Inhibitory IL-10-producing CD4+ T cells are T-bet-dependent and facilitate cytomegalovirus persistence via coexpression of arginase-1
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Evaluation Summary:
This manuscript analyses the inhibitory role of IL-10 producing regulatory T-cells in a mouse cytomegalovirus infection model. The authors report that IL-10 producing CD4+T-cells express genes of chronically activated Th1-cells, are clonally expanded and inhibit anti-viral T-cell responses via arginase, an enzyme that breaks down an essential amino acid for T-cell activation. The manuscript presents some novel and potentially important data; however, it requires the provision of additional experimental data and clarifications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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 T H 1-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.
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Evaluation Summary:
This manuscript analyses the inhibitory role of IL-10 producing regulatory T-cells in a mouse cytomegalovirus infection model. The authors report that IL-10 producing CD4+T-cells express genes of chronically activated Th1-cells, are clonally expanded and inhibit anti-viral T-cell responses via arginase, an enzyme that breaks down an essential amino acid for T-cell activation. The manuscript presents some novel and potentially important data; however, it requires the provision of additional experimental data and clarifications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this study, Clement et al. investigated the functional, phenotypic, and transcriptomic profile of IL-10-producing CD4 T cells induced by chronic infection with murine cytomegalovirus (MCMV). Several published studies showed that viral persistence at various tissue sites was facilitated by IL-10 (Humphreys et al., 2007; Mandaric et al., 2012) and that CD4 T cells also represent an important source of IL-10 (Clement et al., 2016; Humphreys et al., 2007). The present study could be considered a follow-up of the early studies by the same authors showing the accumulation of IL-10+ CD4+ T cells in the salivary gland of mice after MCMV infection. Here they demonstrated clonal expansion of differentiated IL-10-producing CD4 TH1-like T cells that developed in a T-bet-dependent manner and coexpressed arginase-1 …
Reviewer #1 (Public Review):
In this study, Clement et al. investigated the functional, phenotypic, and transcriptomic profile of IL-10-producing CD4 T cells induced by chronic infection with murine cytomegalovirus (MCMV). Several published studies showed that viral persistence at various tissue sites was facilitated by IL-10 (Humphreys et al., 2007; Mandaric et al., 2012) and that CD4 T cells also represent an important source of IL-10 (Clement et al., 2016; Humphreys et al., 2007). The present study could be considered a follow-up of the early studies by the same authors showing the accumulation of IL-10+ CD4+ T cells in the salivary gland of mice after MCMV infection. Here they demonstrated clonal expansion of differentiated IL-10-producing CD4 TH1-like T cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1). The expression of Arg1 also impaired the production of IFN gamma by CD4 T cells. In addition, mice lacking Arg1-expressing CD4 T cells exhibited more efficient virus control. Overall, coexpression of Arg1 in IL-10 producing CD4 T cells facilitates viral persistence in salivary glands.
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Reviewer #2 (Public Review):
The authors present data to show that Il-10 producing regulatory T-cells are clonally expanded, controlled by T-bet and inhibit anti-viral T-cell responses via arginase-1.
Strengths: The authors use appropriate conditional ko and IL-10 reporter mice, perform gene expression and clonotype analysis of Il-10+ T-cells, and provide evidence that these Th1-like regulatory T-cells can suppress via arginase expression, a partially novel finding.
Weaknesses: IL-10+ CD4+T-cells are compared to IL-10-CD4+T-cells with an effector/memory phenotype. The latter may be enriched for cells that are not virus-specific and/or not activated by antigen. This concern questions the value of the gene expression (Figure 1) and clonotype analysis (Figure 2). An unbiased list of differentially expressed genes is missing, the choice of …Reviewer #2 (Public Review):
The authors present data to show that Il-10 producing regulatory T-cells are clonally expanded, controlled by T-bet and inhibit anti-viral T-cell responses via arginase-1.
Strengths: The authors use appropriate conditional ko and IL-10 reporter mice, perform gene expression and clonotype analysis of Il-10+ T-cells, and provide evidence that these Th1-like regulatory T-cells can suppress via arginase expression, a partially novel finding.
Weaknesses: IL-10+ CD4+T-cells are compared to IL-10-CD4+T-cells with an effector/memory phenotype. The latter may be enriched for cells that are not virus-specific and/or not activated by antigen. This concern questions the value of the gene expression (Figure 1) and clonotype analysis (Figure 2). An unbiased list of differentially expressed genes is missing, the choice of the shown genes seems to be arbitrary. CD4Cre expression in T-cells may have unspecific effects in Figure 3, but is not expressed in the control mice. In addition, Arginase is also deleted in Il-10-CD4+T-cells and CD8+T-cells in this setting. The choice to analyse T-bet in Figure 4/5 seems again a bit arbitrary; based on the literature it is expected that the related T-box transcription factors Eomesodermin could have a similar role. T-bet induces Il-10 and Arginase not in a direct manner, but the mechanism was not further elucidated.In my opinion this study is therefore preliminary and does not clarify the relationship to other IL-10 producing regulatory T-cells (Tr1). The fact that FOXP3+Tregs may suppress via Argionase-2 was not mentioned.
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Reviewer #3 (Public Review):
Clement et al. aimed at addressing the functional relevance, along with ontogeny of IL-10-producing CD4+ T cells, in the context of chronic MCMV infection, using bioinformatics approach and in vivo studies with multiple mouse mutants.
The same group of authors, along with others, previously observed the accumulation of IL-10+ CD4+ T cells in the salivary gland in the context of acute/chronic MCMV infection which were dependent on IL-27 and ICOS in a temporal- and site-manner. Accumulation of IL-10+ CD4+ T cells favored virus persistence. In line with that, T cells from IL-10−/− mice were oligoclonal, exhibited a highly activated phenotype, expressed antiviral cytokines, and degranulated in response to cognate Ag encounter ex vivo. Data presented here provide some extended insight into the biology of IL-10 …
Reviewer #3 (Public Review):
Clement et al. aimed at addressing the functional relevance, along with ontogeny of IL-10-producing CD4+ T cells, in the context of chronic MCMV infection, using bioinformatics approach and in vivo studies with multiple mouse mutants.
The same group of authors, along with others, previously observed the accumulation of IL-10+ CD4+ T cells in the salivary gland in the context of acute/chronic MCMV infection which were dependent on IL-27 and ICOS in a temporal- and site-manner. Accumulation of IL-10+ CD4+ T cells favored virus persistence. In line with that, T cells from IL-10−/− mice were oligoclonal, exhibited a highly activated phenotype, expressed antiviral cytokines, and degranulated in response to cognate Ag encounter ex vivo. Data presented here provide some extended insight into the biology of IL-10 producing CD4+ T cells.
Strengths: experiments are properly designed and executed, and address a relevant and interesting research question. Moreover, they provide valuable sequencing data (bulk RNA Seq, TCR Seq, ATAC Seq) for better phenotypical analysis of IL-10-producing CD4+ T cells. The authors describe highly differentiated Th1 cells that express IL-10 and arginase-1, where arginase-1 has an inhibitory effect on the T cell control of the viral load.
Weaknesses: Somehow the study is confusing as some parts relate to IL-10 and some to arginase-1 without clearly referring to the same cells. The study is in part deprived of novelty as the inhibitory effect of arginase-1 as well as the role of IL-10 in CMV control has been studied. Some of the conclusions are not supported by data. Few critical points remain unclear in this manuscript which would provide novelty in relation to previous studies:
1. does the arginase-1 effect take place in the periphery or in the salivary gland, i.e. could arginase-1 play a role at other mucosal sites?
2. what controls the expression of Arginase-1? Can the role for chronic antigen exposure be addressed?
3. how does Arg-1 promote viral persistence and how T cell specific is the observed effect? -
