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TLR7 induces anergy in human CD4+ T cells

Nature Immunology volume 16, pages 118–128 (2015)Cite this article

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Abstract

The recognition of microbial patterns by Toll-like receptors (TLRs) is critical for activation of the innate immune system. Although TLRs are expressed by human CD4+ T cells, their function is not well understood. Here we found that engagement of TLR7 in CD4+ T cells induced intracellular calcium flux with activation of an anergic gene-expression program dependent on the transcription factor NFATc2, as well as unresponsiveness of T cells. As chronic infection with RNA viruses such as human immunodeficiency virus type 1 (HIV-1) induces profound dysfunction of CD4+ T cells, we investigated the role of TLR7-induced anergy in HIV-1 infection. Silencing of TLR7 markedly decreased the frequency of HIV-1-infected CD4+ T cells and restored the responsiveness of those HIV-1+ CD4+ T cells. Our results elucidate a previously unknown function for microbial pattern–recognition receptors in the downregulation of immune responses.

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Figure 1: TLR7 signaling inhibits the proliferation and cytokine secretion of CD4+ T cells.
Figure 2: The inhibitory effect of imiquimod is TLR7 specific.
Figure 3: Mechanism of TLR7-induced anergy.
Figure 4: Imiquimod inhibits the phosphorylation of Jnk.
Figure 5: Imiquimod inhibits the activation of Jnk and Jun after full stimulation of CD4+ T cells through signaling via the TCR and costimulation.
Figure 6: Knockdown of TLR7 or NFAT abolishes infection with HIV-1.
Figure 7: Calcium-induced anergy favors HIV-1 replication.
Figure 8: Inhibition of TLR7 decreases infection in HIV-1+ patients.

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Acknowledgements

We thank L. Devine and Z. Wang for technical assistance; Y. Tsunetsugu-Yokota (Tokyo University of Technology) for HIV-1NL-D proviral DNA; D. Bruce, H. Zapata and B.C. Herold and the laboratory of B.C. Herold for the recruitment of patients; and R. Medzhitov, A. Iwasaki and members of the Hafler laboratory for comments and suggestions. Supported by the National MS Society (CA1061-A-18), the US National Institutes of Health (P01 AI045757, U19 AI046130, U19 AI070352 and P01 AI039671 to D.A.H., and R01 AI065309 to M.J.K.), the Penates Foundation (D.A.H.), the Nancy Taylor Foundation for Chronic Diseases (D.A.H.) and the Race to Erase MS Foundation (M.D.-V.).

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Authors and Affiliations

  1. Department of Neurology and Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA

    Margarita Dominguez-Villar, Anne-Sophie Gautron, Marine de Marcken & David A Hafler

  2. Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, USA

    Marla J Keller

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  1. Margarita Dominguez-Villar
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Contributions

M.D.-V. designed and performed the experiments, analyzed data and wrote the manuscript; A.-S.G. and M.d.M. performed experiments; M.J.K. provided HIV-1 samples; and D.A.H. assisted with the design of experiments, supervised the project and wrote the manuscript.

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Correspondence to Margarita Dominguez-Villar or David A Hafler.

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Integrated supplementary information

Supplementary Figure 1 Costimulatory effects of TLR ligands on CD4+ T cells.

CFSE-labeled CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of different TLR ligands. a. Histograms show the frequency of viable proliferating CD4+ T cells (numbers in histograms). b. Statistical analysis showing the frequency of proliferating CD4+ T cells of 5 experiments performed with 1 donor each. c. IFN-γ and d IL-2 ELISA measurement after 3 days of stimulation. e. CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of IMQ or vehicle as control, RNA was isolated after 12 hours and subjected to gene expression analysis by TaqMan real-time PCR (n=3 donors in 3 independent experiments). *p < 0.05, **p < 0.005, ***p < 0.0005. Error bars represent mean±s.e.m.

Supplementary Figure 2 Imiquimod inhibits the activation of T cell clones.

16 T cell clones were grown from a single donor for 18 days and restimulated with anti-CD3 and anti-CD28 in the presence or absence of IMQ for 3 days. a. IFN-γ secretion measured by ELISA at day 3 after activation. b. CD25 gene expression at day 3 after activation. *p < 0.05, **p < 0.005, ***p < 0.0005.

Supplementary Figure 3 Monocytes are activated with TLR7 ligands.

CD14+ monocytes were stimulated with 5 μg/ml IMQ or vehicle (Veh) for 24 hours. a. Surface staining of HLA-DR, CD80, CD86 and CD25 on imiquimod- (black) or vehicle-treated monocytes (dashed) as compared to isotype control (gray curve). b. IL-1β, TNFα, IL-6 and IL-10 cytokine secretion as measured by ELISA (n=5 in 5 independent experiments). *p < 0.05. Error bars represent mean±s.e.m.

Supplementary Figure 4 Imiquimod induces an increase in intracellular calcium concentration.

a. Bound/unbound calcium ratio on CD4+ T cells stimulated with different doses of IMQ, ssRNA40 (negative control) or ionomycin (positive control) of 6 independent experiments performed. b. Dot plots represent calcium fluxes as measured by INDO-1AM ratio over time on CD4+ T cells stimulated with Poly(I:C) (TLR3 ligand, left) or ODN2006 (TLR9 ligand, right). Representative example of 5 independent experiments performed with one donor each.

Supplementary Figure 5 Expression of anergy-related genes in CD4+ T cells treated with ionomycin or with PMA plus ionomycin.

Gene expression of anergy-related genes on CD4+ T cells treated for 16 hours with Ionomycin (Iono), PMA and ionomycin (P+I) or vehicle. (n=6 donors in 6 independent experiments). *p < 0.05, **p < 0.005, ***p < 0.0005. Error bars represent mean±s.e.m.

Supplementary Figure 6 Imiquimod fails to upregulate anergy-related genes in CD4+ T cells in which the gene encoding NFATc2 is silenced.

CD4+ T cells were transduced with shRNA specific for NFAT1 or a non-target control. a. Transduced cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of IMQ. Bars diagram shows Nfat1 gene expression by TaqMan real-time PCR 24 hours after stimulation. b. Anergy-related gene expression was analyzed on resting NFAT1- or non-target-transduced cells after IMQ treatment for 2 hours, by TaqMan real-time PCR. (n=3 donors in 3 independent experiments). *p < 0.05, **p < 0.005. Error bars represent mean±s.e.m.

Supplementary Figure 7 In vitro infection with HIV-1 induces anergy in CD4+ T cells.

CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for two days and subsequently infected with HIV-1NL-D. a. Frequency of viable HIV-1NL-D+ CD4+ T cells measured every 48 hours for a total of 11 days (n=3 donors in 3 independent experiments). b. Representative example of IL-2 and IFN-γ secretion as measured by intracellular staining after a 4 hour PMA/Ionomycin stimulation at day 7 after infection on mock infected cells (left panel), total CD4+ T cells infected with HIV-1NL-D (middle panel) or HIV-1NL-D+ cells. c. Statistical analysis of IL-2 and IFN-γ (n=3 donors in 3 independent experiments). Error bars represent mean±s.e.m. *p < 0.05, **p < 0.005.

Supplementary Figure 8 Apoptosis after infection of TLR7-deficient cells with HIV-1NL-D.

CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of two TLR7 shRNA (clones 3 and 4) or non-target control shRNA (NT). After two days, the cells were infected with HIV-1NL-D and stained with Annexin V and 7-AAD every 24 hours for 11 days. n=6 donors in 6 independent experiments. Error bars represent mean±s.e.m.

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Dominguez-Villar, M., Gautron, AS., de Marcken, M. et al. TLR7 induces anergy in human CD4+ T cells. Nat Immunol 16, 118–128 (2015). https://doi.org/10.1038/ni.3036

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