Abstract
During chronic viral infection, virus-specific CD8+ T cells become exhausted, exhibit poor effector function and lose memory potential1,2,3,4. However, exhausted CD8+ T cells can still contain viral replication in chronic infections5,6,7,8,9, although the mechanism of this containment is largely unknown. Here we show that a subset of exhausted CD8+ T cells expressing the chemokine receptor CXCR5 has a critical role in the control of viral replication in mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). These CXCR5+ CD8+ T cells were able to migrate into B-cell follicles, expressed lower levels of inhibitory receptors and exhibited more potent cytotoxicity than the CXCR5− subset. Furthermore, we identified the Id2–E2A signalling axis as an important regulator of the generation of this subset. In patients with HIV, we also identified a virus-specific CXCR5+ CD8+ T-cell subset, and its number was inversely correlated with viral load. The CXCR5+ subset showed greater therapeutic potential than the CXCR5− subset when adoptively transferred to chronically infected mice, and exhibited synergistic reduction of viral load when combined with anti-PD-L1 treatment. This study defines a unique subset of exhausted CD8+ T cells that has a pivotal role in the control of viral replication during chronic viral infection.
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Change history
19 October 2016
An Erratum to this paper has been published: https://doi.org/10.1038/nature20107
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Acknowledgements
We thank R. Ahmed (Emory University) for providing the P14 TCR transgenic mice, retroviral vectors and LCMV Armstrong and Cl13 viruses; Y. Zhuang (Duke University) for providing the Id2fl/fl mice; the core facility centre of Third Military Medical University for helping us with cell sorting; T. Wu (NIH) for insightful discussion. The work was supported by National Basic Research Program of China (973 program, 2013CB531500, to L.Y.; 2014CB542501 to H.Q.), the National Natural Science Foundation of China (81220108024 to Y.W.; 81471624 to L.Y.; U1202228 to J.X.; No. 81425011, 81330070 to H.Q.; No.31500733 to Q.B).
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Contributions
L.Y. conceived the project. R.H., S.H., and C.L. performed both in vivo and in vitro experiments. Q.B., M.H., T.S., G.W., and T.N. performed RNA-seq and bioinformatics’ analysis. A.Z., Y.Y., X.Y., L.X., X.C., Y.H., P.W., K.D., Y.C., J.O., Y.L., X.Z., and H.L performed LCMV- and HIV-associated experiments; C.Z performed thymectomy; X.Z performed the reporter assay. L.Y., Y.W., H.Q., and J.X. designed the study, analysed the data and wrote the manuscript; L.Y., and Y.W. supervised the study.
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Extended data figures and tables
Extended Data Figure 1 Virus-specific CXCR5+ CD8+ T cells are not apparent in acutely infected mice and in the non-lymphoid tissues of chronically infected mice and are not Qa-1-restricted.
a, CXCR5 expression in virus-activated CD8+ T cells in the spleens of Arm+-infected mice. b, CXCR5 expression in virus-activated CD8+ T cells in the lungs and livers of Cl13-infected mice. c, Helios and ICOSL expression in virus-activated CXCR5+ CD8+ T cells during Cl13 infection.
Extended Data Figure 2 Virus-specific CXCR5+CD8+ T cells are less exhausted than CXCR5−CD8+ T cells on day 8 after Cl13 infection.
a, b, PD-1, Tim-3 and KLRG1 expression on virus-specific CXCR5+ and CXCR5−CD8+ T cells in the spleens of Cl13-infected mice on day 8 after infection (n = 4 or 5). MFI, mean fluorescence intensity. c, Upon stimulation with the indicated peptides, the cytokine production of CXCR5+ and CXCR5−CD8+ T cells in the spleens of LCMV-Cl13-infected mice was analysed on day 8 post-infection (n = 4 or 5). Data are representative of three independent experiments, and were analysed by two-tailed unpaired t-test (b, c). Error bars (b, c) denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.
Extended Data Figure 3 Virus-specific CXCR5+CD8+ T cells localized in B-cell follicles have minimal effect on germinal centre B and TFH responses.
a, b, Equal numbers of CXCR5+ and CXCR5−CD8+ T cells sorted from Cl13-infected mice were adoptively transferred into infection-matched CD8−/− mice. On day 5 after transfer, frequency and number of germinal centre B cells and TFH cells in the spleens of recipient mice were analysed (n = 3). c, Titration of LCMV-specific IgG in the serum of recipient mice (n = 3). d, The expression levels of PD-L1 and PD-L2 on cell subsets residing in the T-cell zone and in B-cell follicles (n = 4). DC, dendritic cell; FRC, fibroblast reticular cell. The data are representative of three independent experiments, and were analysed by two-tailed unpaired t-test (b–d). Error bars (b–d) denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.
Extended Data Figure 4 The maintenance of functional CXCR5+CD8+ T cells is dependent on follicle structures.
a, Equal numbers of virus-activated CXCR5+CD8+ T and CXCR5−CD8+ T cells obtained from Cl13-infected C57BL/6J (CD45.1) mice were adoptively transferred into infection-matched μMT (CD45.2) or C57BL/6 (CD45.2) (wild-type) mice. Analysis was performed on day 8 after transfer. b, c, Frequency and number of CD45.1+CXCR5+CD8+ T cells in the recipient mice (n = 3). d, e, On stimulation of peptide, surface CD107 expression and cytokine production of CD45.1+CXCR5+CD8+ T cells in the recipient mice (n = 3). f, Viral titers in the indicated tissues obtained from control wild-type and μMT mice without cell transfer and from wild-type and μMT mice receiving CXCR5+CD8+ T cell transfer (n = 3). The data are representative of three independent experiments, and were analysed by two-tailed unpaired t-test (c–e). Error bars (c–e) denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 5 CXCR5 expression is critical for the localization of virus-activated CD8+ T cells to B-cell follicles.
a, Set-up of splenic chimaera mice. Total splenocytes obtained from Cxcr5−/− or wild-type mice were mixed with splenocytes obtained from Cd8−/− mice and then transferred to non-lethally irradiated Cd8−/− recipients and immediately infected with Cl13. Analysis was performed on day 15 after infection. b, The localization of virus-activated CD8+ T cells in the lymph nodes was detected by confocal microscopy on day 15 after infection (blue, IgD; red, CD8; green, CD3) and follicular entry coefficiency was calculated (Cxcr5−/−, n = 15; wild-type, n = 20). Scale bar, 100 μm. c, The CD107 expression and IFN-γ secretion of wild-type and Cxcr5−/− CD8+ T cells upon peptide stimulation (n = 3). d, Viral titers in the indicated tissues from mice that received splenocytes from Cxcr5−/− or wild-type mice (n = 3). Data are representative of three independent experiments, and were analysed by two-tailed unpaired t-test (b–d). Error bars (b–d) denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 6 Distinct transcriptional profiles of CXCR5+ and CXCR5−CD8+ T-cell populations.
a, Transcriptomic profiling of CXCR5+ and CXCR5− cell subsets. b, Gene Ontology (GO) enrichment was analysed using Gene Set Enrichment Analysis (GSEA) and significantly enriched (P value < 0.05) molecular function GO terms were shown with their enrichment scores. c, The enrichment of gene sets containing genes sharing upstream cis-regulatory motifs of transcription factor binding sites were assessed using GSEA. The transcription factor binding sites with significant enrichment (P value < 0.05) in CXCR5+CD8+ cells were listed (left). The GSEA result of the gene set including the E47 (E2A isoform) binding site (denoted as V$E47_02 in the Molecular Signatures Database version 3.0) was shown (right). d, The normalized expression levels of Id2 and E2A isoform E47 in CXCR5+ and CXCR5−CD8+ cells were calculated on the basis of RNA-seq data and was expressed in reads per kilobase per million mapped reads. e, qPCR analysis of the expression levels of Id2 and E2A isoform E47 in CXCR5+ and CXCR5−CD8+ cells. Data are from one experiment with two biological replicates (a–d) or are representative of three independent experiments (e), and were analysed by two-tailed unpaired t-test (e). Error bars (e) denote s.e.m. **P < 0.01. NS, not significant.
Extended Data Figure 7 E2A regulates the transcription of Cxcr5 by directly binding to DNA loci.
a, Kinetic analysis of Id2 expression levels in CXCR5+ and CXCR5−CD8+ T cells during Cl13 infection by qPCR (n = 3). b, Id2 mRNA expression in CD8+ T cells in the spleens of littermate control (control) and Id2−/− mice (n = 3). c, The number of CD44hiCD8+ T cells in the spleens of control and Id2−/− mice on day 25 after Cl13 infection (n = 4). d, An alignment of putative E2A-binding sites in the Cxcr5 intron. The conserved E2A-binding motif ‘CASSTG’ (or ‘GTSSAC’ on the reverse strand) is highlighted in red, and its locations relative to the transcriptional start site (TSS) of Cxcr5 are marked. e, Retroviral reporter constructs containing a wild-type or mutated Cxcr5 regulatory region and the Psv40 promoter, as well as self-inactivating mutations in the long terminal repeats (SIN), a sequence encoding Thy-1.1, and a PGK-EGFP cassette (including P-Pgk1 (a promoter of the gene encoding phosphoglycerate kinase 1) and EGFP). Arrows indicate the transcription start site and orientation, and the numbers shown above indicate the position. f, Thy-1.1 expression levels on GFP+CD8+ T cells transduced with a reporter construct containing wild-type or mutated Cxcr5 regulatory region, MFI of Thy-1.1 was normalized to GFP expression (n = 3). g, CXCR5 expression in non-transduced, E2A-overexpressing, Id2–E2A-co-overexpressing and Id2-overexpressing P14 CD8+ T cells on day 8 after Cl13 infection (n = 4). E2A refers to E47 isoform. h, PD-1 and CD107 surface expression levels and cytokine production in non-transduced P14 cells and E2A-overexpressing P14 cells (n = 4). Data are representative of three independent experiments, and were analysed by two-tailed unpaired t-test (a–c, f–h). Error bars (a–c, f–h) denote s.e.m. *P<0.05; **P<0.01; ***P<0.001. NS, not significant.
Extended Data Figure 8 Virus-activated CXCR5+CD8+ T cells are converted into CXCR5−CD8+ T cells.
a, Schematic map showing the construction of CXCR5–GFP knock-in mice. b, CXCR5-staining and GFP expression in CD19+ cells and in CD44hiCD4+ T cells in CXCR5–GFP knock-in mice and from wild-type mice. c, GFP+CD44hiCD8+ T cells and GFP−CD44hiCD8+ T cells were sorted from day 8 Cl13-infected CXCR5–GFP knock-in mice (CD45.2). The cells were labelled with Celltrace Violet and then transferred into infection-matched wild-type recipients (CD45.1). The presence of GFP and Celltrace Violet in the transferred cells (CD45.2) was detected on days 0, 5, and 12 after transfer. d, Id2 expression levels in GFP+ViolethiCD8+ T cells and in GFP−VioletloCD8+ T cells from recipient mice receiving GFP+CD8+ T cells transfer on day 5 after transfer (n = 3). e, Surface expression of CD107 and IFN-γ production in GFP+CD8+ T cells, newly converted GFP−CD8+ (GFP+/GFP−) T cells and GFP−CD8+ T cells (GFP−, n = 4, GFP+/GFP− and GFP+, n = 3). f, Equal numbers of GFP+CD8+ T cells, GFP+/GFP− T cells and GFP−CD8+ T cells were co-cultured with peptide-coated target cells ex vivo, respectively. Five hours later, the killing efficiency of the effector cells was analysed (n = 3). g, h, The number of CD44hiCD8+ T cells and the frequency of CXCR5+CD8+ T cells in the spleens of control mice infected on day 28 and thymectomized mice (subject to the surgery at day 21 after infection) (n = 4). i, Viral titers in the indicated tissues of control mice, mice received thymectomy and mice received CXCR5+CD8+ T cell transfer after thymectomy (control and CXCR5+ transfer, n = 3; thymectomy, n = 4). Data are representative of three independent experiments, and were analysed by two-tailed unpaired t-test (d–g, i). Error bars (d–g, i) denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.
Extended Data Figure 9 The HIV-specific CXCR5+CD8+ T-cell subset is present in chronically HIV-infected patients.
a, CXCR5 expression in HIV-specific CD8+ T cells in blood of HIV-infected patients. b, The expression levels of PD-1and Tim-3 in HIV-specific CXCR5+ and CXCR5−CD8+ T cells in blood of HIV-infected patients (PD-1, n = 13; Tim-3, n = 12). c, The correlation between viral copy number in serum and CXCR5+CD8+ T cell number in blood in chronic HIV-infected patients prior to anti-retroviral treatment (n = 14). d, HIV-specific (IFN-γ+) CXCR5+CD8+ T cells in lymph nodes of HIV-infected patients. e, CD8+ T-cell localization in the lymph nodes of HIV-infected patients and HIV-negative donors by confocal microscopy (green, CD20; red, CD8). Scale bar, 20μm. f, The expression levels of CD107 and perforin and cytokine production in HIV-specific CXCR5+ and CXCR5−CD8+ T cells in lymph nodes of HIV-infected patients (n = 4). g, The expression levels of E2A isoform E47 and Id2 in IFN-γ+CXCR5+ and IFN-γ+CXCR5−CD8+ T cells in lymph nodes of HIV-infected patients (n = 4). Data are representative of two independent experiments and analysed by two-tailed paired t-test (b, f, g). The correlation between viral load and CXCR5+CD8+ T cell number was analysed by non-parametric Spearman correlation test (c). *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.
Extended Data Figure 10 Diagrammatic summary of the fate of CXCR5+CD8+ T cells during chronic viral infection.
During chronic viral infection, virus-specific exhausted CD8+ T cells differentiate into CXCR5+ and CXCR5− subsets governed by the Id2/–E2A axis. The CXCR5+CD8+ subset migrates into B-cell follicles, where a lesser inhibitory microenvironment prevents the rapid exhaustion and loss of effector functions of these cells. By contrast, the CXCR5− subset undergoes severe exhaustion owing to the inhibitory microenvironment outside B-cell follicles. Follicular CXCR5+CD8+ T cells eventually convert into CXCR5− cells, presumably driven by increased Id2 expression. The de novo converted CXCR5−CD8+ T cells possess better cytotoxicity, hence they are capable of clearing virus-infected cells more efficiently outside of follicles when they exit B-cell follicles.
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He, R., Hou, S., Liu, C. et al. Follicular CXCR5-expressing CD8+ T cells curtail chronic viral infection. Nature 537, 412–416 (2016). https://doi.org/10.1038/nature19317
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DOI: https://doi.org/10.1038/nature19317
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