Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Tumor-necrosis factor impairs CD4+ T cell–mediated immunological control in chronic viral infection

A Publisher Correction to this article was published on 03 July 2020

Abstract

Persistent viral infections are characterized by the simultaneous presence of chronic inflammation and T cell dysfunction. In prototypic models of chronicity—infection with human immunodeficiency virus (HIV) or lymphocytic choriomeningitis virus (LCMV)—we used transcriptome-based modeling to reveal that CD4+ T cells were co-exposed not only to multiple inhibitory signals but also to tumor-necrosis factor (TNF). Blockade of TNF during chronic infection with LCMV abrogated the inhibitory gene-expression signature in CD4+ T cells, including reduced expression of the inhibitory receptor PD-1, and reconstituted virus-specific immunity, which led to control of infection. Preventing signaling via the TNF receptor selectively in T cells sufficed to induce these effects. Targeted immunological interventions to disrupt the TNF-mediated link between chronic inflammation and T cell dysfunction might therefore lead to therapies to overcome persistent viral infection.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Application of inhibitory-pathway RNA fingerprints to CD4+ T cells from HIV-infected subjects.
Figure 2: In vivo evidence of TNFR signaling in late-stage HIV infection.
Figure 3: PD-1 expression on CD4+ T cells is dependent on TNFR signaling in HIV-infected subjects.
Figure 4: cnLCMV-WE mice have a large number of PD-1-expressing T cells.
Figure 5: Neutralization of TNF in cnLCMV-WE mice restores immunity to LCMV.
Figure 6: Neutralization of TNF in cnLCMV-WE mice induces cytokine production in LCMV-specific T cells.
Figure 7: TNFR signaling in CD4+ T cells curtails antiviral immunity.
Figure 8: Constitutive NF-κB activity in mice with acute LCMV infection induces PD-1 expression and loss of helper T cell function.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

References

  1. Rouse, B.T. & Sehrawat, S. Immunity and immunopathology to viruses: what decides the outcome? Nat. Rev. Immunol. 10, 514–526 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. McNab, F., Mayer-Barber, K., Sher, A., Wack, A. & O'Garra, A. Type I interferons in infectious disease. Nat. Rev. Immunol. 15, 87–103 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Hunter, C.A. & Jones, S.A. IL-6 as a keystone cytokine in health and disease. Nat. Immunol. 16, 448–457 (2015).

    CAS  PubMed  Google Scholar 

  4. McFadden, G., Mohamed, M.R., Rahman, M.M. & Bartee, E. Cytokine determinants of viral tropism. Nat. Rev. Immunol. 9, 645–655 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Deeks, S.G., Tracy, R. & Douek, D.C. Systemic effects of inflammation on health during chronic HIV infection. Immunity 39, 633–645 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Crouse, J., Kalinke, U. & Oxenius, A. Regulation of antiviral T cell responses by type I interferons. Nat. Rev. Immunol. 15, 231–242 (2015).

    CAS  PubMed  Google Scholar 

  7. Wherry, E.J. T cell exhaustion. Nat. Immunol. 12, 492–499 (2011).

    CAS  PubMed  Google Scholar 

  8. Kim, P.S. & Ahmed, R. Features of responding T cells in cancer and chronic infection. Curr. Opin. Immunol. 22, 223–230 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Doering, T.A. et al. Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory. Immunity 37, 1130–1144 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Wherry, E.J. et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27, 670–684 (2007).

    CAS  PubMed  Google Scholar 

  11. Youngblood, B., Wherry, E.J. & Ahmed, R. Acquired transcriptional programming in functional and exhausted virus-specific CD8 T cells. Curr. Opin. HIV AIDS 7, 50–57 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Teijaro, J.R. et al. Persistent LCMV infection is controlled by blockade of type I interferon signaling. Science 340, 207–211 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Wilson, E.B. et al. Blockade of chronic type I interferon signaling to control persistent LCMV infection. Science 340, 202–207 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Porichis, F. & Kaufmann, D.E. HIV-specific CD4 T cells and immune control of viral replication. Curr. Opin. HIV AIDS 6, 174–180 (2011).

    PubMed  PubMed Central  Google Scholar 

  15. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sedaghat, A.R. et al. Chronic CD4+ T-cell activation and depletion in human immunodeficiency virus type 1 infection: type I interferon-mediated disruption of T-cell dynamics. J. Virol. 82, 1870–1883 (2008).

    CAS  PubMed  Google Scholar 

  17. Rotger, M. et al. Swiss HIV Cohort Study; Center for HIV/AIDS Vaccine Immunology. Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals. PLoS Pathog. 6, e1000781 (2010).

    PubMed  PubMed Central  Google Scholar 

  18. Vigneault, F. et al. Transcriptional profiling of CD4 T cells identifies distinct subgroups of HIV-1 elite controllers. J. Virol. 85, 3015–3019 (2011).

    CAS  PubMed  Google Scholar 

  19. McKinney, E.F., Lee, J.C., Jayne, D.R., Lyons, P.A. & Smith, K.G. T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection. Nature 523, 612–616 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Oeckinghaus, A., Hayden, M.S. & Ghosh, S. Crosstalk in NF-κB signaling pathways. Nat. Immunol. 12, 695–708 (2011).

    CAS  PubMed  Google Scholar 

  21. Prokunina, L. et al. A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat. Genet. 32, 666–669 (2002).

    CAS  PubMed  Google Scholar 

  22. Salvato, M., Borrow, P., Shimomaye, E. & Oldstone, M.B. Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence. J. Virol. 65, 1863–1869 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Belnoue, E., Fontannaz-Bozzotti, P., Grillet, S., Lambert, P.H. & Siegrist, C.A. Protracted course of lymphocytic choriomeningitis virus WE infection in early life: induction but limited expansion of CD8+ effector T cells and absence of memory CD8+ T cells. J. Virol. 81, 7338–7350 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Doyle, L.B., Doyle, M.V. & Oldstone, M.B. Susceptibility of newborn mice with H-2k backgrounds to lymphocytic choriomeningitis virus infection. Immunology 40, 589–596 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Crawford, A. et al. Molecular and transcriptional basis of CD4+ T cell dysfunction during chronic infection. Immunity 40, 289–302 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Graham, C.M., Christensen, J.R. & Thomas, D.B. Differential induction of CD94 and NKG2 in CD4 helper T cells. A consequence of influenza virus infection and interferon-gamma? Immunology 121, 238–247 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ruan, H., Pownall, H.J. & Lodish, H.F. Troglitazone antagonizes tumor necrosis factor-α-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-κB. J. Biol. Chem. 278, 28181–28192 (2003).

    CAS  PubMed  Google Scholar 

  28. Sasaki, Y. et al. Canonical NF-κB activity, dispensable for B cell development, replaces BAFF-receptor signals and promotes B cell proliferation upon activation. Immunity 24, 729–739 (2006).

    CAS  PubMed  Google Scholar 

  29. Sledzinń´ska, A. et al. TGF-β signalling is required for CD4+ T cell homeostasis but dispensable for regulatory T cell function. PLoS Biol. 11, e1001674 (2013).

    Google Scholar 

  30. Speiser, D.E. et al. T cell differentiation in chronic infection and cancer: functional adaptation or exhaustion? Nat. Rev. Immunol. 14, 768–774 (2014).

    CAS  PubMed  Google Scholar 

  31. Yi, J.S., Du, M. & Zajac, A.J. A vital role for interleukin-21 in the control of a chronic viral infection. Science 324, 1572–1576 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Fröhlich, A. et al. IL-21R on T cells is critical for sustained functionality and control of chronic viral infection. Science 324, 1576–1580 (2009).

    PubMed  Google Scholar 

  33. Elsaesser, H., Sauer, K. & Brooks, D.G. IL-21 is required to control chronic viral infection. Science 324, 1569–1572 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Nokta, M., Rossero, R., Loesch, K. & Pollard, R.B. Kinetics of tumor necrosis factor α and soluble TNFRII in HIV-infected patients treated with a triple combination of stavudine, didanosine, and hydroxyurea. AIDS Res. Hum. Retroviruses 13, 1633–1638 (1997).

    CAS  PubMed  Google Scholar 

  35. De Pablo-Bernal, R.S. et al. TNF-α levels in HIV-infected patients after long-term suppressive cART persist as high as in elderly, HIV-uninfected subjects. J. Antimicrob. Chemother. 69, 3041–3046 (2014).

    CAS  PubMed  Google Scholar 

  36. Aukrust, P. et al. Tumor necrosis factor (TNF) system levels in human immunodeficiency virus-infected patients during highly active antiretroviral therapy: persistent TNF activation is associated with virologic and immunologic treatment failure. J. Infect. Dis. 179, 74–82 (1999).

    CAS  PubMed  Google Scholar 

  37. Aukrust, P. et al. Serum levels of tumor necrosis factor-α (TNF α) and soluble TNF receptors in human immunodeficiency virus type 1 infection--correlations to clinical, immunologic, and virologic parameters. J. Infect. Dis. 169, 420–424 (1994).

    CAS  PubMed  Google Scholar 

  38. Aukrust, P. et al. Effects of intravenous immunoglobulin in vivo on abnormally increased tumor necrosis factor-α activity in human immunodeficiency virus type 1 infection. J. Infect. Dis. 176, 913–923 (1997).

    CAS  PubMed  Google Scholar 

  39. Sade-Feldman, M. et al. Tumor necrosis factor-α blocks differentiation and enhances suppressive activity of immature myeloid cells during chronic inflammation. Immunity 38, 541–554 (2013).

    CAS  PubMed  Google Scholar 

  40. Keir, M.E., Butte, M.J., Freeman, G.J. & Sharpe, A.H. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26, 677–704 (2008).

    CAS  PubMed  Google Scholar 

  41. Han, S., Asoyan, A., Rabenstein, H., Nakano, N. & Obst, R. Role of antigen persistence and dose for CD4+ T-cell exhaustion and recovery. Proc. Natl. Acad. Sci. USA 107, 20453–20458 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Planès, R. et al. HIV-1 Tat protein induces PD-L1 (B7-H1) expression on dendritic cells through tumor necrosis factor α- and toll-like receptor 4-mediated mechanisms. J. Virol. 88, 6672–6689 (2014).

    PubMed  PubMed Central  Google Scholar 

  43. Cepeda, E.J., Williams, F.M., Ishimori, M.L., Weisman, M.H. & Reveille, J.D. The use of anti-tumour necrosis factor therapy in HIV-positive individuals with rheumatic disease. Ann. Rheum. Dis. 67, 710–712 (2008).

    CAS  PubMed  Google Scholar 

  44. Brunasso, A.M.G., Puntoni, M., Gulia, A. & Massone, C. Safety of anti-tumour necrosis factor agents in patients with chronic hepatitis C infection: a systematic review. Rheumatology 50, 1700–1711 (2011).

    CAS  PubMed  Google Scholar 

  45. Kim, S.Y. & Solomon, D.H. Tumor necrosis factor blockade and the risk of viral infection. Nat. Rev. Rheumatol. 6, 165–174 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Habib, S.F., Hasan, M.Z. & Salam, I. Infliximab therapy for HIV positive Crohn's disease: A case report. J. Crohns Colitis 3, 302–304 (2009).

    PubMed  Google Scholar 

  47. Chemnitz, J.M. et al. RNA fingerprints provide direct evidence for the inhibitory role of TGFβ and PD-1 on CD4+ T cells in Hodgkin lymphoma. Blood 110, 3226–3233 (2007).

    CAS  PubMed  Google Scholar 

  48. Beyer, M. et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat. Immunol. 12, 898–907 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Ahmed, R., Salmi, A., Butler, L.D., Chiller, J.M. & Oldstone, M.B. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160, 521–540 (1984).

    CAS  PubMed  Google Scholar 

  50. Lang, P.A. et al. Reactive oxygen species delay control of lymphocytic choriomeningitis virus. Cell Death Differ. 20, 649–658 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank J.L. Riley (University of Pennsylvania) for anti-CD28; J.G. Gribben (Queen Mary University of London) for anti-CTLA-4; M. Schell, M. Kraut, C. Nabakowski, S. Winter and N. Koch for technical assistance; colleagues at the Division of Transfusion Medicine (University Hospital Bonn) for technical support; A. Sharpe for discussions; and the US National Institutes of Health Tetramer Core Facility (contract HHSN272201300006C) for gp66 tetramers. Supported by the Köln Fortune Program of the Faculty of Medicine of the University of Cologne (J.M.C.), the German Research Foundation (SFB 832, SFB 704, INST 217/575-1, INST 217/576-1 and INST 217/577-1 to J.L.S. and M.B.; SFB TRR57 and SFB TRR36 to Z.A., P.A.K. and C.K.; SFB TRR57 to J.T.; and LA2558/3-1, SFB974 and TRR60 to P.A.L. and K.S.L.), the German Research Foundation excellence cluster ImmunoSensation (M.B., Z.A., J.L.S., C.K. and P.A.K.), the German Federal Ministry of Research and Education (01KI0771 and 01KI1017 to C.L., G.F. and P.H.), the German Center for Infection Research (Z.A., P.A.K. and C.K.; partner site Bonn, J.T.), the H. J. & W. Hector Foundation (J.T.), the Alexander von Humboldt Foundation (SKA2008 and SKA2010) and the Jürgen Manchot Foundation (MOI II).

Author information

Authors and Affiliations

Authors

Contributions

M.B., Z.A., J.M.C. and P.H. designed, performed and supervised experiments, analyzed data and wrote the manuscript; D.M., J.S. and A.H. analyzed data; C.L., Y.T., P.V.S., L.S., M.K., J.T., R.S., A.P. and P.A.L. performed experiments; K.S.L., C.K. and G.F. discussed the results; A.O., T.B. and M.H. provided analytical tools; P.A.K. and J.L.S. designed, supervised and analyzed experiments and wrote the manuscript; and all authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Marc Beyer, Percy A Knolle or Joachim L Schultze.

Ethics declarations

Competing interests

M.B., Z.A., J.M.C., P.H., P.A.K. and J.L.S. have applied for patents for the usage of anti-TNF therapy in chronic viral infection.

Integrated supplementary information

Supplementary Figure 1 Analysis of inhibitory signaling in T cells from human HIV-infected subjects.

(a) Workflow for screening of HIV-infected subjects. Boxes in grey indicate the subject groups chosen for further studies. Samples from subjects with at least 106/ml CD4+ T cells were used for further studies (*). Subjects were excluded when RNA amount and quality did not reach necessary quality standards for genomic analysis (**). (b) Flow cytometric analysis of PD-1 expression on CD4+ T cells from HIVloPD-1lo or HIVhiPD-1hi subjects. Mean PD-1 expression of CD4+ T cells from HIVloPD-1lo (n = 26) or HIVhiPD-1hi subjects (n = 37). (c) Relative PDCD1 mRNA expression of CD4+ T cells from HIVloPD-1lo (n = 5) or HIVhiPD-1hi subjects (n = 7) by qPCR. (d) Representative flow cytometry dot plots from one HIVloPD-1lo and one HIVhiPD-1hi subject using current state-of-the-art methodology. (e-g) Flow cytometric analysis of PD-1 expression on CD8+ T cells from HIVloPD-1lo or HIVhiPD-1hi subjects. (e) Representative flow cytometry dot plots from one HIVloPD-1lo and one HIVhiPD-1hi patient. (f) Proportion of PD-1-expressing CD8+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 8). (g) Mean PD-1 expression of CD8+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 8). (h-j) Correlation between CD4+ T cell count of each subject and HIV-RNA (h), CD4+ T cell count and CD4+ T cell PD-1 expression (i), and HIV RNA and CD4+ T cell PD-1 expression (j). White circles: HIVloPD-1lo subjects; grey circles: HIVhiPD-1hi subjects. (k) Representative flow cytometry dot plots from one HIVloPD-1lo or one HIVhiPD-1hi subject for CTLA-4 expression on CD4+ T cells. (l) Proportion of CTLA-4-expressing CD4+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 8). (m) Mean CTLA-4 expression of CD4+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 8). (n) Representative flow cytometry dot plots from one HIVloPD-1lo or one HIVhiPD-1hi subject for CTLA-4 expression on CD8+ T cells. (o) Proportion of CTLA-4-expressing CD8+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 8). (p) Mean CTLA-4 expression of CD8+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 8). (q-s) Generation of RNA fingerprints. (q) Prior to assessment of transcriptional changes the functional impact of all components on purified CD4+ T cells was analyzed. Freshly isolated primary human CD4+ T cells were labeled with CFSE and left unstimulated or were stimulated as indicated. After 4 days, CFSE dilution was analyzed by flow cytometry. The overall percentage of dividing cells is displayed in the corresponding gate. For each condition at least four individual experiments were performed. Shown here are representative results. (r) CD4+ T cells were stimulated as above. After four days the concentration of IFN-γ was determined using flow cytometric bead assays. For each condition at least four individual experiments were performed. Mean ± s.d. (s) Visualization of fold changes and amount of genes significantly altered in CD4+ T cell transcription profiles after indicated stimulations of four different healthy blood donors defining the RNA fingerprint of the particular analyzed component. (t) Schematic overview of analysis of gene expression data for the contribution of RNA fingerprints to the differences between CD4+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 10). (u) Enrichment of inhibitory pathways in HIV-infected individuals. Gene set enrichment analysis (GSEA) using genes regulated by PD-1, CTLA-4, PGE2, TGF-β, and IL-10 as the gene set in CD4+ T cells from HIVhiPD-1hi and HIVloPD-1lo subjects. ES: enrichment score, FDR: false-discovery rate. (b,c,g,m,p) Mean ± s.e.m. (b,c,f,g,l,m,o,p) *P < 0.05 (Student’s t-test). (f,l,o) Bounds of boxes denote interquartile range; lines within boxes denote mean; whiskers indicate interdecile range. Dots represent outliers.

Supplementary Figure 2 TNF-dependent regulation of PD-1 expression.

(a) Relative mRNA expression of CD4+ T cells from HIVloPD-1lo (n = 10) or HIVhiPD-1hi subjects (n = 10) for TNFRI and TNFRII by microarray analysis. (b) Expression of TNFRI and TNFRII on CD4+ T cells from HIVloPD-1lo (n = 5) or HIVhiPD-1hi subjects (n = 6). (c) Schematic representation of the human PDCD1 locus. The PDCD1 promoter predicted by analysis using Genomatix is shown in violet. (d) Luciferase reporter constructs driven by the Genomatix-predicted human PDCD1 promoter (-5.0 kb, red), the region directly upstream of the transcriptional start site (-0.5 kb, green), and an intronic enhancer in intron 4 (intron 4, blue)21 were transfected into HEK293T cells and luciferase activity was assessed after 24 hours in unstimulated cells and cells stimulated with TNF. Control represents the empty pGL4.24 construct. Mean ± s.d. of triplicate cultures are shown. Data are representative of three independent experiments. (e,f) Expression of PD-1 on memory CD4+ T cells from healthy donors pre-stimulated for 3 days with TNF (TNF) or medium alone (US), restimulated with (e) TNF or (f) anti-CD3, IL-2, and TNF (each n = 6). Left, exemplified flow cytometry data, right, cumulative data. (g) Differentiation of CD4+ T cells from HIVloPD-1lo (n = 5) or HIVhiPD-1hi subjects (n = 6) in naïve and memory CD4+ T cells as well as CD7+ or CD7- memory CD4+ T cells. (e,f) *P < 0.05 (Student’s t-test). (a,b,e,f,g) Mean ± s.e.m. n.s. not significant.

Supplementary Figure 3 Analysis of mouse chronic neonatal LCMV infection as model for late-stage HIV-infection.

(a) LCMV serum titers in chronic clone 13 LCMV-infected mice (n = 5). (b) ALT serum concentration in control (n = 3) and chronic neonatal LCMV-WE infected mice (cnLCMV-WE, n = 3). (c,d) Flow cytometric analysis of TIM-3, LILRB4, 2B4, CTLA-4, LAG3, PIR-B, BTLA, CD160, and CD200 co-expression on (c) CD4+PD-1+ and (d) CD8+PD-1+ T cells from cnLCMV-WE mice (n = 5). (e) Left, sorting strategy to isolate PD-1 expressing CD4+ T cells from acute LCMV WE-infected and cnLCMV-WE mice for gene expression analysis. Right, analysis of purities of isolated cell populations. (f) GSEA using genes from the murine chronic clone 13 LCMV-infected gp66+CD4+ T cell RNA fingerprint25 as the gene set in CD4+PD-1+ and CD4+PD-1 T cells from cnLCMV-WE mice. ES: enrichment score, FDR: false-discovery rate. (g) Prediction probability for each sample being classified as HIV-positive (HIV+) or uninfected control (HIV) based on group prediction analysis of the cnLCMV-WE T cell RNA fingerprint using an additional publicly available dataset comparing HIV-infected and uninfected individuals (GSE9927)16. Colors indicate high (red) and low (blue) probability for the cnLCMV-WE RNA fingerprint.

Supplementary Figure 4 Neutralization of TNF in mice with chronic neonatal LCMV strain WE infection restores immunity to LCMV and reverts CD4+PD-1+ T cell gene expression.

(a) Model for TNF neutralization in 8 week old cnLCMV-WE mice. (b) Left, representative, right, cumulative flow cytometric analysis of np396 expression on splenic CD8+ T cells after vehicle treatment or TNF neutralization (each n = 5). (c) Total numbers of splenic np396+CD8+ T cells (each n = 5). (d) PD-1 expression on splenic np396+CD8+ T cells (each n = 5). (e) Left, representative, right, cumulative flow cytometric analysis of PD-1 expression on splenic CD4+ T cells after vehicle treatment (n = 5) or TNF neutralization (n = 4). (f) Left, representative, right, cumulative flow cytometric analysis of PD-1 expression on splenic CD8+ T cells from animals after vehicle treatment (n = 6) or TNF neutralization (n = 5). (g-k) Role of TNFR-signaling in acute murine LCMV strain WE infection. Wild-type mice were acute infected with LCMV strain WE (2 × 104 pfu) and analyzed after 10 days. (g,h) Immunoblot analysis of pIkkα/β (Ser176/180) (top) and β-actin (bottom) in (g) CD4+ and (h) CD8+ T cells from animals after vehicle treatment or TNF neutralization during acute LCMV strain WE infection (n = 3). Data shown are representative of three mice each. (i-k) Left, representative, right, cumulative flow cytometric analysis of (i) PD-1 expression on CD4+ T cells, (j) PD-1 expression on CD8+ T cells, and (k) gp33-specific CD8+ T cells from animals after vehicle treatment or TNF neutralization during acute LCMV strain WE infection (n = 3). (l) Heatmap of z-transformed gene expression data for genes expressed in low amounts in at least one of the inhibitory conditions in human CD4+ T cells, up-regulated in CD4+PD-1+ T cells from mice after TNF neutralization. (m) Heatmap of z-transformed gene expression data for genes highly expressed under at least one of the inhibitory conditions in human CD4+ T cells, down-regulated in CD4+PD-1+ T cells from mice after TNF neutralization. (n) Fold-change-fold-change plot showing the influence of TNF-neutralization on gene expression in CD4+PD-1 and CD4+PD-1+ T cells. The y-axis compares the expression profiles between CD4+PD-1 T cells from mice after vehicle treatment or TNF neutralization, whereas the x-axis compares the expression profiles of CD4+PD-1+ T cells. Highlighted in red are genes assessed in o. (o) Relative mRNA expression of CD4+PD-1 and CD4+PD-1+ T cells from mice after vehicle treatment or TNF neutralization for Ly6c1 and Klrd1 by qPCR. Mean ± s.e.m. of at least triplicates, representative of two independent experiments. *P < 0.05 (Student’s t-test). (p) GSEA using the murine cnLCMV-WE CD4+ T-cell RNA fingerprint as the gene set in CD4+PD-1+ T cells from mice after vehicle treatment or TNF neutralization. (q) Heatmap of z-transformed gene expression data for transcription factors associated with CD4+ T cell exhaustion in LCMV clone 13 infection25 in CD4+PD-1+ T cells from mice after vehicle treatment or TNF neutralization. (r) GSEA using a murine TNF RNA fingerprint (GSE2504)27 as the gene set in CD4+PD-1+ and CD4+PD-1 T cells in mice after vehicle treatment (left) or TNF neutralization (right). (s) GSEA using the human TNF RNA fingerprint genes defined in CD4+ T cells as the gene set in CD4+PD-1+ T cells in mice after vehicle treatment or TNF neutralization. (b-f,i-k) Mean ± s.e.m.*P < 0.05 (Student’s t-test). Data are representative of two independent experiments. n.s. not significant. (p,r,s) ES: enrichment score, FDR: false-discovery rate.

Supplementary Figure 5 Neutralization of TNF in mice infected with LCMV clone 13 partially restores immunity to LCMV.

(a) Model for TNF neutralization of chronic LCMV clone 13-infected mice. (b) Quantification of LCMV titers in serum over time (Control n = 5, anti-TNF n = 9). (c-e) LCMV titers in the liver (c), kidney (d), and lung (e) after TNF neutralization (Control n = 6, anti-TNF n = 11). Box plots showing 25th, mean and 75th percentiles (horizontal bars), 10th and 90th percentage (whiskers), and outliers (dots). (c-e) *P < 0.05 (Student’s t-test). n.s. not significant. Data are representative of two independent experiments.

Supplementary Figure 6 Neutralization of TNF in mice with chronic neonatal LCMV strain WE infection restores cytokine production by CD4+ and CD8+ T cells.

(a) Numbers of IL-2 and (b) IFN-γ expressing splenic gp33+CD8+ T cells after TNF neutralization in cnLCMV-WE mice. Numbers of (c) IL-2, (d) IFN-γ, (e) IL-21, and (f) CD40L expressing splenic gp66+CD4+ T cells. (g) Cumulative flow cytometric analysis of TNF expression in splenic gp66+CD4+ T cells. (h) Numbers of TNF expressing splenic gp66+CD4+ T cells. (a-f,h) *P < 0.05 (Student’s t-test). (a-h) Mean ± s.e.m. Each n = 5. Data are representative of two independent experiments. n.s. not significant.

Supplementary Figure 7 Role of TNFR-signaling in T cells in mice with chronic neonatal LCMV strain WE infection.

(a) Protocol used to determine the effect of TNF on T cells in cnLCMV-WE mice. 2 × 106 CD8+ T cells from Thy1.2 congenic wild-type or TNFRI-TNFRII-deficient mice 8 days after acute infection with LCMV strain WEwere transferred to Thy1.1 mice with chronic neonatal LCMV infection and assessed after 10 days. (b,c) Left, sorting strategy to isolate Thy1.2+CD4+ and CD8+ T cells from acute LCMV strain WE-infected (b) wild-type and (c) TNFRI-TNFRII-deficient mice for adoptive transfer in cnLCMV-WE Thy1.1+ mice. Right, analysis of purities of isolated cell populations. (d,e) Flow cytometric analysis of gp33-specific Thy1.2+CD8+ T cells from mice receiving wild-type (n = 3) or TNFRI-TNFRII-deficient CD8+ T cells (n = 3). (d) Cumulative percentage of splenic gp33-specific CD8+ T cells. (e) Cumulative percentage of splenic PD-1+ gp33-specific CD8+ T cells. (f) ALT serum concentration in mice receiving no transfer (n = 4), wild-type (n = 3), or TNFRI-TNFRII-deficient CD8+ T cells (n = 3). (g) Quantification of LCMV titers in serum as in f. (h) Protocol used to determine the effect of TNF on T cells in cnLCMV-WE mice. 2 × 106 CD8+ T cells and 2 × 106 CD4+ from Thy1.2+ congenic wild-type or TNFRI-TNFRII-deficient mice 8 days after acute infection with LCMV strain WE were transferred to Thy1.1+ mice with chronic neonatal LCMV infection and assessed after 10 days. (i-k) Flow cytometric analysis of gp66-specific Thy1.1+CD4+ T cells from mice receiving no transfer (n = 4), wild-type (n = 4), or TNFRI-TNFRII-deficient CD4+ and CD8+ T cells (n = 4). (i) Cumulative percentage of splenic gp66-specific Thy1.1+CD4+ T cells. (j) Cumulative numbers of splenic gp66-specific Thy1.1+CD4+ T cells. (k) Cumulative percentage of splenic PD-1+ gp66-specific Thy1.1+ CD4+ T cells. (l) Left, representative flow cytometric analysis of PD-1 expression on Thy1.2+CD4+ T cells from mice receiving wild-type or TNFRI-TNFRII-deficient CD4+ and CD8+ T cells. Right, cumulative data. (m,n) Flow cytometric analysis of PD-1 expression on Thy1.1+CD4+ T cells as in i. (m) Cumulative percentage of splenic Thy1.1+CD4+PD-1+ T cells. (n) Cumulative numbers of splenic Thy1.1+CD4+PD-1+ T cells. (o) Left, representative flow cytometric analysis of PD-1 expression on Thy1.2+CD8+ T cells as in l. Right, cumulative data. (p,q) Flow cytometric analysis of PD-1 expression on Thy1.1+CD8+ T cells as in I. (p) Cumulative percentage of splenic Thy1.1+CD8+PD-1+ T cells. (q) Cumulative numbers of splenic Thy1.1+CD8+PD-1+ T cells. Data from 4 mice per group are shown. (d,e,i-q) Mean ± s.e.m. (d,e,l,o) *P < 0.05 (Student’s t-test). (i-k,m,n,p,q) *P < 0.05 vs. wild-type (one-way ANOVA with Bonferroni FDR correction). n.s. not significant. Data are representative of two independent experiments.

Supplementary Figure 8 Constitutive NF-κB activity in mice acutely infected with LCMV strain WE induces PD-1 expression and loss of T cell helper function.

(a) Model for induction of constitutive Ikk activity in oil- (Control) or tamoxifen-treated CD4-Cre-ERt2 × R26StopFLIkk2ca mice (IkkE/E) before acute WE LCMV infection. (b) Flow cytometric analysis of successful recombination of the transgenic allele in CD4+ T cells from oil (Control) or tamoxifen-treated mice (Tam) as evidenced by GFP expression 14 days after infection. Left, exemplary flow cytometric data, right, cumulative data from tamoxifen-treated mice (n = 6). (c) Flow cytometric analysis of gp66-specific CD4+ T cells from oil (Control) or tamoxifen-treated mice (Tam) (each n = 6). (d) Flow cytometric analysis of gp33-specific CD8+ T cells from oil (Control) or tamoxifen-treated mice (Tam) (each n = 6). (e) PD-1 expression on splenic gp33+CD8+ T cells from oil (Control) or tamoxifen-treated mice (Tam) (each n = 6). (f) IL-2 and IFN-γ expression on splenic gp33+CD8+ T cells from oil (Control) or tamoxifen-treated mice (Tam) (each n = 6). (b-f) *P < 0.05 (Student’s t-test). Mean ± s.e.m. Data are representative of two independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1 and 3, 7–10 (PDF 1473 kb)

Supplementary Table 2

Genes comprising the different RNA fingerprints (XLSX 33 kb)

Supplementary Table 4

Tabular output from IPA upstream regulator analysis. The output is sorted by predicted "activity" of the analyzed molecule. (XLSX 81 kb)

Supplementary Table 5

Genes associated with PD-1 expression in CD4+ T cells from chronic neonatal WE LCMV-infected mice. (XLSX 16 kb)

Supplementary Table 6

Genes identified as differentially expressed between anti-CD3 and anti-CD28-stimulated CD4+ T cells and any of the five inhibitory molecules expression in human CD4+ T cells (FC | 2.0 |, p < 0.05, Diff > 100) which show a counter-regulation in CD4+ PD-1+ T cells from chronic neonatal WE LCMV-infected mice after TNF neutralization (XLSX 14 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Beyer, M., Abdullah, Z., Chemnitz, J. et al. Tumor-necrosis factor impairs CD4+ T cell–mediated immunological control in chronic viral infection. Nat Immunol 17, 593–603 (2016). https://doi.org/10.1038/ni.3399

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3399

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing