IFNAR1-signalling simultaneously limits splenic Th1 and Tfh cell responses.

<p>(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 <i>and Ifnar1</i>^<i>-/-</i> mice (n = 6/group), 6 days <i>p</i>.<i>i</i> with <i>Pc</i>AS. 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 <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (D) Numbers of ICOS⁺ Tfh cells (PD1⁺CXCR5⁺ CD4⁺ T cells), 16 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL infection. (E) Proportions and absolute numbers of splenic CD4⁺ T-cells expressing Ki-67 in naïve, WT and <i>Ifnar1</i>^<i>-/-</i> mice 6 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (F) Proportions and absolute numbers of splenic CD4⁺ T-cells expressing Ki-67 in naïve mice, and WT mice, 6 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL 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 <i>Ifnar1</i>^<i>-/-</i> 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)) <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (H) Absolute numbers of splenocytes, CD4⁺ T-cells and B-cells in WT naïve, infected WT and <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL (n = 17–18, pooled from three experiments (n = 5–6 per expt)) Mann-Whitney U-test **P<0.01, *P<0.05.</p

Antibody-mediated IFNAR1 blockade boosts humoral immune responses during blood-stage infection.

<p>WT mice (n = 5/group) were treated with anti-IFNAR1 blocking antibody (α-Ifnar1) or control IgG prior to and during infection with <i>Pc</i>AS. (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 <i>p</i>.<i>i</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 <i>p</i>.<i>i</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 <i>p</i>.<i>i</i>. Data representative of 2 independent experiments. Mann-Whitney U test *P<0.05; **P<0.01.</p

IFNAR1-signalling limits splenic Tfh cell responses.

<p>(A) WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 5/group) were infected with <i>Py</i>17XNL, and splenic Tfh (PD1⁺ CXCR5⁺) cell proportions and absolute numbers were assessed at day 16 <i>p</i>.<i>i</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 <i>and Ifnar1</i>^<i>-/-</i> mice (n = 5-6/group) were infected with <i>Pc</i>AS, and splenic Tfh (PD1⁺ CXCR5⁺ or Bcl-6⁺ CXCR5⁺) cell proportions and absolute numbers were assessed at day 8 <i>p</i>. <i>i</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.</p

IFNAR1-signalling obstructs B-cell and parasite-specific antibody responses during blood-stage infection.

<p>(A) Time-course analysis of parasitemia in WT and <i>Ifnar1</i>^<i>-/-</i>mice (n = 5/group) infected with <i>Py</i>17XNL. (B) ELISA quantitation of <i>Py</i>17XNL-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 <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</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 (IgD^lo IgM^lo) in naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</i>. (F) Representative FACS plots (gated on B220⁺ CD19⁺ live singlets), proportions and absolute numbers of splenic plasmablasts (IgD^loCD138^hi) in naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 6), 6 days <i>p</i>.<i>i</i>. (G) Time course analysis of IgM, total IgG, IgG2b and IgG3 levels in naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> 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).</p

IFNAR1-signalling via cDCs limits Tfh and GC B-cell responses.

<p>(A) Schematic for the mixed bone marrow chimeric approach: 50:50 mixed WT (<i>Ifnar1</i>^<i>+/+</i>; CD45.1) and <i>Ifnar1</i>^<i>-/-</i> (CD45.2) bone marrow was transferred into lethally-irradiated <i>rag1</i>^<i>-/-</i> 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 <i>Ifnar1</i>^<i>-/-</i> (CD45.2⁺) CD8⁺ (TCRβ^- B220^- CD11c^hi MHC-II^hi CD8α⁺) and CD8^- (TCRβ^- B220^- CD11c^hi MHC-II^hi Sirpα⁺ CD8^-) conventional DCs. (C) Paired analysis between splenic WT and <i>Ifnar1</i>^<i>-/-</i> cDC subsets in individual mice for cell-surface expression of CD86, PD-L1, PD-L2, CD40 and ICOS-L, 2 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL. Data representative of two independent experiments. Statistics: Wilcoxon test, *P<0.05. (D) Representative FACS plots and proportions for WT (CD45.1⁺) <i>Ifnar1</i>^<i>+/+</i> and <i>Ifnar1</i>^<i>-/-</i> plasmablasts, 7 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS infection. Data representative of 2 independent experiments. Statistics: Wilcoxon test. (E) Representative FACS plots and proportions for WT (CD45.1⁺) <i>Ifnar1</i>^<i>+/+</i> and <i>Ifnar1</i>^<i>-/-</i> CD4⁺ T cells expressing ICOS and CXCR5, 7 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS 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^+/- <i>ifnar1</i>^<i>f/f</i> and CD11cCre^-/- <i>ifnar1</i>^<i>f/f</i> littermates (n = 6) (WT naïve mice as staining controls), on day 6 <i>p</i>.<i>i</i>. with <i>Py</i>17XNL; Statistics: Mann-Whitney U-test **P<0.01, *P<0.05. Data representative of 2 independent experiments.</p

ICOS-signalling promotes humoral immune responses during blood-stage infection.

<p>(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 <i>Py</i>17XNL 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 <i>Py</i>17XNL. (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 (IgD^lo IgM^lo) 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 <i>p</i>.<i>i</i>. (F) <i>Py</i>17XNL-specific IgM, total IgG, IgG2b and IgG3 levels in serum of naïve and infected, α-ICOSL and control IgG-treated mice, 16 days <i>p</i>.<i>i</i>. (G) Parasitemias in WT mice (n = 6/group) infected with <i>Py</i>17XNL, and treated every three days with α-ICOSL or control IgG until day 21 <i>p</i>.<i>i</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 <i>p</i>.<i>i</i>., and one control-IgG treated mouse succumbed on day 20 <i>p</i>.<i>i</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.</p

IFNAR1-signalling restricts CD4⁺ T-cell expression of ICOS and proximity to B cell areas of the spleen.

<p>(A) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and absolute numbers of splenic ICOS⁺ CD4⁺ T-cells in naïve, and <i>Py</i>17XNL-infected WT and <i>Ifnar1</i>^<i>-/-</i>mice (n = 6 per group) 6 days <i>p</i>.<i>i</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 <i>Pc</i>AS-infected WT and <i>Ifnar1</i>^<i>-/-</i>mice (n = 6 per group) 6 days <i>p</i>.<i>i</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 <i>Pc</i>AS-infected WT and <i>Ifnar1</i>^<i>-/-</i>mice (n = 5) on day 5 <i>p</i>.<i>i</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 <i>Ifnar1</i>^<i>-/-</i> mice). Scale bar: 100μm. Mann-Whitney U test *P<0.05; **P<0.01.</p

IFNAR1-deficiency boosts humoral immunity via enhanced ICOS-signalling.

<p>WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 6/group), infected with <i>Pc</i>AS, and treated with αICOSL (100μg) or control IgG2a throughout infection (days 0, 2, 4 and 6 <i>p</i>.<i>i</i>.), were assessed on day 8 <i>p</i>.<i>i</i>. for (A) parasite-specific IgM and IgG, as well as (B) Parasitemia. (C&D) WT and <i>Ifnar1</i>^<i>-/-</i> mice were infected with <i>Pc</i>AS, 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 <i>p</i>.<i>i</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.</p

Elevated Blimp-1 levels in CD4⁺ T cells from VL patients were associated with increased IL-10 production.

<p><b>(A)</b><i>PRDM1</i> and (<b>B</b>) IL-10 mRNA accumulation in purified CD4⁺ cells isolated from the PBMCs, as well as (<b>C</b>) plasma IL-10 levels from VL patients before (closed circles; Pre) and 28 days after (open circles; Post) drug treatment. Data are from the same 10 paired samples, **p<0.01, *p<0.05, Wilcoxon signed-rank test.</p

Blimp-1 inhibits the control of parasite growth during experimental visceral leishmaniasis.

<p><i>Prdm1</i>^fl/fl and <i>Prdm1</i>^ΔT C57BL/6 mice were infected with <i>L</i>. <i>donovani</i> and spleen (<b>A</b>) and liver (<b>D</b>) parasite burdens were measured at times indicated. The frequency of Th1 and Tr1 cells in the spleen (<b>B</b>) and liver (<b>E)</b> were measured by flow cytometry. Organ weights and frequencies of TNF producing CD4⁺ T cells in the spleen (<b>C</b>) and liver (<b>F)</b> were also assessed, as were serum TNF and IFNγ levels (<b>G</b>). Antigen-specific Th1 cell frequency (<b>H</b>), IFNγ, TNF and IL-10 production (<b>I</b>) were measured in splenocytes after 24 hours of culture in the presence of parasite antigen at times indicated. In all panels, closed shapes represent <i>Prdm1</i>^fl/fl mice, while open shapes indicate <i>Prdm1</i>^ΔT littermates. Representative of 5 similar experiments, mean ±SEM, n = 5–6 in each group in each experiment, **p<0.01, *p<0.05, Mann-Whitney U test.</p