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Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells

Abstract

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.

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Fig. 1: VSV and LCMV infections result in distinct antiviral CD4+ T cell polarization and in vivo dynamics.
Fig. 2: Characterization of the antiviral CD4+ T cell priming niche.
Fig. 3: Spatiotemporal regulation of type I IFN expression determines antiviral CD4+ T cell polarization.
Fig. 4: Early type I IFN sensing by DCs and IL-6 are essential for antiviral TFH differentiation.
Fig. 5: scRNA-seq analysis of LN DCs upon viral infection.

Data availability

All data are available in the main text or the Supplementary Information. RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession GSE130009. Source data for Figs. 14 and Extended Data Figs. 1, 2, 57 and 9 are presented with the paper.

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Acknowledgements

We thank M. Mainetti and M. Freschi for technical support; M. Silva for secretarial assistance; J.C. de la Torre (The Scripps Research Institute) for providing rLCMV; D. Pinschewer (University of Basel) for providing rVSV; M. Linterman (Babraham Institute) for providing Cd11c-Cre; Ifnar1fl/fl bone marrow; R. Pardi and A. Mondino for critical reading of the manuscript; and the members of the Iannacone laboratory for helpful discussions. Confocal immunofluorescence histology was carried out at Alembic, San Raffaele Scientific Institute, and the Vita-Salute San Raffaele University. Flow cytometry was carried out at FRACTAL, San Raffaele Scientific Institute. We would like to acknowledge the PhD program in Basic and Applied Immunology and Oncology at Vita-Salute San Raffaele University, as V.C. and E.S. conducted this study as partial fulfillment of their PhD in Molecular Medicine within this program. M.I. is supported by European Research Council (ERC) Consolidator grant 725038, Italian Association for Cancer Research (AIRC) grants 19891 and 22737, Italian Ministry of Health (MoH) grant GR-2011-02347925, Lombardy Foundation for Biomedical Research (FRRB) grant 2015-0010, the European Molecular Biology Organization Young Investigator Program and a Career Development award from the Giovanni Armenise-Harvard Foundation. M.K. is supported by Italian Ministry of Education grants SIR-RBSI14BAO5 and PRIN-2017ZXT5WR.

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M.D.G., V.C. and M.K. designed and performed experiments, analyzed data, performed the statistical analyses and drafted the manuscript. E.S., C.G.M., P.D.L., E.B., C.C., E.C., L.G. and A.F. performed experiments and analyzed data. A.G., C.M. and I.A. performed the NICHE-seq and scRNA-seq analyses and prepared the related figures. S.E. and W.K. performed the Xcr1-DTR experiments and analyzed data. M.K. provided funding, conceptual advice and supervision. M.I. designed and coordinated the study, provided funding and wrote the manuscript.

Corresponding authors

Correspondence to Mirela Kuka or Matteo Iannacone.

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Peer review information Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 VSV and LCMV infections result in distinct antiviral CD4+ T cell polarization and in vivo dynamic behavior, independently of viral strain, viral dose, infection route and TCR signal strength.

a, Schematic representation of experimental procedure for the results described in Fig. 1a–c. 1 × 106 purified Ag-specific (Tg7 when VSV-Ind was used, SMARTA cells in all other cases) CD45.1+ CD4+ T cells were injected into CD45.2+ WT recipients 1 day before intrafootpad infection. dLNs were collected at the indicated time points after infection and analyzed by flow cytometry. TFH were defined as either Bcl-6+ CXCR5+ or CXCR5+ T-bet- cells (in the latter case we always verified that cells were also Bcl-6+); TH1 were defined as T-bet+ CXCR5- cells. b, Representative flow cytometry plots showing TFH and TH1 cells (out of CD44high endogenous CD4+ T cells) in dLNs 7 days after footpad infection with the indicated virus. Numbers indicated the percentage of cells within the indicated gate. Results are representative of at least 2 independent experiments. c, Quantification of TFH (top) and TH1 cells (bottom)—expressed as percentages of CD44high endogenous CD4+ T cells—in dLNs of mice described in b. Results are representative of at least 2 independent experiments. Mean± SEM is shown. PBS n = 2, all other conditions n = 3. A one-way Anova test was applied; *** p value<0.001. d, Quantification of TFH (left) and TH1 (right) cells (expressed as percentages out of total transferred cells) in the spleens of CD45.2+ WT recipients injected with 1 × 106 Ag-specific (Tg7 when VSV-Ind was used, SMARTA in all other cases) CD45.1+ T cells one day prior to intravenous infection with VSV-Ind (left), LCMV-Arm (right) or LCMV-Cl13 (not shown), respectively. Mean + /- SEM is shown. An unpaired two-tailed t test was applied. *** p value<0.001. e, Quantification of TFH (left) and TH1 (right) cells (expressed as percentages out of total transferred cells) in dLNs 5 days after infection of CD45.2+ WT recipients injected with 1 × 106 Ag-specific (Tg7 for VSV-Ind, SMARTA for LCMV-WE) CD45.1+ T cells one day prior to intrafootpad infection with the indicated doses of VSV-Ind (black) or LCMV-WE (red). Results are pooled from 2 independent experiments. Mean + /- SEM is shown.n = 6. A one-way Anova test was applied. *** p value<0.001. f-g, Quantification of TFH (left) and TH1 (right) cell absolute numbers (f) or TFH/TH1 absolute number ratios (g) at 0,3,5,7 and 14 days after VSV-Ind (black), rVSV (blue) or rLCMV (red) infection. Mean + /- SEM is shown. Day 0 n = 3 (VSV), 4 (rVSV and rLCMV); Day 3 n = 5 (VSV), 7 (rVSV and rLCMV); Day 5 n = 5 (VSV), 9 (rVSV), 10 (rLCMV); Day 7 n = 3; Day 14 n = 3 (VSV and rVSV), 6 (rLCMV). Black and blue stars indicate significance of respectively VSV and rVSV samples towards LCMV samples. A two-way Anova with LSD post-test was applied. * p value<0.05; ** p value<0.01; *** p value<0.001; **** p value<0.0001.

Source data

Extended Data Fig. 2 Spatiotemporal dynamics and activation of Ag-specific CD4+ T cells within dLNs upon VSV or LCMV infection.

(a, b) Track speed mean (a) and meandering index (b) of GFP+ Ag-specific (Tg7 when VSV-Ind was used, SMARTA in all other cases) CD4+ T cells in the mice described in Fig. 1d–g and Supplementary Movie 1, 3 days after PBS, VSV-Ind, rVSV or rLCMV injection. Data are pooled from 2 independent experiments. PBS, n = 395; VSV-Ind n= 11219; rVSV n=6692; rLCMV n = 3537. One-way Anova test was applied. **** p value<0.0001; (c, d) Mean fluorescent intensity of CD69 (c) and CD25 (d) within Ag-specific (SMARTA) CD4+ T cells in dLNs, 2 days after PBS, rVSV or rLCMV injection. Data are representative of 2 independent experiments. Mean + /- SEM is shown. n = 2. One-way Anova test was applied. ** p value<0.01; *** p value<0.001 (e, f) Methods used to determine the normalized distance from T area centre (e) and percentages of clustered T cells / section (f). e, T cell area volume was defined based on polyclonal B cell positioning and its centre was geometrically identified in Imaris. Ag-specific CD4+ T cells were localized using Imaris built-in spot detection function and distance from T cell area centre was calculated and normalized for T cell are volumes. f, A T cell cluster was defined as a minimum of 3 T cells aggregating within closest distance of 15 μm measured from cell centroids (see Materials and Methods). Cell clusters of less than 3 cells were manually removed.

Source data

Extended Data Fig. 3 Confocal analysis of the CD4+ T cell priming niche.

Confocal imaging of murine dLNs collected 2 days after rVSV or rLCMV infection. Dashed lines represent the edges of B cell follicles and were depicted based on B220 staining. a, Ag-specific GFP+ CD4+ T cells (SMARTA, depicted in purple) and Ag-specific CFP+ B cells (KL25, depicted in cyan) were adoptively transferred into WT mice. b, Ag-specific CFP+ CD4+ T cells (SMARTA, depicted in purple) were adoptively transferred into CX3CR1-GFP x CCR2-RFP mice. A colocalization channel for GFP and RFP was used to depict inflammatory monocytes (cells positive for both CX3CR1 and CCR2, in green). c, Ag-specific CFP+ CD4+ T cells (SMARTA, depicted in purple) and Ag-specific GFP+ CD8+ T cells (P14, depicted in green) were adoptively transferred into WT mice. d, Ag-specific CFP+ CD4+ T cells (SMARTA, depicted in purple) were adoptively transferred into NKp46-ZsGreen mice. Scale bars represent 50 μm or 30 μm (zoom). The dotted square represents the zoomed area in the IFA where CD4+ T cell clusters are found. All images are representative of at least 2 independent experiments.

Extended Data Fig. 4 Early antiviral CD4+ T cell localization is independent of Ag-specific B cells, Ag-specific CD8+ T cells and CCR2+ monocytes.

Confocal imaging of dLNs of VI10YEN (a), Cor93 Tg TCR (b), and CCR2−/− (c) mice collected either 3 (a) or 5 (b, c) days after rVSV (left) or rLCMV (right) infection. Ag-specific CD4+ T cells are depicted in green. Dashed lines represent the edges of B cell follicles and were depicted based on polyclonal B cell positioning (b, c) or B220 staining (a) (both in grey). Scale bars represent 50 μm. Results are representative of at least 2 independent experiments.

Extended Data Fig. 5 Antiviral CD4+ T cell are primed by cDC2 cells and differentiate independently of NK cells.

a, Schematic representation of the experimental procedure for the results described in panels b and c. 1 × 106 purified CD45.1+ Ag-specific (SMARTA) CD4+ T cells were injected into NKp46-DTR mice treated with PBS or DT as indicated. dLNs were collected 5 days after rVSV (blue) or rLCMV (red) infection. Percentages of NK cells (b), TFH (c, left) and TH1 (c, right) in dLNs were quantified by flow cytometry. Data are representative of 2 independent experiments. Mean+/- SEM is shown. n = 3. A one-way Anova with Bonferroni’s post-test was applied. * p value<0.05; **** p value<0.0001 (d) Schematics of the experimental setup for the results described in panel e. 1 × 106 purified CD45.1+ Ag-specific (SMARTA) CD4+ T cells were transferred to WT mice treated with anti-ICOS blocking antibody or with isotype control, as indicated, prior to rVSV (blue) or rLCMV (red) infection. dLNs were collected 3 days after infection. e, ICOSL expression (mean fluorescent intensity) within CD11c+ MHC-IIhigh CD8+ (cDC1) and CD11c+ MHC-IIhigh CD11b+ (cDC2) cell subsets in dLNs of the mice described in d. Data are representative of 2 independent experiments. Mean + /- SEM is shown. PBS conditions n= 2, all other conditions n=3. A one-way Anova with Bonferroni’s post-test was applied. ** p value<0.01; **** p value<0.0001. f, 1 × 106 purified CD45.1+ Ag-specific (SMARTA) CD4+ T cells were transferred to WT and DT-treated XCR1-DTR mice prior to rVSV (blue, left) or rLCMV (red, right) infection. Quantification of TFH (left) and TH1 (right)—expressed as percentages of the total transferred cells—in dLNs 5 days after infection is shown. Mean ± SEM is shown. Data are representative of 2 independent experiments. n= 4-6.

Source data

Extended Data Fig. 6 Measurement of IFN-α isoforms upon rVSV and rLCMV infection.

Analysis of Ifna2, Ifna5, Ifna6, Ifna7, Ifna9, Ifna12, Ifna13, Ifna14 gene expression in dLN at 0, 4, 8, 16, 24 and 48 hours after rVSV (blue) or rLCMV (red) infection by qPCR. Data are pooled from 2 independent experiments. Mean± SEM is shown. 0 hours n = 3; 4 hours n = 4; 8 hours n = 3 (rLCMV), 4 (rVSV); 16 hours n = 3 (rLCMV), 4 (rVSV); 24 hours n = 2 (rLCMV), 4 (rVSV); 48 hours n = 4. A two-way Anova with LSD post-test was applied. * p value<0.05. The same sample was measured repeatedly for the 4 genes.

Source data

Extended Data Fig. 7 Early type I IFN signalling promotes germinal centre B cells and antiviral antibody responses.

a, Quantification of IgD- CD95+ germinal centre (GC) B cells (left)—expressed as percentage of B220+ cells—and of IgG1+ cells (right)—expressed as percentage of B220+ B cells—in the dLNs of mice treated with anti-IFNAR blocking antibody (or isotype control), and infected with rVSV 14 days earlier. Mean + /- SEM is shown. n=3. An unpaired two-tailed t test was applied. *** p value<0.001. b, GP–binding IgG1 Abs (expressed as fold induction over uninfected controls) were measured in the sera of mice described in panel A, 14 days after rVSV infection. Data are pooled from 2 independent experiments. Mean ± SEM is shown. n = 7. An unpaired two-tailed t test was applied. * p value<0.05. c, Schematic representation of experimental procedure for the results described in Fig. 3d–f. 1 × 106 purified CD45.1+ Ag-specific (SMARTA) CD4+ T cells were transferred to CD45.2+ WT recipients and treated anti-IFNAR1 blocking antibody either 1 day prior to (light blue) or 1 day after (yellow) rVSV infection.

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Extended Data Fig. 8 Expression of interferon stimulated genes within the cellular components of the CD4+ T cell priming niche.

Expression of the indicated interferon-stimulated genes (ISGs) within the cellular components of the photoactivated CD4+ T cell priming niches of the mice described in Fig. 2d–f. The colour bar on the bottom indicates each cell’s origin (blue: photoactivated cells from rVSV; red: photoactivated cells from rLCMV).

Extended Data Fig. 9 Blocking IL-6 impairs antiviral antibody responses.

a, WT mice were treated with anti-IL-6 blocking antibody (or isotype control) and sera were collected 14 days after rVSV infection. GP–binding IgG2b Abs were measured in the sera and expressed as fold induction over uninfected controls. Data are representative of 2 independent experiments. Mean ± SEM is shown. An unpaired two-tailed t test was applied. *** p value<0.001. b, Schematic representation of experimental procedure for the results described in Fig. 4e. 1 × 106 purified CD45.1+ Ag-specific (SMARTA) CD4+ T cells were transferred to CD45.2+ WT recipients and treated with anti-IL-6 blocking antibody starting either 1 day prior to (yellow) or 1 day after rVSV infection (orange).

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Extended Data Fig. 10 Expression of interferon stimulated genes in dendritic cells.

Expression of different ISGs across 2179 single QC positive CD11c+ MHC-IIhigh cells grouped in 5 clusters as described in the legend to Fig. 5a.

Supplementary information

Reporting Summary

Supplementary Video 1

Multiphoton intravital microscopy in dLNs of wild-type mice injected with GFP+ antigen-specific CD4+ T cells (green) and Deep Red-labeled polyclonal B cells (gray) 3 d after intrafootpad infection with VSV-Ind, rVSV or rLCMV. Dashed lines denote B cell follicles based on polyclonal B cell positioning. Scale bars, 50 μm. Time is given in min:s. For four-dimensional analysis of cell migration, ten z stacks (spacing of 5 μm) of 50 μm were acquired every 10 s for 25–50 min. Data are representative of at least three independent experiments.

Supplementary Video 2

Multiphoton intravital microscopy in dLNs of wild-type mice injected with GFP+ antigen-specific CD4+ T cells (green) and Deep Red-labeled polyclonal B cells (gray) 2 d after infection with rVSV or rLCMV. Dashed lines denote B cell follicles based on polyclonal B cell positioning. Scale bars, 50 μm. Time is given in min:s. For four-dimensional analysis of cell migration, ten z stacks (spacing of 5 μm) of 50 μm were acquired every 10 s for 25–30 min. Data are representative of at least three independent experiments.

Supplementary Video 3

Multiphoton intravital microscopy in dLNs of wild-type mice injected with GFP+ antigen-specific CD4+ T cells (green) and Deep Red-labeled polyclonal B cells (gray) and treated with anti-IFNAR blocking antibody (or isotype control) 3 d after infection with rVSV. Dashed lines denote B cell follicles based on polyclonal B cell positioning. Scale bars, 50 μm. Time is given in min:s. For four-dimensional analysis of cell migration, ten z stacks (spacing of 5 μm) of 50 μm were acquired every 10 s for 25–50 min. Data are representative of at least three independent experiments.

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De Giovanni, M., Cutillo, V., Giladi, A. et al. Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells. Nat Immunol 21, 321–330 (2020). https://doi.org/10.1038/s41590-020-0596-6

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