Immunology and Inflammation

An IL-21R hypomorph circumvents functional redundancy to define STAT1 signaling in germinal center responses

Christoph Jandl
Joanna Warren
Samantha Owens
Marcel Batten
Howard Wang
Cecile King author has email address

Department of Immunology, Garvan Institute of Medical Research, Darlinghurst, Australia
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia

https://doi.org/10.7554/eLife.109834.1

Open access
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Figures and data

Selective defect in IL-21 induced STAT1 activation in Il21r^EINS 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 naïve WT, Il21r^EINS and Il21r-/- mice were stimulated with 80ng/ml rmIL-21, rmIL-6 or incubated in media for unstimulated controls for 15min. 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 shown. For flow cytometric analysis we gated on TCRβ⁺ CD4⁺ cells (E) to assessed levels of intracellular P-STAT1 (F) and P-STAT3 (G) expression on CD4⁺ T cells after stimulation for 15 mins in vitro with recombinant murine IL-21. 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. Graphs show individual mice, Data are shown as mean ± SD, 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.

The Il21r^EINS mutation reduces the differentiation of T follicular helper cells.
WT, Il21r^−/− and Il21r^EINS mice were immunized with SRBC and the kinetics of the immune response was observed by analysing the response on the timepoints indicated. Analysis of the GC response in the spleen in WT, Il21r^−/−and Il21r^EINS mice on day 7 following SRBC immunization. (A) Histological sections of the spleen on day 7 of SRBC immunisation showing CD4+ cells (red), IgD (green) and IgG1 (blue). (B) FACS dot plots shows gating strategy for CXCR5^high and PD-1^high CD4⁺ T cells and FoxP3⁺ Tfr cells. (C) Percentage of T follicular helper (Tfh) cells (CXCR5^high PD-1^high FoxP3⁻ CD4⁺ T cells) as a percentage of CD4⁺ T cells population. (D) Percentage of FoxP3⁺ T follicular regulatory (Tfr) cells of the CXCR5^high PD-1^high CD4⁺ T cell population. (E) Percentage of FoxP3⁺ Treg cells within the CD4⁺ T cell population. Values shown from individual mice n=4-8 per group, from 2 pooled experiments including means +/− SD with 2 experimental replicates. Kinetics of the (F) T follicular helper (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 2 experimental replicates. 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.

The Il21r^EINS mutation reduces germinal centre output.
WT, Il21r^−/− and Il21r^EINS mice were immunized with 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), 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 mean +/− SD, n=5-8 mice with 2 experimental replicates. Kinetics of the GC B cell response (E) and the IgG1⁺ FAS⁺ B cell response (F) in the spleen of WT and Il21r^EINS mice. Values are shown from individual mice, including means +/− SD from 3 experimental repeats where n=4-5 per group. 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.

T cell intrinsic effect of Il21r^EINS on the germinal centre response.
Mixed BM chimeras were reconstituted with equal ratios of WT CD45.1⁺ BM cells and Il21r^EINS CD45.2⁺ BM cells. 8 weeks after transfer, the mice were immunized with SRBC and analysed 7 days later. Gating strategy employed to differentiate Tfh and Tfr cells from WT CD45.1⁺ and Il21r^EINS CD45.2⁺ donor mice (A). Percentage of CD45.2⁺ donor cells within the total CD4⁺ T cell population alongside the CD45.2⁺ CXCR5^hi PD1^hi FoxP3⁻ Tfh cell population (B), percentage of CD45.2⁺ donor cells within the total CD4⁺ T cell population alongside CD45.2⁺ CXCR5^hi PD1^hi FoxP3⁺ Tfr cells (C). Relative percentage of WT CD45.1⁺ and CD45.2⁺ Il21r^EINS cells of Tfh cells (D), relative percentage of WT CD45.1⁺ and CD45.2⁺ Il21r^EINS cells of Tfr cells (E) relative percentage of CD45.1⁺ versus CD45.2⁺ Il21r^EINS Treg cells (F). 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 (G), percentage of CD45.2⁺ donor B cells within the IgG1⁺ FAS⁺ B cell population (H). Relative percentage of WT CD45.1⁺ and Il21r^EINS CD45.2⁺ GC B cells (I), relative percentage of CD45.1⁺ and CD45.2⁺ IgG1⁺ FAS⁺ B cells (J). Data shown as individual mice n=5 from 3 separate experiments with similar results. Statistical significance was assessed by students T test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

The Il21r^EINS mutation affects the expression of Tfh functional molecules.
Staining for ICOS and IL-21 on Tfh and Tfr cells from WT and Il21r^EINS 7 days after SRBC immunization. Percentage of IL-21+ Tfh cells (A), histogram showing IL-21 expression in Tfh cells (B), quantification of IL-21 expression (MFI) in Tfh cells (C). Percentage of ICOS⁺ Tfh cells (D), Histogram showing ICOS expression on Tfh cells (E), levels of ICOS expression on Tfh cells (MFI) (F). Percentage ICOS⁺ Tfr cells (G), histogram showing ICOS expression on Tfr cells (H), levels of ICOS expression on Tfr cells (I). Data are shown from one representative experiment as individual mice and means +/− SD, from 3 experimental replicates with similar results. Statistical significance was assessed by students T test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Genetic loss of IL-21:IL-21R signalling, but not IL-21 mediated STAT1 activation, drives 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 Il21r^EINS mice. Percentage of IL-6Rα expressing CD4⁺ T cells (A) and mean fluorescence intensity (MFI) of IL-6Rα on CD4⁺ T cells (B). Percentage (C) and MFI (D) of IL-6Rα expressing naïve CD44^lo CD4⁺ T cells. Percentage (E) and MFI (F) of IL-6Rα expressing activated/memory phenotype CD44^hi CD4⁺ T cells. Graphs show data for individual mice, means +/− SD from 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 Il21r^EINS mice containing phosphorylated STAT1 (G) and phosphorylated STAT3 (H) after stimulation with 40 ng/ml rmIL-6 over a 2-hour time course detected by immunostaining of phosphorylated STAT proteins, flow cytometry and FACS analyses. Statistical significance was assessed by two-way ANOVA with multiple comparisons; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

The effect of Il21r^EINS on the contribution of IL-6 to the GC response.
Analysis of the germinal centre (GC) response in the spleen in WT, Il21r^−/− and Il21r^EINS mice on day 7 following SRBC immunization. IL-6 receptor (IL-6Rα) expression on CXCR5^hi PD1^hi FoxP3⁻ T follicular helper (Tfh) cells from the spleen of WT, Il21r^−/−and Il21r^EINS 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 0.5mg neutralizing anti-IL-6 mAb or isotype mAb and 0.25mg on day 0, the day of SRBC immunisation, and then day 2, day 4 and day 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=5 from 2 experimental repeats with similar results. Statistical significance was assessed by 1-way ANOVA using Bonferroni’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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