An IL-21R STAT hypomorph circumvents functional redundancy in germinal centre responses
- Department of Immunology, Garvan Institute of Medical Research, Australia
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia
Figures
Figure 1 with 1 supplement
Dominant defect in IL-21-induced STAT1 activation in Il21rEINS cells.
The structure of murine IL-21R generated by alphaFOLD, showing the einseitig mutation leading to a change from aspartic acid to valine at amino acid position 375 (A). The aspartic acid at amino acid position 375 is highly conserved (B). Splenocytes from naive WT, Il21rEINS, and Il21r−/− mice were stimulated with 80 ng/ml rmIL-21, rmIL-6, or incubated in media for unstimulated controls for 15 min. Western blots of total splenocytes either unstimulated or stimulated for 15 mins with rmIL-21, lysed with RIPA buffer and stained with antibodies against phosphorylated (p) STAT1 (PY701) and STAT1 (C), pSTAT3 (PY705) and STAT3 (D) are representative of four similar individual experiments. For flow cytometric analysis we gated on TCRβ+ CD4+ cells (E) to assess levels of intracellular P-STAT1 (F) and P-STAT3 (G) expression on CD4+ T cells from individual mice after stimulation for 15 min in vitro with recombinant murine IL-21 where n = 5–8 mice per group. Titration curves showing phosphorylation of (H) STAT1 and (I) STAT3 determined by flow cytometry and FACS analyses after 15 min stimulation of spleen derived CD4+ T cells with rmIL-21. Data are shown as mean ± SD where n = 5 mice per group, representative of 4 similar individual experiments. Statistical significance was assessed by one-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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Figure 1—source data 1
Western blot analyses of STAT1 and STAT3.
Source data showing uncropped Western blots with proteins and molecular weight markers indicated and the relevant sections of the cropped blots as shown in Figure 1 for phosphorylated STAT1 and total STAT1 as shown in Figure 1 panel C and phosphorylated STAT3 and total STAT3 as shown in Figure 1 panel D.
- https://cdn.elifesciences.org/articles/109834/elife-109834-fig1-data1-v1.pdf
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Figure 1—source data 2
Western blot analyses of phosphorylated STAT1.
Source data showing the original uncropped Western blots at multiple serial exposures.
- https://cdn.elifesciences.org/articles/109834/elife-109834-fig1-data2-v1.tif
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Figure 1—source data 3
Western blot analyses of total STAT1.
Source data showing the original uncropped Western blots at multiple serial exposures.
- https://cdn.elifesciences.org/articles/109834/elife-109834-fig1-data3-v1.tif
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Figure 1—source data 4
Western blot analyses of phosphorylated STAT3.
Source data showing the original uncropped Western blots at multiple serial exposures.
- https://cdn.elifesciences.org/articles/109834/elife-109834-fig1-data4-v1.tif
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Figure 1—source data 5
Western blot analyses of total STAT3.
Source data showing the original uncropped Western blots at multiple serial exposures.
- https://cdn.elifesciences.org/articles/109834/elife-109834-fig1-data5-v1.tif
Figure 1—figure supplement 1
The Il21rEINS mutation has little to no effect on STAT5 activation in CD4+ T cells.
Splenocytes from naive WT, Il21rEINS, or Il21r−/− mice were stimulated with 100 ng/ml rmIL-21, rmIL-6, or rmIL-2 for 15 min. Cells were fixed and stained for intracellular phosphorylated (p) STAT3 (A) and pSTAT1 (B) using the BD Phosphlow system and pSTAT expression was analysed in CD4+ T cells via flow cytometry. Data shown are representative of 3 similar experiments. Splenocyte derived total CD4+ T cells or CD44hi CD4+ T cells from WT, Il21rEINS, and Il21r−/− mice were stimulated with rmIL-2, rmIL-21, or incubated in media for unstimulated controls for 15 min. Cells were fixed and stained for intracellular pSTAT5 using the BD Phosphlow system. Mean fluorescence intensity (MFI) of pSTAT5 in CD4+ T cells (C) and percentage pSTAT5+ CD44hi CD4+ T cells (D) were assessed. CD4+ T cells from the spleen of WT, Il21rEINS, or Il21r−/− mice were stimulated with decreasing concentrations of rmIL-21, expression levels of pSTAT5 (E) and percent CD4+ T cells containing pSTAT5 (F). Data are shown as mean ± SD, n = 4–5 mice per group with 3 experimental repeats. Statistical analyses were performed by two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2 with 2 supplements
The Il21rEINS mutation reduces the differentiation of T follicular helper (Tfh) cells.
Analysis of the germinal centre (GC) response in the spleen in WT, Il21r−/−, and Il21rEINS mice on day 7 following sheep red blood cell (SRBC) immunization. (A) Histological sections of the spleen on day 7 of SRBC immunization showing CD4+ cells (red), IgD (green), and IgG1 (blue). (B) FACS dot plots showing gating strategy for CXCR5high and PD-1high CD4+ T cells and FoxP3+ Tfr cells. (C) Percentage of Tfh cells (CXCR5high PD-1high FoxP3− CD4+ T cells) as a percentage of CD4+ T cells population. (D) Percentage of FoxP3+ T follicular regulatory (Tfr) cells of the CXCR5high PD-1high CD4+ T cell population. (E) Percentage of FoxP3+ Treg cells within the CD4+ T cell population. Values shown from individual mice (n = 8–12 per group) including means ± SD from 2 pooled experiments with 2 experimental replicates. Kinetics of the (F) Tfh cell response and (G) the T follicular regulatory (Tfr) cell response over a 21-day time course data is shown as means ± SD, n = 4–8 with 3 experimental repeats. Statistical significance was assessed by one-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2—figure supplement 1
Lymphocyte development appears unaffected by the Il21rEINS mutation.
Analysis of thymocytes and splenocytes from naive WT and Il21rEINS mice, representative dot plot showing the gating of CD4+, CD8+ and CD4+ CD8+ double positive (DP) thymocytes (A), as well as percentages of CD4+ CD8+ DP thymocytes (B), CD8+ single positive thymocytes (C), CD4+ single positive thymocytes (D), and FoxP3+ CD4+ T cells (E) in the thymus. Representative dot plot showing the gating of CD4+, CD8+ T cells in the spleen (F), as well as percentages of CD8+ T cells (G), CD4+ T cells (H), and FoxP3+ CD4+ T cells (I) in the spleen of naive mice. Also shown are percentages of B cells (J), CD138+ plasma cells (K), CD21med CD23+ follicular B cells (L), CD21+ CD23− marginal zone B cells (M). NK1.1+ NK cells (N), NK1.1+ TCRb+ NKT cells (O), CD11c+ CD11low TCRβ- B220- dendritic cells (DC) (P), and Ly6G+ CD11c− CD11b+ TCRβ− B220− neutrophils (Q). Values shown from individual mice, including means ± SD from 3 replicate experiments where n = 4–5 per group. Statistical analyses performed by Student’s t-test.
Figure 2—figure supplement 2
Decreased numbers of follicular T cell populations in Il21rEINS mice.
Il21rEINS mice were immunized with 2 × 108 sheep red blood cells (SRBCs) and lymphocyte populations were analysed 7 days later by flow cytometry and FACS analyses. Numbers of CXCR5hi PD1hiT FoxP3− follicular helper (Tfh) cells (A), CXCR5hi PD1hiT FoxP3+ T follicular regulatory (Tfr) cells (B), as well as the overall CXCR5+ PD-1+ follicular CD4+ T cell population (C) of WT, Il21rEINS, and Il21r−/− mice are shown. Values shown are from individual mice, including means ± SD from 3 pooled experiments where n = 3–5 per group.
Figure 3
The Il21rEINS mutation reduces germinal centre (GC) output.
WT, Il21r−/−, and Il21rEINS mice were immunized with sheep red blood cell (SRBC), and the kinetics of the immune response was observed by analysing the response on the timepoints indicated. FAS+ GL7+ GC B cells as a percentage of total B cells (A) and IgG1+ FAS+ C cells as a percentage of total B cells (B). Absolute numbers of GC B cells (C) and IgG1+ FAS+ B cells (D) per spleen. Analysis of splenocytes on day 7 following SRBC immunization. Data are shown as individual mice (n = 8–12 per group) and mean ± SD with 2 experimental repeats. Kinetics of the GC B cell response (E) and the IgG1+ FAS+ B cell response (F) in the spleen of WT and Il21rEINS mice. Values are shown as means ± SD (n = 4–5 per group), with 3 experimental repeats. Statistical significance was assessed by one-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 4
T cell intrinsic effect of Il21rEINS on the germinal centre (GC) response.
Mixed bone marrow (BM) chimeras were reconstituted with equal ratios of WT CD45.1+ BM cells and Il21rEINS CD45.2+ BM cells. Eight weeks after transfer, the mice were immunized with sheep red blood cell (SRBC) and analysed 7 days later. Gating strategy employed to differentiate Tfh and Tfr cells from WT CD45.1+ and Il21rEINS CD45.2+ donor mice (A). Percentage of CD45.2+ donor cells within the total CD4+ T cell population alongside the CD45.2+ CXCR5hi PD1hi FoxP3− Tfh cell population (B), percentage of CD45.2+ donor cells within the total CD4+ T cell population alongside CD45.2+ CXCR5hi PD1hi FoxP3+ Tfr cells (C). Relative percentage of WT CD45.1+ and CD45.2+ Il21rEINS cells of Tfh cells (D), relative percentage of WT CD45.1+ and CD45.2+ Il21rEINS cells of Tfr cells (E) relative percentage of CD45.1+ versus CD45.2+ Il21rEINS Treg cells (F). Control mixed BM chimeras were reconstituted with equal ratios of WT CD45.2+ BM cells and WT CD45.1+ BM cells. Eight weeks after transfer, chimeras were immunized with SRBC and analysed 7 days later. Percentage of CD45.2+ donor cells within the total CD4+ T cell population and percentage of CD45.2+ donor cells within the Treg population (G). Percentage CD45.2+ within the CXCR5hi PD1hi FoxP3+ Tfr population (H), percentage of CD45.2+ CXCR5hi PD1hi FoxP3− Tfh cell population (I). Percentage of CD45.2+ donor cells within the total B cell population alongside the percentage of CD45.2+ donor cells within the GC B cell population (J), percentage of CD45.2+ donor B cells within the IgG1+ FAS+ B cell population (K). Relative percentage of WT CD45.1+ and Il21rEINS CD45.2+ GC B cells (L), relative percentage of CD45.1+ and CD45.2+ IgG1+ FAS+ B cells (M). Data are shown as individual mice (n = 4–7 per group), 3 separate experiments with similar results. Statistical significance was assessed by paired Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 5
The Il21rEINS mutation affects the expression of Tfh functional molecules.
Staining for ICOS and IL-21 on Tfh and Tfr cells from WT and Il21rEINS 7 days after sheep red blood cell (SRBC) immunization. Percentage of IL-21+ Tfh cells (A), histogram showing IL-21 expression in Tfh cells (B), and quantification of IL-21 expression (MFI) in Tfh cells (C). Percentage of ICOS+ Tfh cells (D), histogram showing ICOS expression on Tfh cells (E), and levels of ICOS expression on Tfh cells (MFI) (F). Percentage ICOS+ Tfr cells (G), histogram showing ICOS expression on Tfr cells (H), and levels of ICOS expression on Tfr cells (I). Data are shown from individual mice (n = 4–8 per group) and means ± SD, 3 experimental replicates with similar results. Statistical significance was assessed by Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 6
Genetic loss of IL-21:IL-21R signalling, but not IL-21-mediated STAT1 activation, influences IL-6Rα expression and responsiveness to IL-6.
IL-6 receptor (IL-6Rα) expression on CD4+ T cells from the spleen of WT, Il21r−/−, and Il21rEINS mice. Percentage of IL-6Rα expressing CD4+ T cells (A) and mean fluorescence intensity (MFI) of IL-6Rαon CD4+ T cells (B). Percentage (C) and MFI (D) of IL-6Rα expressing naive CD44lo CD4+ T cells. Percentage (E) and MFI (F) of IL-6Rα expressing activated/memory phenotype CD44hi CD4+ T cells. Graphs show individual mice (n = 5 per group) including means ± SD, two experiments with similar results. Statistical significance was assessed by one-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Flow cytometric analyses of the percentage of CD4+ T cells from the spleen of WT, Il21r−/−, and Il21rEINS mice containing phosphorylated STAT1 (G) and phosphorylated STAT3 (H) after stimulation with 40 ng/ml rmIL-6 over a 2-hr time course detected by immunostaining of phosphorylated STAT proteins, flow cytometry, and FACS analyses. Graphs show individual mice (n = 5 per group) including means ± SD, two experiments showing similar results. Statistical significance was assessed by two-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 7 with 1 supplement
The effect of Il21rEINS on the contribution of IL-6 to the germinal centre (GC) response.
Analysis of the GC response in the spleen in WT, Il21r−/−, and Il21rEINS mice on day 7 following sheep red blood cell (SRBC) immunization. IL-6 receptor (IL-6Rα) expression on CXCR5hi PD1hi FoxP3− T follicular helper (Tfh) cells from the spleen of WT, Il21r−/−, and Il21rEINS mice showing percentage (A) and mean fluorescence intensity (MFI) (B) and quantitation of MFI of IL-6Rα on Tfh cells (C). IL-6 signalling was blocked in vivo by the injection of 0.5 mg neutralizing anti-IL-6 mAb or isotype mAb and 0.25 mg on day 0, the day of SRBC immunization, and then days 2, 4, and 6. GC populations were analysed in the spleen on day 7. (D) Percentage of Tfh cells of CD4+ T cells, (E) percent IgG1+FAS+ B cells of total B cells. Data shown as individual mice (n = 4–5 per group) with 2 experimental repeats showing similar results. Statistical significance was assessed by one-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 7—figure supplement 1
Il21rEINS mutation does not affect functional CD4+ T cell responses to IL-6.
(A) Th17 cell conversion efficiency was compared using splenic CD4+ T cells from WT and Il21rEINS mice assessed on day 3 of stimulation after 4 hr of restimulation with PMA and ionomycin. IL-17 containing CD4+ T cells were assessed by intracellular immunostaining and flow cytometry. IL-6 signalling was blocked in vivo by the injection of 0.5 mg neutralizing anti-IL-6 mAb or isotype control mAb on day 0 followed by 0.25 mg on days 2, 4, and 6. Splenocytes from WT, Il21r−/− , and Il21rEINS mice were stimulated with rmIL-6 for 15 min at the concentrations shown and phosphorylated STAT1 (B) and phosphorylated STAT3 (C) detected in CD4+ T cells by intracellular immunostaining and flow cytometry. Mice were immunized with sheep red blood cell (SRBC) on day 0 and analysed on day 7 by flow cytometry and FACS analyses. Shown here are the percentages of (D) Tfh cells within the CD4+ T cells population and (E) the germinal centre (GC) B cell population as a percentage of total B cells. Data are shown as individual mice with n = 5 per group from 2 separate experiments with similar results. Statistical significance was assessed by (A–C) one-way ANOVA using Bonferroni’s multiple comparisons test and Student’s t-test (D, E); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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An IL-21R STAT hypomorph circumvents functional redundancy in germinal centre responses
eLife 15:RP109834.
https://doi.org/10.7554/eLife.109834.3
