Fig 1.
Splenic Germinal Centre B-cell and plasmablast responses are strongly dependent on CD4+ cells during blood-stage Plasmodium infection.
(A) Parasitemia and (B) survival of WT mice (n = 6) treated with CD4-depleting monoclonal antibody (αCD4) or control IgG 1 day prior to infection with Py17XNL. (C) Flow cytometric gating strategy employed to analyze splenic B-cell responses throughout the manuscript. (D-F) WT mice (n = 5) were administered αCD4 or control-IgG prior to Py17XNL infection. Presented are representative FACS plots (gated on B220+ CD19+ live singlets), proportions and absolute numbers in the spleen of (D) plasmablasts (IgDlo CD138hi), (E) GC B-cells (GL-7+ Fas+), and (F) Ig-switched B-cells (IgDlo IgMlo cells) from naïve and infected, control IgG and αCD4-treated WT mice, 10 days p.i. Statistics: Mann-Whitney U test, **P<0.01, except (B) in which Log-rank test applied (** p = 0.0012). Experiment in A&B done once, C-F representative of two independent experiments.
Fig 2.
ICOS-signalling promotes humoral immune responses during blood-stage infection.
(A) Flow cytometric gating strategy employed to analyze splenic CD4+ T-cell responses throughout the manuscript. (B) Representative FACS plots and time-course analysis of cell-surface ICOS expression on splenic CD4+ T-cells from WT mice (n = 3/time point) during Py17XNL infection. (C-F) WT mice (n = 6/group) were treated with anti-ICOSL blocking monoclonal antibody (α-ICOSL) or its isotype control (rat-IgG2a) prior to and during infection with Py17XNL. (C) Representative FACS plots (gated on B220+ CD19+ live singlets), proportions and numbers of splenic GC B-cells (GL-7+ Fas+), (D) numbers of splenic Ig-class switched (IgDlo IgMlo) B-cells, and (E) representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and numbers of splenic Tfh cells (PD1+ CXCR5+), in naïve mice and infected, α-ICOSL and control IgG-treated mice, 16 days p.i. (F) Py17XNL-specific IgM, total IgG, IgG2b and IgG3 levels in serum of naïve and infected, α-ICOSL and control IgG-treated mice, 16 days p.i. (G) Parasitemias in WT mice (n = 6/group) infected with Py17XNL, and treated every three days with α-ICOSL or control IgG until day 21 p.i. (depicted by arrows, with estimated period of cover highlighted with shaded grey box—an α-ICOSL-treated mouse succumbed to infection on each of days 15, 17 and 30 p.i., and one control-IgG treated mouse succumbed on day 20 p.i. Data representative of two independent experiments in (B-E), and two pooled independent experiments in (F), with experiment in (G) being conducted once. Statistics: Mann-Whitney U test, *P<0.05; **P<0.01.
Fig 3.
IFNAR1-signalling obstructs B-cell and parasite-specific antibody responses during blood-stage infection.
(A) Time-course analysis of parasitemia in WT and Ifnar1-/-mice (n = 5/group) infected with Py17XNL. (B) ELISA quantitation of Py17XNL-specific IgM, total IgG, IgG1, IgG2b, IgG3 in serum diluted from 1 in 400 in two-fold sequential dilutions to 1 in 3200, and (C) IgG2c at 1 in 400 in the sera of naïve and infected WT and Ifnar1-/- mice, 16 days p.i. (D&E) Representative FACS plots (gated on B220+ CD19+ live singlets), proportions and absolute numbers of (D) splenic GC B-cells (GL7+ Fas+) and (E) Ig-class-switched B-cells (IgDlo IgMlo) in naïve and infected WT and Ifnar1-/- mice, 16 days p.i. (F) Representative FACS plots (gated on B220+ CD19+ live singlets), proportions and absolute numbers of splenic plasmablasts (IgDloCD138hi) in naïve and infected WT and Ifnar1-/- mice (n = 6), 6 days p.i. (G) Time course analysis of IgM, total IgG, IgG2b and IgG3 levels in naïve and infected WT and Ifnar1-/- mice (n = 6). Statistics: Mann-Whitney U test (A & C-G), Two way ANOVA and Tukey’s test for multiple comparisons in (B), *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data representative of three independent experiments for (A), (D) and (E), two for (B) and (F) and one for (C) and (G).
Fig 4.
IFNAR1-signalling limits splenic Tfh cell responses.
(A) WT and Ifnar1-/- mice (n = 5/group) were infected with Py17XNL, and splenic Tfh (PD1+ CXCR5+) cell proportions and absolute numbers were assessed at day 16 p.i. Representative FACS plots gated on CD4+ TCRβ+ live singlets. Data representative of three independent experiments; Mann-Whitney U test *P<0.05; **P<0.01. (B) WT and Ifnar1-/- mice (n = 5-6/group) were infected with PcAS, and splenic Tfh (PD1+ CXCR5+ or Bcl-6+ CXCR5+) cell proportions and absolute numbers were assessed at day 8 p. i. Representative FACS plots gated on CD4+ TCRβ+ live singlets. Data representative of three independent experiments; Mann-Whitney U test *P<0.05; **P<0.01.
Fig 5.
IFNAR1-signalling restricts CD4+ T-cell expression of ICOS and proximity to B cell areas of the spleen.
(A) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and absolute numbers of splenic ICOS+ CD4+ T-cells in naïve, and Py17XNL-infected WT and Ifnar1-/-mice (n = 6 per group) 6 days p.i. Experiment performed once. Mann-Whitney U test **P<0.01. B) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and absolute numbers of splenic ICOS+ CD4+ T-cells in naïve, and PcAS-infected WT and Ifnar1-/-mice (n = 6 per group) 6 days p.i. Data representative of three independent experiments. Mann-Whitney U test **P<0.01. C) Representative confocal microscopy images showing spleen sections from naïve (n = 2) and PcAS-infected WT and Ifnar1-/-mice (n = 5) on day 5 p.i., stained for ICOS (Green), CD3 (Blue), and B220 (Red). T-B borders are outlined by white dotted lines. Summary graphs illustrate ICOS+ CD3+ cell densities for individual T/B borders and B cell follicles, with data pooled from four T-B borders or follicles per mouse, (n = 2 for naïve and n = 5 for WT and Ifnar1-/- mice). Scale bar: 100μm. Mann-Whitney U test *P<0.05; **P<0.01.
Fig 6.
Antibody-mediated IFNAR1 blockade boosts humoral immune responses during blood-stage infection.
WT mice (n = 5/group) were treated with anti-IFNAR1 blocking antibody (α-Ifnar1) or control IgG prior to and during infection with PcAS. (A) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and absolute numbers of splenic ICOS+ CD4+ T cells in naïve and infected mice on days 6 and 7 p.i. (B) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and numbers of splenic Tfh cells (as PD1+CXCR5+ CD4+ T cells) in naïve and infected and antibody-treated mice, 7 days p.i. Bcl-6 expression is also shown in histograms for Tfh (PD1+CXCR5+; red gate) and non-Tfh cells (PD1-CXCR5-; blue gate), alongside Geometric Mean Bcl-6 expression by these populations in individual mice. (C and D) Numbers of splenic (C) plasmablasts and (D) GC B cells in naïve, and infected and treated mice, 7 days p.i. Data representative of 2 independent experiments. Mann-Whitney U test *P<0.05; **P<0.01.
Fig 7.
IFNAR1-signalling simultaneously limits splenic Th1 and Tfh cell responses.
(A & B) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and absolute numbers of splenic (A) Th1 (IFNγ+ Tbet+) and (B) emerging Tfh (PD1+ CXCR5+) cells in WT and Ifnar1-/- mice (n = 6/group), 6 days p.i with PcAS. Data representative of two independent experiments. (C) Numbers of splenic ICOS+ Th1 cells (Tbet+ IFNγ+ CD4+ T cells) and Tfh cells (PD1+CXCR5+ CD4+ T cells) 6 days p.i. with PcAS. (D) Numbers of ICOS+ Tfh cells (PD1+CXCR5+ CD4+ T cells), 16 days p.i. with Py17XNL infection. (E) Proportions and absolute numbers of splenic CD4+ T-cells expressing Ki-67 in naïve, WT and Ifnar1-/- mice 6 days p.i. with PcAS. (F) Proportions and absolute numbers of splenic CD4+ T-cells expressing Ki-67 in naïve mice, and WT mice, 6 days p.i. with Py17XNL and treatment with α-IFNAR1 or Control IgG. (G) Absolute numbers of splenocytes, CD4+ T-cells and B-cells, in WT naïve, infected WT and Ifnar1-/- mice 6 days (n = 17–18, pooled from three independent experiments (n = 5–6 per expt)) and 8 days (n = 29, pooled from five experiments (n = 5–6 per expt)) p.i. with PcAS. (H) Absolute numbers of splenocytes, CD4+ T-cells and B-cells in WT naïve, infected WT and Ifnar1-/- mice, 16 days p.i. with Py17XNL (n = 17–18, pooled from three experiments (n = 5–6 per expt)) Mann-Whitney U-test **P<0.01, *P<0.05.
Fig 8.
IFNAR1-signalling via cDCs limits Tfh and GC B-cell responses.
(A) Schematic for the mixed bone marrow chimeric approach: 50:50 mixed WT (Ifnar1+/+; CD45.1) and Ifnar1-/- (CD45.2) bone marrow was transferred into lethally-irradiated rag1-/- mice (to avoid potential issues with residual radio-resistant T- and B-cells), and subsequently left for 8–12 weeks prior to infection studies. (B) Gating strategy for splenic WT (CD45.1+) and Ifnar1-/- (CD45.2+) CD8+ (TCRβ- B220- CD11chi MHC-IIhi CD8α+) and CD8- (TCRβ- B220- CD11chi MHC-IIhi Sirpα+ CD8-) conventional DCs. (C) Paired analysis between splenic WT and Ifnar1-/- cDC subsets in individual mice for cell-surface expression of CD86, PD-L1, PD-L2, CD40 and ICOS-L, 2 days p.i. with Py17XNL. Data representative of two independent experiments. Statistics: Wilcoxon test, *P<0.05. (D) Representative FACS plots and proportions for WT (CD45.1+) Ifnar1+/+ and Ifnar1-/- plasmablasts, 7 days p.i. with PcAS infection. Data representative of 2 independent experiments. Statistics: Wilcoxon test. (E) Representative FACS plots and proportions for WT (CD45.1+) Ifnar1+/+ and Ifnar1-/- CD4+ T cells expressing ICOS and CXCR5, 7 days p.i. with PcAS infection. Experiment performed once. Statistics: Wilcoxon test, *P<0.05. (F) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and absolute numbers of splenic Tfh cells and (G) splenic GC B-cells (gated on B220+ CD19+ live singlets) in CD11cCre+/- ifnar1f/f and CD11cCre-/- ifnar1f/f littermates (n = 6) (WT naïve mice as staining controls), on day 6 p.i. with Py17XNL; Statistics: Mann-Whitney U-test **P<0.01, *P<0.05. Data representative of 2 independent experiments.
Fig 9.
IFNAR1-deficiency boosts humoral immunity via enhanced ICOS-signalling.
WT and Ifnar1-/- mice (n = 6/group), infected with PcAS, and treated with αICOSL (100μg) or control IgG2a throughout infection (days 0, 2, 4 and 6 p.i.), were assessed on day 8 p.i. for (A) parasite-specific IgM and IgG, as well as (B) Parasitemia. (C&D) WT and Ifnar1-/- mice were infected with PcAS, and treated as in (A & B) with α-ICOSL or control IgG2a: (C) Representative FACS plots (gated on CD4+ TCRβ+ live singlets), proportions and absolute numbers of splenic Tfh cells, and (D) proportions and absolute numbers of GC B-cells (gated on B220+ CD19+ live singlets) on day 6 p.i. Statistics: One-way ANOVA, Tukey’s test for multiple comparisons, *P<0.05; **P<0.01; ****P<0.0001. (A) & (B) representative of two independent experiments. (C) & (D) representative of three independent experiments.