled analyses from a minimum of n = 5 mice per group representing three independent experiments. (C) Representative flow cytometry plots showing the expression of Arg1 versus Thy1.1 among CD4+ T cells isolated from the SGs and spleens on day 14 p.i. (A, C) Data are shown as pooled analyses from a minimum of n = 7 mice per group representing three independent experiments. (D) Representative flow cytometry plots showing the expression of Arg1 versus IL-10 among OT-II-specific CD4+ T cells generated in vitro in the absence or presence of IL-12 (3 ng/ml) ± OVA323–339 for 7 days and then stimulated with anti-CD3/CD28 for 4 hr. (E) Summary bar graph showing Arg1 protein concentrations in culture supernatants from (D) after stimulation with anti-CD3/CD28 for 48 hr. Up arrows indicate the higher concentration of OVA323–339 (0.5 μM), and down arrows indicate the lower concentration of OVA323–339 (0.01 μM). Data are shown as mean ± standard error of the mean (SEM). Figure 3—source data 1 Interleukin (IL)-10-producing CD4+ T cells are enriched for expression of arginase-1 (Arg1). Source data Figure 3A: expression of Arg1 among leukocytes isolated via magnetic separation from the salivary glands (SGs) and spleens of Cd4+/+Arg1flox/flox or Cd4Cre/+Arg1flox/flox mice on day 14 p.i. detected by Western blot. Original blots are shown for each antibody. Uncut blots are shown with black boxes to delineate the images used in Figure 3A. https://cdn.elifesciences.org/articles/79165/elife-79165-fig3-data1-v1.zip Download elife-79165-fig3-data1-v1.zip Figure 3—source data 2 Interleukin (IL)-10-producing CD4+ T cells are enriched for expression of arginase-1 (Arg1). Source data Figure 3E: Arg1 protein concentrations in culture supernatants after stimulation of leukocytes with anti-CD3/CD28 for 48 hr measured via ELISA. https://cdn.elifesciences.org/articles/79165/elife-79165-fig3-data2-v1.xlsx Download elife-79165-fig3-data2-v1.xlsx IL-10-producing TH1 cells can be induced experimentally via high-dose antigen stimulation in the presence of IL-12 (Saraiva et al., 2009). Accordingly, we hypothesized that Arg1+ IL-10-producing CD4+ T cells might develop under similar conditions in vitro, given the corresponding TH1-like profile observed during chronic infection with MCMV. To test this notion, we stimulated ovalbumin (OVA)-specific transgenic CD4+ T cells from OT-II mice with high or low doses of the cognate peptide (OVA323–339) in the absence or presence of IL-12. Despite constitutively high expression levels of Arg1, only high-dose OVA323–339 in combination with IL-12 induced the development of CD4+ T cells that expressed Arg1 and IL-10, and importantly, all IL-10-producing CD4+ T cells coexpressed Arg1 (Figure 3D). Arg1 protein concentrations in culture supernatants also increased substantially after stimulation with high-dose OVA323–339 in the presence of IL-12 (Figure 3E). Collectively, these data suggested that Arg1 expression was a hallmark of IL-10-producing CD4+ T cells, which developed almost exclusively in the SGs during chronic infection with MCMV. CD4+ T cells promote viral persistence via expression of Arg1 To explore the biological relevance of our findings, we infected Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice with MCMV. Lineage-specific deletion of Arg1 did not impact the function or phenotype of CD4+ T cells in naive mice (Figure 3—figure supplement 1C, D). Higher numbers of IFNγ-expressing CD4+ T cells (Figure 4A) and higher frequencies of proliferating (Ki-67+) CD4+ T cells (Figure 4B) were nonetheless observed after viral antigen stimulation in the SGs of Cd4Cre/+Arg1flox/flox versus Cd4+/+Arg1flox/flox mice during the chronic phase of infection with MCMV. These data suggested that Arg1 inhibited the proliferation of CD4+ T cells in vivo, consistent with a previous in vitro study (Munder et al., 2006). Similarly, higher numbers of virus-specific CD8+ T cells, quantified using tetrameric antigen probes, were detected in the spleens of Cd4Cre/+Arg1flox/flox versus Cd4+/+Arg1flox/flox mice on day 30 p.i. (Figure 4C). In contrast, Arg1 deletion had no significant impact on the development of virus-specific IL-10-producing CD4+ T cells in the SGs (Figure 4D). Figure 4 with 1 supplement see all Download asset Open asset CD4+ T cells promote viral persistence via expression of arginase-1 (Arg1). Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice were infected with 3 × 104 pfu of murine cytomegalovirus (MCMV). (A) MCMV-specific CD4+ T cell responses in the salivary glands (SGs) on days 7 and 14 p.i. measured using flow cytometry to detect interferon (IFN)γ. Immunodominant peptides were pooled for stimulation. Data are shown as mean ± standard error of the mean (SEM; n = 4–6 mice per group representing three independent experiments). (B) Expression of Ki-67 among CD4+ T cells isolated from the SGs on day 14 p.i. measured via flow cytometry. Data are shown as mean ± SEM (n = 4–5 mice per group representing two independent experiments). (C) MCMV tetramer+ CD8+ T cells quantified in spleens on day 30 p.i. via flow cytometry. Data are shown as mean ± SEM (n = 4 mice per group representing two independent experiments). (D) MCMV-specific CD4+ T cell responses in the SGs on days 7 and 14 p.i. measured using flow cytometry to detect interleukin (IL)-10. Immunodominant peptides were pooled for stimulation. Data are shown as mean ± SEM (n = 8–10 mice per group representing two independent experiments). (A–D) *p < 0.05 (Mann–Whitney U test). (E) Viral genomes in saliva on days 7, 14, 21, and 28 p.i. measured via qPCR. Data are shown as mean ± SEM (n = 8 mice per group representing two independent experiments). *p < 0.05 (Mann–Whitney U test). (F) MCMV replication in SG homogenates on day 30 p.i. measured via plaque assay. Data are shown as individual points with median values (n = 6–7 mice per group representing two or three independent experiments). **p < 0.01 (Mann–Whitney U test). Figure 4—source data 1 CD4+ T cells promote viral persistence via expression of arginase-1 (Arg1). Source data Figure 4A: murine cytomegalovirus (MCMV)-specific CD4+ T cell responses in the salivary glands (SGs) on days 7 and 14 p.i. measured using flow cytometry to detect interferon (IFN)γ (Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice). Source data Figure 4B: percent expression of Ki-67 among CD4+ T cells isolated from the SGs on day 14 p.i. measured via flow cytometry (Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice). Source data Figure 4C: MCMV tetramer+ CD8+ T cells quantified in spleens on day 30 p.i. via flow cytometry (Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice). Source data Figure 4D: MCMV-specific CD4+ T cell responses in the SGs on days 7 and 14 p.i. measured using flow cytometry to detect interleukin (IL)-10 (Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice). Source data Figure 4E: viral genomes in saliva (DNA copies/μl) on days 7, 14, 21, and 28 p.i. measured via qPCR (Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice). Source data Figure 4F: MCMV replication in SG homogenates (pfu/organ) on day 30 p.i. measured via plaque assay (Cd4+/+Arg1flox/flox and Cd4Cre/+Arg1flox/flox mice). https://cdn.elifesciences.org/articles/79165/elife-79165-fig4-data1-v1.xlsx Download elife-79165-fig4-data1-v1.xlsx IFNγ-expressing CD4+ T cells are known to restrict MCMV replication in the SGs (Walton et al., 2011). Accordingly, we found that viral shedding in the saliva (Figure 4E) and viral replication in the SGs (Figure 4F) were reduced in Cd4Cre/+Arg1flox/flox versus Cd4+/+Arg1flox/flox mice during the chronic phase of infection with MCMV. Importantly, no differences in vir" @default.
• W4384385597 created "2023-07-15" @default.
• W4384385597 creator A5038051461 @default.
• W4384385597 date "2022-05-16" @default.
• W4384385597 modified "2023-09-25" @default.
• W4384385597 title "Editor's evaluation: Inhibitory IL-10-producing CD4+ T cells are T-bet-dependent and facilitate cytomegalovirus persistence via coexpression of arginase-1" @default.
• W4384385597 doi "https://doi.org/10.7554/elife.79165.sa0" @default.
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