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