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 (T_FH) or type 1 helper T (T_H1) 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 T_FH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into T_H1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4⁺ T cell differentiation and might instruct vaccine design strategies.

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 x 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. T_FH 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⁺); T_H1 were defined as T-bet⁺ CXCR5^- cells. (b) Representative flow cytometry plots showing T_FH and T_H1 cells (out of CD44^high 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 T_FH (top) and T_H1 cells (bottom) – expressed as percentages of CD44^high 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 T_FH (left) and T_H1 (right) cells (expressed as percentages out of total transferred cells) in the spleens of CD45.2⁺ WT recipients injected with 1 x 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 T_FH (left) and T_H1 (right) cells (expressed as percentages out of total transferred cells) in dLNs 5 days after infection of CD45.2⁺ WT recipients injected with 1 x 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 T_FH (left) and T_H1 (right) cell absolute numbers (f) or T_FH/T_H1 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.

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.

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 x 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), T_FH (c, left) and T_H1 (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 x 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-II^high CD8⁺ (cDC1) and CD11c⁺ MHC-II^high 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 x 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 T_FH (left) and T_H1 (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.

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.

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 x 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.

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 x 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).

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

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

Fig. 1. VSV and LCMV infections result in distinct antiviral CD4⁺ T cell polarization and in vivo dynamics

(a) Representative flow cytometry plots of transferred CD45.1⁺ Tg7 CD4⁺ T cells or CD45.1⁺ SMARTA CD4⁺ T cells (1 x 10⁶ each) in the footpad-draining popliteal LNs (dLNs) of CD45.2⁺ wild-type recipient mice 5 days post intrafootpad infection with VSV Ind (left) or LCMV WE (right), respectively (Extended Data 1a for schematic representation of experimental set up). Numbers indicate percentages within the indicated gates. Plots are representative of at least 5 independent experiments. (b) Representative flow cytometry plots of transferred CD45.1⁺ SMARTA CD4⁺ T cells (1 x 10⁶) in the footpad-draining popliteal LNs (dLNs) of CD45.2⁺ wild-type recipient mice 5 days post intrafootpad infection with rVSV (left) or rLCMV (right). Numbers indicate percentages within the indicated gates. Plots are representative of at least 5 independent experiments. (c) Quantification of T_FH cells (left) or T_H1 cells (right) as percentages of the transferred Tg7 (in case of VSV Ind infection) or SMARTA (in case of infection with rVSV or rLCMV) CD45.1⁺ CD4⁺ T cells in dLNs of CD45.2⁺ wild-type recipients at 0, 3, 5, 7 and 14 days post-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 VSV and rVSV samples, respectively, compared to rLCMV samples. A two-way Anova with LSD post-test was applied. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (d) Confocal micrographs of dLNs in wild-type (WT) mice injected with 1 x 10⁶ purified GFP⁺ Tg7 (VSV Ind) or SMARTA (all other groups) CD4⁺ T cells (green) and 3 x 10⁷ purified deep red-labelled polyclonal B cells (grey), 1 day prior to injection with PBS, VSV Ind, rVSV and rLCMV. The images on the bottom are the same as the images on top except that the channel pertaining to polyclonal B cells was removed to improve clarity. Images were collected 3 days after infection and are representative of at least 3 independent experiments. Scale bars represent 200 μm. (e) Quantification of follicular Ag-specific CD4⁺ T cells in dLNs of mice as in d. Mean+/- SEM is shown. n=2 (PBS), 4 (VSV Ind, rVSV, rLCMV). A one-way Anova with Bonferroni’s post-test was applied. *** p < 0.001 (f) Snapshots from multiphoton intravital imaging of dLNs in WT mice injected with 1 x 10⁶ purified GFP⁺ Tg7 (VSV Ind) or SMARTA (all other groups) CD4⁺ T cells (green) and 3 x 10⁷ purified deep red-labelled polyclonal B cells (grey), injected 1 day prior to injection with PBS, VSV Ind, rVSV and rLCMV (see Supplementary Movie 1). Cell tracks are coloured based on track speed mean values (blue = 0 μm/sec; red =1 μm/sec; see Supplementary Movie 1). Dashed lines define B cell follicles and were depicted based on polyclonal B cell positioning. Data are representative of at least 2 independent experiments. (g) Intravital cytometry plots of the movies in g showing either Tg7 (VSV Ind) or SMARTA (all other groups) CD4⁺ T cell XY cell positions over time (top), or meandering index versus track speed mean (bottom, see Supplementary Movie 1). Gates represent percentages of follicular CD4⁺ T cell tracks (top) and meandering index^hitrack speed mean^hi CD4⁺ T cell tracks (bottom). Numbers indicate the percentage of tracks within the indicated gates. Data are representative of at least 2 independent experiments.

Fig. 2. Characterization of the antiviral CD4⁺ T cell priming niche.

(a) Confocal micrographs of dLNs in mice injected with 5 x 10⁶ purified GFP⁺ Ag-specific (SMARTA) CD4⁺ T cells (green) and 3 x 10⁷ purified deep red-labelled polyclonal B cells (grey), 1 day before injection with PBS, rVSV or rLCMV. Images were acquired 2 days after infection and are representative of at least 2 independent experiments. The top panels depict the positioning of GFP⁺ Ag-specific (SMARTA) T cells whereas the bottom panels indicate the clustered Ag-specific (SMARTA) T cells (purple). A T cell cluster was defined as a minimum of 3 T cells within a distance of 15 μm measured from the centroid of each cell (see Materials and Methods). Scale bars represent 200 μm. See also Supplementary Movie 2. (b, c) Quantification of the normalized distance of Ag-specific (SMARTA) T cells from the T cell area centre (b) and percentages of clustered T cells / section (c) in the mice described in a. Mean+/- SEM is shown. PBS n =772, rVSV n=1025, rLCMV n=1127 (b); PBS n = 3, rVSV n=5, rLCMV n=4 (c). Results are pooled from 2 independent experiments. A one-way Anova with Bonferroni’s post-test was applied. * p < 0.05; **** p < 0.0001. (d) Multiphoton intravital micrographs depicting the photoactivation of the CD4⁺ T cell priming niche upon rVSV or rLCMV infection. Images were acquired 2 days after infection. Ag-specific (SMARTA) CFP⁺ CD4⁺ T cells are depicted in green and PA-GFP⁺ in blue. Top panels, before photoactivation; bottom panels, after photoactivation. Scale bars represent 50 μm. Images are representative of at least 2 independent experiments. (e) Gene expression profiles of 2406 single cells from photoactivated priming niches, grouped in 7 clusters. The colour bar on the bottom indicates each cell’s origin (blue: photoactivated cells from rVSV infected mice; red: photoactivated cells from rLCMV infected mice). (f) Relative abundance of different cell clusters within CD4⁺ T cell priming niches. Data represent cell counts from two biological replicates. Two-sided FDR-adjusted Fisher’s exact test. * p < 0.05; *** p < 0.001; **** p < 0.0001. (g-i) 1 x 10⁶ purified CD45.1⁺ Ag-specific (SMARTA) CD4⁺ T cells were injected into CD45.2⁺ WT recipients and into either VI10YEN (g, n=6), Cor93 Tg TCR (h, n=4 or 6), or Ccr2^-/- (I, n=4 or 6) mice. dLNs were collected 3 (g) or 5 (h-i) days after rVSV (blue) or rLCMV (red) infection. Percentages of T_FH (g-i, left) and T_H1 (h-i, right) Ag-specific CD4⁺ T cells out of total transferred cells in dLNs were quantified by flow cytometry. Data are representative of at least 2 independent experiments. Mean+/- SEM is shown. A one-way Anova with Bonferroni’s post-test was applied. ** p < 0.01; *** p < 0.001

Fig. 3. Spatiotemporal regulation of type I IFN expression determines antiviral CD4⁺ T cell polarization.

(a) Expression profile of selected ISGs (Irf7, Cxcl9, Cxcl10, Oasl1, Ifitm3, Oas2, Isg15) in 2406 single cells from the photoactivated CD4⁺ T cell priming niches described in Fig. 2 d-f. Data are pooled from 2 independent experiments. Kolmogorov–Smirnov test was applied. **** p < 0.0001. (b) Analysis of Ifna4, Ifnb (top) and Isg15, Oas2 (bottom) gene expression in dLN at 0, 4, 8, 16, 24 and 48 hours after rVSV (blue) or rLCMV (red) infection by qPCR. 0 hours n = 3; 4 hours n = 4; 8 hours n = 4; 16 hours n = 4; 24 hours n = 3 (rLCMV), 4 (rVSV); 48 hours n = 4. Data are pooled from 2 independent experiments. Mean+/- SEM is shown. A two-way Anova with LSD post-test was applied. * p < 0.05; ** p < 0.01; *** p < 0.001. The same sample was measured repeatedly for the 4 genes. (c) Confocal micrographs of dLNs in REX3 reporter mice injected with 1 x 10⁷ purified GFP⁺ Ag-specific (SMARTA) CD4⁺ T cells (green) and 3 x 10⁷ purified deep-labelled polyclonal B cells (grey), 12 hours after rVSV or rLCMV infection. CXCL10⁺ (blue) and CXCL9⁺ (red) cells are depicted. Data are representative of at least 2 independent experiments. (d) Representative flow cytometry plots show T_FH and T_H1 within Ag-specific CD4⁺ T cells, 5 days after infection of CD45.2⁺ WT recipients injected with 1 x 10⁶ purified CD45.1⁺ SMARTA CD4⁺ T cells and treated with anti-IFNAR1 blocking antibody (or isotype control) 1 day prior to rVSV (blue, left) or rLCMV (red, right) infection. Numbers represent the percentage of cells within the indicated gate. (e) Quantification of T_FH (top) and T_H1 (right) – expressed as percentages of Ag-specific CD4⁺ T cells out of total transferred cells – in dLNs of mice described in d. Mean+/- SEM is shown. n=6 (rVSV), 8 (rLCMV). Data are representative of at least 2 independent experiments. A one-way Anova with Bonferroni’s post-test was applied. *** p < 0.001. (f) Quantification of the percentages of T_FH (left) and T_H1 (right) Ag-specific CD4⁺ T cells (out of total transferred cells) in dLNs 5 days after infection of CD45.2⁺ WT recipients injected with 1 x 10⁶ purified CD45.1⁺ Ag-specific (SMARTA) CD4⁺ T cells and treated anti-IFNAR1 blocking antibody either 1 day prior to (light blue) or 1 day after (yellow) rVSV infection. Data are representative of at least 2 independent experiments. Mean +/- SEM is shown. n=4. A one-way Anova with Bonferroni’s post-test was applied. ** p < 0.01; *** p < 0.001. (g) Quantification of the percentages of T_FH (left) and T_H1 (right) Ag-specific CD4⁺ T cells (out of total transferred cells) in dLNs 5 days after infection (bottom) of CD45.2⁺ WT recipients injected with 1 x 10⁶ purified CD45.1⁺ Ag-specific (SMARTA) CD4⁺ T cells, infected with rLCMV and treated or not with Poly(I:C).. Data are representative of at least 2 independent experiments. PBS n=7, Poly(I:C) n=9. An unpaired two-tailed t test was applied. ** p < 0.01.

Fig. 4. Early type I IFN sensing by DC and IL-6 are essential for antiviral T_FH differentiation.

(a) Quantification of the percentages of T_FH and T_H1 Ag-specific CD4⁺ T cells (out of total transferred cells) in dLNs 5 days after rVSV infection of CD45.2⁺ WT mice injected with 1 x 10⁶ purified CD45.1⁺ WT or Ifnar1^-/- Ag-specific (SMARTA) CD4⁺ T cells. Mean +/- SEM is shown. n=3 (b) Quantification of the percentages of T_FH and T_H1 Ag-specific (SMARTA) CD4⁺ T cells (out of total transferred cells) in dLNs 5 days after rVSV infection of either CD45.2⁺ CD11c-Cre or CD45.2⁺ CD11c-Cre x Ifnar1^fl/fl recipients injected with 1 x 10⁶ purified CD45.1⁺ Ag-specific (SMARTA) CD4⁺ T cells. dLNs were collected 5 days after rVSV infection. Data are representative of 3 independent experiments. Mean+/- SEM is shown. CD11c-Cre n=3, CD11c-Cre x Ifnar1^fl/fl n=4. An unpaired two-tailed t test was applied. *** p < 0.001; **** p < 0.0001. (c) qPCR analysis of Il6 gene expression profile at 0, 4, 8, 16, 24 and 48 hours in dLNs of rVSV (blue)- or rLCMV (red)-infected mice. Mean+/- SEM is shown. 0 hours n = 4; 4 hours n = 4; 8 hours n = 4; 16 hours n = 4; 24 hours n = 3 (rLCMV), 4 (rVSV); 48 hours n = 4. Data are pooled from 2 independent experiments. A two-way Anova with LSD post-test was applied. * p < 0.05; ** p < 0.01. (d) qPCR analysis of Il6 gene expression in either wild-type or Ifnar1^-/- mice in dLNs 8 hours after rVSV infection. Data are pooled from 2 independent experiments. Mean +/- SEM is shown. An unpaired two-tailed t test was applied. * p < 0.05. (e) Quantification of the percentages of T_FH (left) and T_H1 (right) Ag-specific CD4⁺ T cells (out of total transferred cells) in CD45.2⁺ wild-type recipients injected with 1 x 10⁶ purified CD45.1⁺ Ag-specific (SMARTA) CD4⁺ T cells and treated with anti-IL-6 blocking antibody starting either 1 day prior to (yellow) or 1 day after rVSV infection (orange). Data are representative of at least 2 independent experiments. Mean +/- SEM is shown. No Ab n=5, IL-6 Ab d -1 n=5, IL-6 Ab d +1 n=3. A one-way Anova with Bonferroni’s post-test was applied. * p < 0.05; ** p < 0.01.

Fig. 5. scRNA-seq analysis of lymph node DCs upon viral infection.

(a) Gene expression profiles of 2179 single QC-positive lymph node CD11c⁺ MHC-II^high cells grouped in 5 clusters. The colour bars on the bottom indicates each cell’s origin (blue: cells from the popliteal lymph node of mice 8 hours after intrafootpad rVSV infection; pink: cells from the popliteal lymph node of mice 8 hours after intrafootpad rLCMV infection; red: cells from the popliteal lymph node of mice 48 hours after intrafootpad rLCMV infection; light grey: WT mice; dark grey: Ifnar1^-/- mice). (b) Relative abundance of different DC clusters 8 hours after rVSV infection, 8 hours after rLCMV infection and 48 hours after rLCMV infection. Dashed lines show changes in cluster abundances between WT and Ifnar1^-/- mice under the same infection condition. Significant changes in abundances are highlighted. Two-sided FDR-adjusted Fisher’s exact test. * p < 0.05; *** p < 0.001 (c) Colour bar shows total size-normalized expression of interferon stimulated genes (ISGs) in cells in (a). Detailed expression of individual ISG genes is shown in Extended Data Fig. 10. (d) Distribution of total size-normalized expression of ISGs in CD11c⁺ MHC-II^high cells grouped by experimental condition. (e) Total expression of the ISG Ifit1, and the T_FH cell-promoting cytokine Il6 in different experimental conditions. Values represent size-normalized total transcripts per 100 cells. Colours represent relative contribution from the five different DC subsets.