1. Immunology and Inflammation

An IL-21R STAT hypomorph circumvents functional redundancy in germinal centre responses

  1. Christoph Jandl
  2. Joanna Warren
  3. Samantha Owens
  4. Marcel Batten
  5. Howard Wang
  6. Cecile King  Is a corresponding author
  1. Department of Immunology, Garvan Institute of Medical Research, Australia
  2. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia
https://doi.org/10.7554/eLife.109834.3
Share this article
Cite this article
  1. Christoph Jandl
  2. Joanna Warren
  3. Samantha Owens
  4. Marcel Batten
  5. Howard Wang
  6. Cecile King
(2026)
An IL-21R STAT hypomorph circumvents functional redundancy in germinal centre responses
eLife 15:RP109834.
https://doi.org/10.7554/eLife.109834.3
  2. Download BibTeX
  3. Download .RIS

eLife Assessment

These results add to a growing body of literature demonstrating a role for IL-21 in T follicular helper cell expansion and differentiation, as well as in germinal center formation and maintenance. The authors use an IL21R ENU-induced mutant, together with complementary in vitro and in vivo experiments, to investigate the roles of STAT1 and STAT3 signaling downstream of IL-21R. The experimental approach is valuable, but the main conclusion is incompletely supported by the evidence, as the mutant exhibits a broad reduction in STAT signaling rather than a selective impairment of STAT1 signaling.

https://doi.org/10.7554/eLife.109834.3.sa0

Abstract

Interleukin-21 receptor engagement initiates a Janus kinase–signal transducer and activator of transcription (STAT) signalling cascade that activates several STAT proteins that drive the germinal centre (GC) response. However, the relative effects of IL-21 on individual STAT proteins during the differentiation of T follicular helper (Tfh) cells and GC B cells has been difficult to distinguish. Here, we characterize a novel mutation in the interleukin-21 receptor (IL-21REINS) that creates a dominant defect in the activation of STAT1. Our findings provide evidence that IL-21-mediated activation of STAT1 has a non-redundant role in the differentiation of Tfh cells following T-dependent immunization. IL-21REINS Tfh cells were even more impaired than Tfh cells genetically deficient in IL-21R, questioning our current understanding of the role of IL-21 derived from protein knockout mice. The observation that functional compensation fails in the presence of the IL-21R hypomorph provides insight into how underlying compensation can impact our interpretation of a complex biological system.

Introduction

Signals delivered by cytokines are known to shape the humoral immune response to T-dependent antigen, acting on multiple cell subsets involved in this process (Vinuesa et al., 2016). Interleukin-21 signals through the IL-21 receptor (IL-21R), which is a dimeric receptor consisting of the IL-21Rα chain and the γc belonging to the family of common γ chain (γc) receptors (Ozaki et al., 2000; Parrish-Novak et al., 2000; Asao et al., 2001; Habib et al., 2002; Leonard and Wan, 2016). IL-21:IL-21R engagement leads to the activation of Janus kinase (JAK)1 and JAK3, which, in turn, results in the phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3 and to a lesser extent, STAT5 (Asao et al., 2001; Avery et al., 2010; Zeng et al., 2007; Villarino et al., 2017). Previous studies have shown the importance of signalling mediated by the cytokine interleukin (IL)-21 for the differentiation of T follicular helper (Tfh) cells and germinal centre (GC) B cells (Nurieva et al., 2008; Vogelzang et al., 2008; Linterman et al., 2010; Zotos et al., 2010; Quast et al., 2022; Petersone et al., 2023), with some studies questioning a T cell intrinsic role for IL-21 (Linterman et al., 2010; Zotos et al., 2010; Karnowski et al., 2012).

IL-21, produced in abundance by Tfh cells (Vogelzang et al., 2008), activates STAT1 and STAT3 in both Tfh cells and B cells, influencing humoral immune responses. IL-21 co-stimulates the antigen signal in CD4+ T cells and can induce the production of IL-21, amplifying its own effects (Caprioli et al., 2008). IL-21-mediated activation of the JAK–STAT pathway is required for the establishment of long-lasting immune responses in both humans and mice (Avery et al., 2010; Nurieva et al., 2008; Vogelzang et al., 2008; Fornek et al., 2006; Rasheed et al., 2013). Analyses of IL-12-induced expression of IL-21 by human CD4+ T cells demonstrated a non-redundant role of STAT3 in human Tfh cell differentiation, whereas STAT1 was dispensable (Ma et al., 2012). By contrast, in mice, STAT1 activity was required for Bcl6 induction and early Tfh differentiation following LCMV infection (Choi et al., 2013). In B cells, STAT3 plays a critical role in generating memory cells and plasma cells from naive murine precursors in vivo and in human cells in response to IL-10 and IL-21 in vitro (Avery et al., 2010). Analyses of mice containing a selective Stat3 deficiency in CD4+ T cells demonstrate reduced Tfh and GC B cell differentiation following viral infection and Stat3-deficient Tfh cells exhibited a transcriptome similar to Th1 cells (Ray et al., 2014).

Similar to IL-21, the cytokine IL-6 induces the activation of STAT1 and STAT3 and promotes the differentiation of Tfh cells. A reduction in the magnitude of the GC response and Tfh cell numbers can be observed in mice deficient in the cytokine IL-6 (Il6−/− mice) (Nurieva et al., 2008; Kopf et al., 1998), and the absence of both IL-6 and IL-21 in vivo led to an even greater decrease in Tfh cell numbers in response to infection than the absence of either cytokine alone, indicating that IL-21 and IL-6 have redundant functions for Tfh cell differentiation (Karnowski et al., 2012; Eto et al., 2011). Both IL-6 and IL-21 drive the expression of molecules important for Tfh cell differentiation including the Tfh cell master transcription factor Bcl-6 (Choi et al., 2013; Nurieva et al., 2009; Yu et al., 2009; Lee et al., 2011). Furthermore, IL-6 has not only been shown to compensate for a lack of IL-21 but is also able to induce the production of IL-21, further positively influencing Tfh cell development (Dienz et al., 2009). In this context, the degree to which IL-21 is crucial for Tfh cell differentiation is affected by the infection (Karnowski et al., 2012) and the type of adjuvant applied to immunization, where IL-6 and type-I interferon inducing Pathogen-Associated Molecular Patterns, such as found in sheep red blood cell (SRBC) immunization (Loetsch et al., 2017), might compensate for the lack of IL-21 signalling in Tfh cells (Vogelzang et al., 2008; Quast et al., 2022; Bessa et al., 2010).

Previous studies analysing STAT3 and STAT1 mutations in humans and Stat1- and Stat3-deficient mice have shown that both STAT1 and STAT3 are important for IL-21-mediated gene regulation in CD4+ T cells (Wan et al., 2015), with Stat3 heterozygosity adequate to drive a profound effect on Th17 cell differentiation (Andreani et al., 2025). However, the relative importance of the activation of STAT1 and STAT3 downstream of the IL-21R for the GC reaction remains incompletely understood. In this study, which is based in part upon the dissertation of Jandl and King, 2016, we analyse the effect of a single point mutation in the cytosolic region of the IL-21R that limits IL-21-mediated STAT1 activation and show that this partial loss of function mutation inhibits the differentiation of Tfh cells and reduces the magnitude of the GC reaction to T-dependent antigen. Tfh cells that harboured a defective IL-21R were more impaired in response to SRBC immunization than Tfh cells deficient in the IL-21R, offering insights into IL-6-mediated compensatory mechanisms that occur when the protein is completely ablated.

Results

The einseitig (EINS) mutation in IL-21R leads to a dominant STAT1 signalling defect

Tfh cell differentiation in mice made genetically deficient in the full-length IL-21R protein has revealed that IL-6 can, to varying degrees, compensate for the absence of IL-21:IL-21R signalling (Karnowski et al., 2012; Eto et al., 2011). We queried whether the same level of compensation would operate in the presence of a mutant IL-21R. To test this, we analysed a novel mouse line that was generated on a C57BL/6 background by N-ethyl-N-nitrosourea (ENU) mutagenesis, a method to chemically induce point mutations in the DNA of murine spermatogonial stem cells (Justice et al., 1999). A single point mutation in IL-21R (Figure 1A), that we named einseitig, was identified in these mice (Il21rEINS mice), leading to a change from the highly conserved aspartic acid to valine on amino acid position 375 of the receptor protein (Figure 1B). This change from a negatively charged to a neutral amino acid in the cytoplasmic tail region that controls STAT activation resulted in a PolyPhen-2 score of 0.99, predicting a high likelihood of a functional disruption of the IL-21R.

Figure 1 with 1 supplement see all
Download asset Open asset
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.

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
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
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
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
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

Since the engagement of IL-21R with its ligand IL-21 predominantly leads to phosphorylation of both STAT1 and STAT3 (Asao et al., 2001; Habib et al., 2003), we first characterized the impact of the einseitig mutation (Figure 1A) on the phosphorylation of STAT proteins in response to IL-21. Splenocytes from WT, Il21rEINS, and Il21r−/− mice were stimulated with recombinant murine (rm) IL-21 for 15 min in vitro and the phosphorylation of STAT1 and STAT3 assessed by Western blot. IL-21-dependent STAT1 phosphorylation was reduced in Il21rEINS splenocytes compared with WT (Figure 1C, Figure 1—source data 1–3), whereas STAT3 phosphorylation in Il21rEINS splenocytes in response to IL-21 appeared intact (Figure 1D, Figure 1—source data 1 and 4). Control Il21r−/− splenocytes showed an absence of IL-21-dependent phosphorylation of both STAT1 (Figure 1C, Figure 1—source data 1 and 2 and source data) and STAT3 (Figure 1D, Figure 1—source data 1, 4, and 5).

To further interrogate the STAT1 defect, splenocytes from WT, Il21rEINS, and Il21r−/− mice were stimulated with rmIL-21 in vitro and the phosphorylation levels of STAT1 and STAT3 in CD4+ T cells were assessed by intracellular immunostaining and flow cytometry (Figure 1E). Consistent with our Western blot analyses, we observed a marked decrease in the mean fluorescence intensity (MFI) of IL-21-induced STAT1 phosphorylation in Il21rEINS CD4+ T cells compared with WT CD4+ T cells, whereas no pSTAT1 could be detected in Il21r−/− CD4+ T cells (Figure 1E, F). By contrast, the levels of STAT3 phosphorylation were comparable in Il21rEINS and WT CD4+ T cells. As expected, no pSTAT3 could be detected in Il21r−/− cells in response to rmIL-21 (Figure 1E, G).

To exclude the possibility that the ability of the mutant IL-21R to mediate STAT1 and STAT3 phosphorylation was dependent on the intensity of the IL-21-mediated signal, we also assessed pSTAT1 and pSTAT3 levels in response to decreasing concentrations of rmIL-21. Corresponding to observations with a single concentration of rmIL-21, the levels of pSTAT1 in Il21rEINS CD4+ T cells were reduced in comparison to WT across a wide range of rmIL-21 concentrations (Figure 1H), whereas the levels of phosphorylated STAT3 in CD4+ T cells were significantly less affected by the Il21rEINS mutation (Figure 1I). As anticipated Il21r−/− CD4+ T cells did not activate either STAT1 or STAT3 following stimulation with rmIL-21 (Figure 1H, I). Since IL-6 activates both STAT1 and STAT3 through a different receptor than IL-21, stimulation with IL-6 served as a positive control for STAT activation (Choi et al., 2013; Eto et al., 2011; Heinrich et al., 1998). Despite a STAT1 defect in response to IL-21, percentages of Il21rEINS CD4+ T cells showed equivalent STAT1 activation to WT cells when stimulated with rmIL-6 (Figure 1—figure supplement 1). The percentages of CD4+ T cells that activated STAT3 in response to IL-6 were also similar between Il21rEINS, Il21r−/−, and WT CD4+ T cells (Figure 1—figure supplement 1).

In addition to STAT1 and STAT3, IL-21 has also been shown to activate STAT5, albeit to a lesser extent than IL-2 (Zeng et al., 2007). To assess STAT5 phosphorylation in Il21rEINS CD4+ T cells, we stimulated splenocytes from WT, Il21rEINS, and Il21r−/− mice either with rmIL-6, rmIL-21, or rmIL-2 in vitro and subsequently measured the expression levels of pSTAT5 in CD4+ T cells by flow cytometry. Expression levels and percentages of pSTAT5 in WT and Il21rEINS CD4+ T cells were equivalent in response to all three stimulatory conditions (Figure 1—figure supplement 1). Furthermore, titration of rmIL-21 activated STAT5 to a similar extent in WT and Il21rEINS CD4+ T cells shown both as an increased intensity of pSTAT5 (Figure 1—figure supplement 1) and percentage of CD4+ T cells containing pSTAT5 (Figure 1—figure supplement 1), whereas no effect was observed in Il21r−/− cells. Taken together, our findings provide evidence that the einseitig mutation in IL-21R leads to a distinct STAT1 activation defect that does not affect either STAT3 or STAT5 activation in response to IL-2. Il21rEINS mice thus provide a unique opportunity to distinguish the contribution of IL-21-mediated STAT1 activation in the GC response.

The einseitig mutation leads to a defective GC response to T-dependent antigen

Prior to immunization, lymphoid and myeloid cell populations in the thymus and the spleen of naive Il21rEINS mice were analysed, indicating no major impact of the einseitig mutation on the development of lymphoid cell populations when compared with WT mice (Figure 2—figure supplement 1). To assess the influence of the einseitig mutation on the GC reaction, we compared the response of Il21rEINS, Il21r−/− , and WT mice following immunization with the T-dependent antigen, SRBCs. Histological analyses of spleen sections from mice 7 days after SRBC immunization showed that GCs were still able to form when IL-21-dependent STAT1 signalling was diminished (Figure 2A). However, the reduction of IL21-mediated STAT1 phosphorylation led to decreased percentages (Figure 2B, C) and numbers (Figure 2—figure supplement 2) of FoxP3− CXCR5high PD-1high CD4+ Tfh cell population 7 days after SRBC immunization despite intact STAT3 phosphorylation downstream of the IL-21R. Interestingly, this defect was more pronounced in the presence of limited signalling through the mutant IL-21R, since Il21r−/− Tfh cells did not exhibit an equivalent reduction in response to SRBC immunization (Figure 2, Figure 2—figure supplement 2). These data suggest that whilst IL-6 has been shown to compensate for a lack of IL-21 in Tfh cell differentiation (Karnowski et al., 2012; Eto et al., 2011), no such redundancy operated when a defective IL-21R was present.

Figure 2 with 2 supplements see all
Download asset Open asset
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.

We have shown previously that the FoxP3+ CXCR5high PD-1high CD4+ Tfr cell population was increased in Il21r−/− mice (Jandl et al., 2017). We similarly observed an increase in the percentages (Figure 2D), but not the numbers (Figure 2—figure supplement 2), of Tfr cells in Il21rEINS mice in comparison to WT mice 7 days after SRBC immunization. However, FoxP3+ CD4+ T regulatory cells (Tregs) were observed at similar frequencies in wild-type and Il21rEINS mice, but were increased in Il21r−/− mice, following SRBC immunization (Figure 2E). Assessment of the kinetics of T follicular cells showed the percentages of Tfh cells were significantly decreased 5 and 7 days after SRBC immunization (Figure 2F), and the percentage of Tfr cells showed a slight increase 5 days after SRBC immunization (Figure 2G) in Il21rEINS mice compared with WT mice. Taken together, these findings suggested that IL-21-mediated STAT1 phosphorylation supported the differentiation of Tfh cells and inhibited the differentiation of Tfr cells.

Consistent with these findings, the percentages of Il21rEINS GC B cells (Figure 3A) and IgG1+ FAS+ B cells (Figure 3B) were significantly reduced relative to WT GC B cells but were slightly increased when compared with Il21r−/− GC B cells. These data were supported by a decrease in absolute numbers of GC B cells (Figure 3C) and IgG1+ FAS+ B cells (Figure 3D) in both Il21rEINS and Il21r−/− mice compared with WT mice. Further analyses of the kinetics of the GC populations by flow cytometry from days 3 to 21 after SRBC immunization showed that the percentages of GC B cells were decreased in Il21rEINS mice relative to their WT counterparts on days 5 and 7 (Figure 3E). Despite an increase in the IgG1+ FAS+ B cell population from day 3 in response to immunization, the percentages of Il21rEINS IgG1+ GC B cells were significantly lower relative to WT cells 7 days after SRBC immunization (Figure 3F). These findings demonstrate that IL-21-mediated STAT1 activation supports the differentiation of GC B cells following SRBC immunization.

Figure 3
Download asset Open asset
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.

IL-21-dependent activation of STAT1 has a T cell intrinsic component

To determine the cell intrinsic effects of the STAT1-specific IL21-signalling defect caused by the einseitig mutation on GC populations, we transferred equal amounts of bone marrow (BM) cells from WT mice expressing the congenic marker CD45.1 and BM from either Il21rEINS or WT expressing CD45.2 into lethally irradiated RAG-1 deficient mice. After 8 weeks of rest to allow for reconstitution, we checked for chimerism in the blood and subsequently immunized the BM chimera mice with SRBC and the relative contribution of WT and Il21rEINS cells to T and B cell populations was examined.

Analyses of the spleens of chimeric mice following immunization with SRBC showed that CD45.2+ Il21rEINS cells contributed only an average of 41.2% of the total donor CD4+ T cell population, suggesting that the Il21rEINS mutation negatively affected CD4+ T cell proliferation or survival (Figure 4A, B). However, the Il21rEINS Tfh cell population showed a small but consistent decrease relative to CD45.2+ CD4+ T cells deriving from Il21rEINS donor mice, indicating decreased differentiation of Tfh cells (Figure 4A, B). By contrast, the Il21rEINS FoxP3+ Tfr population showed an increased proportion relative to CD4+ T cells deriving from Il21rEINS donor mice (Figure 4A, C). Parallel analyses of the T cell subsets within each donor population derived either from CD45.1+ WT or CD45.2+ Il21rEINS donor BM within individual chimeric mice confirmed a decrease of Il21rEINS Tfh cells (Figure 4D) and showed small increases in both CD45.2+ Il21rEINS FoxP3+ Tfr cells (Figure 4E) and Treg cells (Figure 4F) in comparison with WT cells. Control chimeras reconstituted with 50% WT CD45.1 BM and 50% WT CD45.2 BM exhibited similar contributions of CD45.1+ and CD45.2+ cells to the total CD4+ T cell, Treg cell (Figure 4G), Tfr cell (Figure 4H), and Tfh cell (Figure 4I) populations after immunization with SRBC, therefore excluding a potential impact of the congenic markers on reconstitution. Taken together, these findings provide evidence for a cell intrinsic contribution of IL-21-dependent STAT1 signalling to the differentiation of both Tfh and Tfr cells.

Figure 4
Download asset Open asset
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.

Parallel analyses of the donor B cell populations after immunization with SRBC showed that within the total B cell population approximately 53.1% are CD45.2+ Il21rEINS (Figure 4J). In contrast to what was observed for the Il21rEINS T follicular cell subsets, we did not detect a cell intrinsic effect of the einseitig mutation in either GC B cells (Figure 4J) or IgG1+ FAS+ B cells (Figure 4K). Similarly, comparison of B cell subsets derived from Il21rEINS and WT donors’ side by side showed no difference between Il21rEINS GC B cells (Figure 4L) or IgG1+ FAS+ B cells (Figure 4M) in comparison with WT GC B cells. Collectively, our findings indicate a cell intrinsic contribution of IL21-dependent STAT1 signalling to the differentiation of T follicular subsets, but not GC B cells.

Reduced IL-21-dependent STAT1 phosphorylation leads to quantitative and qualitative differences in follicular CD4+ T cell populations

Whilst the frequency of Tfh cells were reduced by the einseitig mutation, it was unclear whether the expression of Tfh functional molecules was also affected. IL-21R signalling within Tfh cells increases the proportion of Tfh cells that produce IL-21 in vivo, thereby establishing a feed-forward loop (Quast et al., 2022). When we assessed IL-21 production in Tfh cells 7 days after SRBC immunization, we observed a decrease in the percentages of IL-21+ Tfh cells in IL21rEINS in comparison to their WT counterparts (Figure 5A). Furthermore, the levels of IL-21 produced by Il21rEINS Tfh cells were decreased when compared with WT Tfh cells (Figure 5B, C).

Figure 5
Download asset Open asset
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.

T follicular cells express the costimulatory molecule inducible T cell co-stimulator (ICOS), which is important for Tfh cell differentiation and function in humans and mice (Grimbacher et al., 2003; Akiba et al., 2005). The vast majority of WT Tfh cells express ICOS, which were reduced in Il21rEINS CXCR5hi PD1hi CD4+ Tfh cells (Figure 5D) and, more notably, the intensity of ICOS expression was lower on Il21rEINS Tfh cells compared with WT Tfh cells (Figure 5E, F). The observed decrease in ICOS expression was not limited to the follicular helper population, since the ICOS-expressing fraction of the Il21rEINS Tfr population was also reduced relative to WT cells (Figure 5G), as well as the levels of ICOS expression on Il21rEINS Tfr cells (Figure 5H, I). These observations demonstrated that IL-21-induced STAT1 signalling was important for optimal IL-21 production by Tfh cells and for the expression of ICOS on T follicular cell subsets. Therefore, IL-21:IL-21R-induced STAT1 activation plays an important role in both the differentiation and function of Tfh cells.

IL-21-induced activation of STAT1 does not control CD4+ T cell expression of IL-6Rα

As discussed earlier, IL-6 is produced following SRBC immunization (Loetsch et al., 2017) and has been shown to compensate, to varying degrees, for IL-21 during the differentiation of Il21r−/− Tfh cells (Karnowski et al., 2012; Choi et al., 2013). Both cytokines play a role in Tfh cell differentiation, shown by the severe reduction of Tfh cells in mice made deficient in both IL-21 and IL-6 in response to virus infection (Karnowski et al., 2012). Therefore, we were intrigued that whilst IL-6 can compensate for Tfh cell differentiation in the absence of IL-21R, it could not compensate in the presence of a mutant IL-21R, potentially due to a niche-filling effect of the mutant IL-21R (Papathanasiou et al., 2003). To test this, we first assessed expression of the alpha chain of the IL-6 receptor (IL-6Rα) on CD4+ T cells. Flow cytometric analyses indicated increased expression of IL-6Rα (Figure 6A) and an increased percentage of IL-6Rα expressing Il21r−/− CD4+ T cells (Figure 6B), compared with both Il21rEINS and WT CD4+ T cells.

Figure 6
Download asset Open asset
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.

Since IL-6Rα is expressed on both CD44lo naive and CD44hi activated/memory phenotype CD4+ T cells, we analysed naive CD4+ T cells within the total CD4+ T cell population in the spleen, which demonstrated a greater percentage of IL-6Rα expressing Il21r−/− CD4+ T cells than WT cells or Il21rEINS cells (Figure 6C). Whereas Il21rEINS IL-6Rα+ naive cells were intermediate; with a greater percentage relative to WT cells, but less than Il21r−/− cells (Figure 6C). By contrast, the levels of IL-6Rα expression were higher on naive CD4+ T cells that lack IL-21:IL-21R signalling relative to both WT and Il21rEINS cells (Figure 6D). In all genotypes, the expression of IL-6Rα was lower on activated/memory phenotype CD4+ T cells than naive as has been shown previously (Briso et al., 2008). However, IL-6Rα expressing Il21r−/− CD44hi CD4+ T cells, in turn, were even further reduced when compared with WT, but not Il21rEINS CD44hi CD4+ T cells (Figure 6E). Similar to naive CD4+ T cells, the levels of IL-6Rα expression on Il21r−/− CD44hi cells were markedly higher when compared with both Il21rEINS and WT CD44hi CD4+ cells (Figure 6F). Taken together, these findings indicate that IL-21-mediated STAT1 activation affected the percentages of IL-6Rα expressing naive CD+ T cells but not the level of expression of IL-6Rα on CD4+ T cells.

Analyses of the activation of STAT1 and STAT3 in WT, Il21r−/− , and Il21rEINS CD4+ T cells exposed to low dose rmIL-6 indicated that each genotype responded similarly (Figure S5). We then extended these analyses to test the level of STAT1 and STAT3 activation in WT, Il21r−/− , and Il21rEINS CD4+ T cells over a 2-hr time course. These findings demonstrated that Il21r−/− CD4+ T cells maintained higher levels of phosphorylated STAT1 (Figure 6G) and STAT3 (Figure 6H) at 60 min following exposure to rmIL-6. By contrast, WT and Il21rEINS CD4+ T cells showed equivalent levels of both phosphorylated STAT1 (Figure 6G) and STAT3 (Figure 6H). These findings indicate that the absence of the IL-21R, but not the presence of an IL-21R STAT1 hypomorph, alters the kinetics or maintenance of STAT1 and STAT3 activation over time in response to IL-6.

IL-6 is unable to compensate for IL-21 in the presence of an IL-21R STAT1 hypomorph

The observation of increased expression of IL-6Rα on Il21r−/− CD4+ T cells and delayed STAT1 and STAT3 activation in response to IL-6 may reflect differences in the capacity to ulitize IL-6 in the absence of IL-21:IL-21R signalling. In classic IL-6 signalling, the soluble cytokine binds to its membrane bound IL-6Rα, which then associates with signal-transducing β-receptor (gp130) resulting in the activation of STAT1 and STAT3. To test responsiveness to IL-6, we first assessed Th17 differentiation, which is strongly dependent on signals from IL-6 (Kimura and Kishimoto, 2010), by culturing WT, Il21r−/−, and Il21rEINS CD4+ T cells under Th17 polarizing conditions. On day 3 of culture, we observed similar percentages of Il21rEINS and WT IL-17+ Th17 cells, but reduced differentiation of Il21r−/− Th17 cells, as has been previously reported (Nurieva et al., 2007; Korn et al., 2007) indicating that the biological function of IL-6 was intact in Il21rEINS CD4+ T cells (Figure 7—figure supplement 1). Whilst IL-6 was able to support Th17 differentiation in Il21rEINS CD4+ T cells, Th17 differentiation was reduced in Il21r−/− CD4+ T cells despite increased levels of IL-6Rα. These observations suggested that responsiveness to IL-6 may be altered in the absence of normal IL-21:IL-21R signalling.

To test whether IL-21-mediated STAT1 activation affected IL-6-mediated compensation, we utilized a previously published method (Eto et al., 2011) to block IL-6 signalling in vivo by treating WT, Il21rEINS, and Il21r−/− mice with neutralizing anti-IL6 monoclonal antibody (mAb). Analyses of untreated mice 7 days after SRBC immunization showed that the percentage of Il21r−/− CXCR5hi PD1hi T follicular cells expressing IL-6Rα was comparable between genotypes (Figure 7A), but the level of expression of IL-6Rα was greater on Il21r−/− Tfh cells compared with WT and Il21rEINS cells (Figure 7B, C).

Figure 7 with 1 supplement see all
Download asset Open asset
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.

Neutralization of IL-6 with anti-IL6 mAb did not significantly reduce the FoxP3− Tfh cell population in either WT or Il21rEINS but did reduce the percentage of Il21r−/− Tfh cells 7 days after immunization with SRBC (Figure 7D). By contrast, we could not detect a significant change upon in vivo neutralization of IL-6 on the FoxP3+ Tfr population in either WT, Il21rEINS, or Il21r−/− mice (Figure 7—figure supplement 1). Similarly, we did not observe a significant difference in the percentage of GC B cells in WT, Il21rEINS, and Il21r−/− mice (Figure 7—figure supplement 1). However, IgG1+ FAS+ B cells were decreased in Il21r−/− but not Il21rEINS or WT mice upon in vivo blockade of IL-6 (Figure 7E). Our in vivo IL-6 blocking experiments indicated a significantly greater contribution of IL-6 to GC output in Il21r−/− than Il21rEINS or WT mice. Taken together these findings demonstrate that, despite the strong ability of IL-6 to activate STAT1, IL-6 is ineffective at fully compensating for the geminal centre response in the presence of an IL-21 receptor mutant that predominantly affects IL-21 activation of STAT1.

Discussion

The importance of IL-21-induced JAK–STAT signalling for the humoral immune response to T-dependent antigen has been confirmed by a wide body of data (Nurieva et al., 2008; Vogelzang et al., 2008; Linterman et al., 2010; Zotos et al., 2010; Wan et al., 2015). However, the relative role of IL21-dependent STAT1, STAT3, and STAT5 activation on the GC response has been difficult to tell apart. Here, we identify an IL-21R hypomorph and take advantage of its selective defect in STAT1 activation to elucidate the role of IL-21-induced STAT1 activation in the differentiation of GC T and B cells.

The ENU-induced mutation in Il21rEINS mice caused a change from a negatively charged aspartic acid to a neutral, hydrophobic, valine at the highly conserved amino acid position 375 in the cytoplasmic tail region of the IL-21R receptor protein. Whilst previous studies have demonstrated that the main STAT1 docking tyrosine is Y510 (Zeng et al., 2007), the Il21rEINS mutation may affect the geometry or stability of the IL-21R, leading to diminished STAT1 phosphorylation. Comparison of the pSTAT1 levels of IL-21 stimulated cells from Il21rEINS and WT mice revealed diminished STAT1 activation by mutant IL-21R in CD4+ T cells, whereas STAT3 and STAT5 activation were unaffected. In contrast to the STAT3 dominance observed following interferon-gamma stimulation of STAT1 deficient cells (Wan et al., 2015), we did not observe a compensatory increase in STAT3 phosphorylation in Il21rEINS CD4+ T cells. Moreover, activation of STAT1 by cytokines acting through different receptors, such as IL-6, remained undisturbed in Il21rEINS CD4+ T cells.

Previous studies have demonstrated an important role for STAT1 and STAT3 activation on GC populations. For instance, STAT3 has a non-redundant role in human Tfh cell differentiation (Ma et al., 2012) and IL-21-dependent STAT3 phosphorylation in human B cells is crucial for the development of a strong high-affinity plasmablast response (Avery et al., 2010; Fornek et al., 2006). In murine CD4+ T cells, STAT3 expression is required for both Tfh cell and GC B cell differentiation and generation of virus-specific antibody responses (Ray et al., 2014). IL-6, in turn, mediates STAT1 activation that is important for the induction of Bcl-6 in early Tfh cell differentiation (Choi et al., 2013). This study has revealed a previously unappreciated role for IL-21-mediated STAT1 activation in the both the differentiation of Tfh cells and the expression of molecules important to Tfh cell function. Il21rEINS Tfh cells exhibited reduced IL-21 production and ICOS expression was reduced on both Il21rEINS Tfh and Il21rEINS Tfr cells. This finding indicates that STAT1 activated by IL-21 is important for optimal Tfh cell differentiation.

Analyses of mixed BM chimeras immunized with SRBC, identified a T cell intrinsic contribution for IL-21-dependent STAT1 activation to both the differentiation of Tfh cells and the inhibition of Tfr cells. However, the T cell intrinsic effect was small, and additional factors may contribute to the reduced differentiation of Il21rEINS Tfh cells, including the effect of IL-21:IL-21R signalling on non-lymphoid compartments. By contrast, there was no B cell intrinsic role for the mutant IL-21R for the differentiation of GC B cell populations. The absence of a B cell intrinsic effect of the reduced IL-21-mediated STAT1 phosphorylation might be explained by a stronger dependency of B cells on IL-21-mediated STAT3 activation (Avery et al., 2010; Fornek et al., 2006; Berglund et al., 2013).

We, and others, have previously observed a T cell intrinsic role for IL-21 signalling in the GC response in mice immunized with OVA delivered in alum (Vogelzang et al., 2008; Quast et al., 2022). By contrast, immunization with the T-dependent antigen SRBC, that induces ample IL-6 expression (Loetsch et al., 2017), did not reveal a significant reduction in Il21r−/− Tfh cells, as has been previously reported (Linterman et al., 2010). The observation that IL-6 can act redundantly, to varying degrees, to support Il21r−/− Tfh cell differentiation (Karnowski et al., 2012; Eto et al., 2011; Poholek et al., 2010), delivering an important STAT1-mediated signal (Choi et al., 2013), suggested that IL-6 may contribute to Tfh cell differentiation in the absence of the Il21r but not in the presence of a defective receptor. This notion was supported by increased expression of IL-6Rα on Il21r−/− CD4+ T cells compared with WT cells with an intact IL-21R or an Il21rEINS hypomorph.

IL-6Rα is highly expressed on naive and central memory T cells (Briso et al., 2008; Jones et al., 2010), but decreases as IL-6Rα undergoes cleavage following T cell activation and IL-6 ligation, resulting in the release of soluble IL-6Rα (Briso et al., 2008). In this context, the increased levels of IL-6Rα on Il21r−/− naive and memory phenotype CD4+ T cells in unmanipulated mice might suggest a reduced level of T cell activation in Il21r−/− mice. The reason for increased expression of IL-6Rα on cells that lack IL-21:IL-21R signalling is not known, but may reflect IL-21’s role as a costimulatory molecule and that IL-21 and IL-6 each work optimally when both cytokines are present (Dienz et al., 2009; Suto et al., 2008). Despite clear differences in the level of expression of IL-6Rα on Il21r−/− CD4+ T cells, we did not find a marked difference in the levels of either activated STAT1 or STAT3 in response to stimulation with IL-6 in vitro. However, we did observe that Il21r−/− CD4+ T cells had a delayed peak, or more sustained levels, of phosphorylated STAT1 and STAT3 over time and were more affected by neutralization of IL-6 in vivo. This indicates that expression of IL-6Rαis affected by IL-21:IL-21R signalling, which may influence utilization of IL-6 in Il21r−/− CD4+ T cells following SRBC immunization.

Identification of the Il21rEINS mutation has enabled the study of the distinct contribution of IL-21-mediated STAT1 activation in the GC response. Despite the potential input of other STAT1 inducing cytokines during T-dependent immunization, IL-21-induced STAT1 activation could not be entirely compensated for. Functional compensation has been reported in several settings to explain differences in the observed effects of genetic deletion versus protein mutants or gene knockdown (Kok et al., 2015; Rossi et al., 2015; Oliver et al., 2007; Rouf et al., 2023). Thus, whilst IL-6 has been shown to compensate for a lack of IL-21 in Tfh cell differentiation (Karnowski et al., 2012; Eto et al., 2011), no such redundancy operated to compensate for a IL-21R STAT1 hypomorph. The remaining function of the IL-21EINS receptor likely reduced the need for compensatory pathways that are activated in complete protein knockouts. Future experiments that selectively restore IL-21-induced STAT1 signalling in Il21rEINS CD4+ T cells would provide definitive evidence linking the Il21rEINS mutation to impaired STAT1 activation in GC responses.

Materials and methods

Mice

C57/BL6 (B6), Ly5.1 congenic mice (B6 background), as well as RAG1−/− mice bred to B6 N10 were purchased from the Animal Research Centre in Perth, Australia. Il21r−/− mice were obtained from Dr Warren Leonard (NIH) via Dr Mark Smyth (Melbourne) at B6 N6 and backcrossed to N12 for experimental use. Il21rEINS mice were purchased from the Australian Phenomics facility ANU (Prof Christopher Goodnow). The Garvan Institute and St. Vincent’s Hospital Animal Ethics Committee granted permission to perform animal experiments. Animals were housed under specific pathogen-free conditions and handled in accordance with the Australian code of practice for the care and use of animals for scientific purposes. Age and sex matched (male and female) littermate mice between 8 and 14 weeks of age were used for experimental purposes.

Immunizations and IL-6 neutralization

Request a detailed protocol

Mice were immunized (i.v.) with 2 × 108 SRBCs (IMVS, Australia) and spleens were analysed at timepoints shown. For in vivo neutralization of IL-6: Mice initially received 0.5 mg neutralizing anti-IL-6 (clone MP5-20F3, rat IgG1; BioXCell) or isotype mAb (anti-rat IgG1; BioXCell), and subsequently given 0.25 mg mAb (i.v.) every other day following the initial dose through the end of the experiment (Eto et al., 2011).

BM chimeras

Request a detailed protocol

Cohorts of RAG1−/− mice were lethally irradiated by a 137Cs source (single dose of 0.6 Gy) and reconstituted the following day by i.v. injection with BM cells isolated from femurs and tibiae obtained by flushing bone with lymphocyte isolation media in sterile conditions. After irradiation, mice were maintained on antibiotic water containing cotrimoxazole (Roche) for 7 days. Irradiated RAG1−/− mice were reconstituted with 1x107 BM cells from Ly5.1 congenic mice (B6 background) expressing the congenic marker CD45.1 to allow detection, combined with equal proportions BM from CD45.2+ WT or CD45.2+ Il21rEINS mice. Since no mature lymphocytes develop in RAG1−/− mice, no radio-resistant donor T cells were present in our analysis. Our reconstitution protocol typically resulted in an approximate 35–65:65–35 ratio of WT:Il21rEINS lymphocytes. Mice were used for immunization protocols as described above at 8 weeks post-reconstitution.

Immunofluorescence and FACS analyses

Request a detailed protocol

For flow cytometric analysis of mouse lymphocytes, red blood cells were removed by hypotonic lysis. 50 μl of a single-cell suspension at 2 × 107 cells/ml from spleen and lymph nodes were stained in FACS buffer containing pre-diluted antibodies in 96-well V-bottomed microtitre plates (Nunc, Roskilde, Denmark). Antibodies against surface molecules purchased from BD Bioscience were anti-B220-APC and APC-Cy7 (RA3-6B2, dilution 1/300), anti-CD4-PE-Cy7 and A488 (RM4-5, dilution 1/300), anti-CD21-FITC (7G6, dilution 1/200), anti-CD23-PE (B3B4, dilution 1/200), anti-CD25-APC and BV605 (PC61, dilution 1/100), anti-CD45.1-PE-Cy7 (A20, dilution 1/200), anti-CD45.2-APC-Cy7 (104, dilution 1/200), anti-CXCR5-biotin (2G8, dilution 1/100), anti-GL7-PE (GL7, dilution 1/300), anti-FAS-FITC (Jo2, dilution 1/200), anti-IgG1-biotin (A85-1, dilution 1/100), and anti-Ly6G-PB (1A8, 1/300). Antibodies purchased from eBioscience were anti-CD8-APC (53–6.7, dilution 1/200), anti-CD11b-FITC (M1/70, dilution 1/200), anti-CD11c-APC (N418, dilution 1/200), anti-CD19-eF450 (1D3, dilution 1/200), anti-CD38-PE-Cy5.5 (90, dilution 1/200), anti-CD44-APC-eFluor780 (IM7, dilution 1/200), anti-ICOS-PE (C398.4A, dilution 1/300), anti-NK1.1-PE (PK136, dilution 1/200), anti-PD-1-FITC (RMP1-30, dilution 1/100), Streptavidin-PerCP-Cy5.5 and APC (dilution 1/300), anti-TCRβ-APC, FITC and PerCP-Cy5.5 (H57-597, dilution 1/300). Intracellular immunostaining for FoxP3 carried out using the anti-Foxp3-eF450 (FJK-16s, dilution 1/100). Antibodies against intracellular proteins were purchased from eBioscience using the BD intracellular staining kit (Cat#554714) according to the manufacturer’s instructions. Intracellular phosphorylated STAT proteins were detected with anti-STAT1 (PY701)-A647 (4a, dilution 1/5), anti-STAT3 (PY705)-PE (4/P-STAT3, dilution 1/5) and anti-P-STAT5-PerCP-Cy5.5 (47, dilution 1/5) mAbs using the BD Phosphlow buffer III (Cat#558050) according to the manufacturer’s instructions. Cytokines were detected either directly ex vivo or after 4-hr stimulation at 37°C in cell culture media with Phorbol 12-myristate 13 acetate (PMA 500 ng/ml, BIOMOL USA), ionomycin (500 ng/ml, Invitrogen) and GolgiStop (1:1000, BD Biosciences, CA). Intracellular IL-21 was detected using IL-21rFc (manufactured in house Herber et al., 2007), conjugated to A647 using the Molecular Probes A647 Antibody Labeling Kit (Thermo Fisher) according to the manufacturer’s instructions. Intracellular IL-17 was detected using anti-IL-17a-APC-Cy7 (TC11-18H10, dilution 1/100) purchased from BD Biosciences and staining for intracellular cytokines was carried out using the BD intracellular staining kit (Cat#554714) according to the manufacturer’s instructions. Cells were acquired using either a FACS Canto II or a Fortessa cytometer (BD Biosciences, CA) and analysed using Flowjo (Treestar, CA).

Immunohistochemistry

Request a detailed protocol

5 μm frozen OCT (Tissue Tek, Australia) spleen sections were fixed in ice-cold acetone for 8 min, washed and incubated with blocking reagent (Avidin/biotin (Dako)) at room temperature. Primary biotin or fluorochrome-conjugated antibodies were incubated at RT overnight. Abs against surface molecules purchased from BD Biosciences were anti-CD4-PE (RM4-5, dilution 1/100) and anti-IgG1-biotin (A85-1, dilution 1/100). Streptavidin-APC (dilution 1/200) was purchased from eBioscience and IgD was detected using anti-IgD-A488 (11-26c2a, dilution 1/100) Ab purchased from Biolegend. Sections were analysed using a Leica DM I6000 SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany). The images were processed using the Leica acquisition and analysis software ImageJ (Freeware NIH Bethesda, USA) or Adobe Photoshop, version 7 (San José, CA).

SDS–PAGE and immunoblotting

Request a detailed protocol

Splenocytes were stimulated at a concentration of 3 × 106 cells/ml with rmIL-21 (Peprotech, NJ) at 80 ng/ml for or incubated in media 15 min. Cells were washed and lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma, CA) and run by SDS polyacrylamide-gel electrophoresis using a 4–12% gradient gel and nitrocellulose membranes (Invitrogen). Membranes were probed with anti-STAT1 (42H3, Cell Signalling, USA) or anti-STAT3 (F-2, Santa Cruz, USA) and anti-β-actin (AC-74, Sigma-Aldrich, USA). Antibody binding was detected using goat anti-rabbit IgG-HRP (cat# SC-2004, Santa Cruz, USA), goat anti-mouse IgG-HRP (cat # 31439, Thermo Fisher, USA), or rabbit anti-goat IgG-HRP (cat# 31433, Thermo Fisher, USA) and developed using ECL (Perkin-Elmer, MA). After the first round of detection membranes were stripped using stripping buffer (0.015% glycine, 0.001% SDS, 0.01% Tween20 in water, pH = 2.2) for 30 min at 50°C and after two washes with PBS-T re-probed with anti phospho-STAT1 (D4A7, Cell Signalling, USA) or anti phospho-STAT3 (3E2, Cell Signalling, USA).

Th17 cell conversion in vitro

Request a detailed protocol

CD4+ T cells from the spleens were isolated by positive selection using magnetic cell sorting by staining CD4+ T cells in MACS buffer with anti-CD4 magnetic beads according to the manufacturers instructions and collected using LS columns (both from Milentenyi Biotec, Auburn, USA) giving a cell purity of 90–95%. In brief lymphocytes were resuspended at 2 × 108 cells/ml and incubated with 10 µl anti-CD4 magnetic beads (Milentenyi Biotec, Auburn USA) per 100 µl of labelled splenocytes for 20 min at 4°C. These cells were washed twice in MACS buffer and CD4+ T cells were isolated using LS columns. To induce Th17 cell conversion, CD4+ T cells were activated in the presence of 2 µg/ml plate-bound anti-CD3 mAb (clone 145-2C11, BD Pharmingen), with soluble 5 µg/ml anti-CD28 (clone 37.51, eBioscience), as well as rmIL-6 (50 ng/ml, Peprotech, USA), rmIL-23 (5 ng/ml, Peprotech, USA), recombinant human TGFβ1 (1 ng/ml, Peprotech, USA), anti-IL-4 mAb (10 µg/ml, BD Bioscience), and anti-IFNγ mAb (10 µg/ml, BD Bioscience) for 3 days. On day 3, cells were washed in PBS and subsequently stimulated for 4 hr with 500 ng/ml PMA (BIOMOL, USA) and 500 ng/ml ionomycin (Invitrogen, USA) in the presence of Golgi Stop (1:1000, BD Biosciences, CA). Conversion efficiency was assessed by flow cytometry on day 3 after surface staining for CD4 followed by intracellular staining for IL-17a.

In vitro stimulation

Request a detailed protocol

For intracellular pSTAT staining via flow cytometry: 5 × 107 splenocytes were stimulated at a concentration of 3 × 106 cells/ml with 80 ng/ml rmIL-21 (Peprotech, NJ), rmIL-6 (BD Biosciences, CA) or rmIL-2 (R&D Systems, MN), or at the concentrations indicated, for 15 min, or across a 2-hr time course as indicated, before intracellular immunofluorescence staining, flow cytometry and FACS analysis.

Data analysis and statistics

Request a detailed protocol

Data were analysed using Prism software (GraphPad software, CA) to calculate unpaired, two-way Student’s t-test, with an F test to compare variances. Analysis of more than two groups was performed using one-way ANOVA followed by Bonferroni’s test to compare groups or two-way ANOVA as indicated.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for all figures.

References

Article and author information

Author details

  1. Christoph Jandl

    Department of Immunology, Garvan Institute of Medical Research, Darlinghurst, Australia
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared

  2. Joanna Warren

    Department of Immunology, Garvan Institute of Medical Research, Darlinghurst, Australia
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  3. Samantha Owens

    School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
    Contribution
    Validation, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6661-4150

  4. Marcel Batten

    Department of Immunology, Garvan Institute of Medical Research, Darlinghurst, Australia
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared

  5. Howard Wang

    School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
    Contribution
    Formal analysis, Writing – review and editing
    Competing interests
    No competing interests declared

  6. Cecile King

    1. Department of Immunology, Garvan Institute of Medical Research, Darlinghurst, Australia
    2. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    c.king@unsw.edu.au
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2193-2563

Funding

National Health and Medical Research Council (1148051)

  • Cecile King

Freiburg Institute for Advanced Studies, Albert-Ludwigs-Universität Freiburg (Marie Skłodowska-Curie FCFP fellowship)

  • Cecile King

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

This project was supported by project grant funding from NHMRC APP1148051 and Senior Fellowship in the Marie Skłodowska-Curie FCFP at the Freiburg Institute for Advanced Studies (FRIAS), The University of Freiburg, Germany. Our thanks to Professor Bodo Grimbacher Institute for Immunodeficiency, Center for Chronic Immunodeficiency (CCI), Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Ethics

The Garvan Institute and St. Vincent's Hospital Animal Ethics Committee granted permission to perform animal experiments under Animal Ethics protocol 18_39. Animals were housed under specific pathogen-free conditions and handled in accordance with the Australian code of practice for the care and use of animals for scientific purposes.

Version history

  1. Sent for peer review:
  2. Preprint posted:
  3. Reviewed Preprint version 1:
  4. Reviewed Preprint version 2:
  5. Version of Record published:

Cite all versions

You can cite all versions using the DOI https://doi.org/10.7554/eLife.109834. This DOI represents all versions, and will always resolve to the latest one.

Copyright

© 2026, Jandl et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 330
    views
  • 4
    downloads
  • 0
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Christoph Jandl
  2. Joanna Warren
  3. Samantha Owens
  4. Marcel Batten
  5. Howard Wang
  6. Cecile King
(2026)
An IL-21R STAT hypomorph circumvents functional redundancy in germinal centre responses
eLife 15:RP109834.
https://doi.org/10.7554/eLife.109834.3

Share this article

https://doi.org/10.7554/eLife.109834

Categories and tags

Research organism