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

Reviewer #1 (Public review):

Summary:

King and colleagues generated a mouse with a point mutation in IL21R and investigated the influence on IL-21-mediated T and B cell activation and differentiation. They found that mutant mice show a reduced T and B cell response, with CD4 T cell differentiation into T follicular helper cells being primarily affected.

Strengths:

The authors combined in vitro and in vivo analysis, including bone-marrow chimeric mice.

Weaknesses:

The effect of the IL21R EINS mutant does not specifically affect STAT1, as clearly shown in Figure 1 H, I. Particularly at lower doses of IL21, which may be more relevant in vivo, the effects are very similar. A second key weakness is the very small Tfh response, a not very clear PD-1 and CXCR5 staining to identify Tfh, and a lack of a steady-state (prior to immunisation) comparison of Tfh numbers in the different mouse strains. The latter makes it impossible to know what fraction of the response is antigen-specific.

Reviewer #2 (Public review):

Summary:

In the manuscript, "An IL-21R hypomorph circumvents functional redundancy to define STAT1 signaling in germinal center responses," Cecile King and colleagues identify a cytoplasmic site of the IL-21 receptor that differentially regulates STAT1 and STAT3 activation upon IL-21 stimulation. They further examine the immunological consequences of this site-specific alteration on Tfh differentiation and Tfh-dependent humoral immunity, raising important questions about how gene-knockout models may obscure nuanced functional roles of signaling molecules.

Strengths:

The study convincingly highlights a non-redundant role for STAT1 downstream of IL-21-IL-21R signaling in the Tfh differentiation pathway. This conclusion is supported by in vitro analyses of STAT1 and STAT3 activation in CD4 T cells stimulated with IL-21 or IL-6; by in vivo assessments of Tfh and germinal center B cell responses in WT and IL21R-EINS mutant mice, including bone-marrow chimera systems; and by investigating the expression of Tfh-related molecules in WT versus IL21R-EINS CD4 T cells.

Weaknesses:

Although the experiments were carefully executed with appropriate controls, a key question remains unresolved: whether the Tfh differentiation defect in IL21R-EINS mice is directly attributable to reduced STAT1 activation. Rescue experiments that restore STAT1 signaling in IL21R-EINS TCR-transgenic CD4 T cells would provide strong evidence linking the mutation to impaired STAT1 activation and, consequently, defective Tfh differentiation. Without such evidence, it remains formally possible that additional, uncharacterized mutations introduced during ENU mutagenesis contribute to the phenotypes observed, particularly given the discrepancies between IL21R knockout and IL21R-EINS mutant mice.