A structural blueprint for interleukin-21 signal modulation

Abstract

Interleukin-21 (IL-21) plays a critical role in generating immunological memory by promoting the germinal center reaction, yet clinical use of IL-21 remains challenging because of its pleiotropy and association with autoimmune disease. To better understand the structural basis of IL-21 signaling, we determine the structure of the IL-21-IL-21R-γc ternary signaling complex by X-ray crystallography and a structure of a dimer of trimeric complexes using cryo-electron microscopy. Guided by the structure, we design analogs of IL-21 by introducing substitutions to the IL-21-γc interface. These IL-21 analogs act as partial agonists that modulate downstream activation of pS6, pSTAT3, and pSTAT1. These analogs exhibit differential activity on T and B cell subsets and modulate antibody production in human tonsil organoids. These results clarify the structural basis of IL-21 signaling and offer a potential strategy for tunable manipulation of humoral immunity.

Keywords: CP: Immunology; CP: Molecular biology; IL-21; cytokine; germinal center; humoral immunity; immunology; receptor; signaling; signaling complex; structural biology; vaccination.

Graphical abstract

Figure 1

Structure of the IL-21 receptor complex (A and B) Two views of the 2.8 Å resolution structure of the ternary IL-21 receptor complex, showing IL-21 in green, IL-21R in blue, and γc in pink (PDB: 8ENT). (C) Close-up view of the IL-21–γc binding interface at site IIa. Hydrogen bonds are shown as black dashed lines. (D) Close-up view of the IL-21R–γc binding interface at site IIb. Hydrogen bonds are shown as black dashed lines. (E and F) Two views of the 3.7 Å resolution reconstruction of the 2:2:2 IL-21–IL-21R–γc complex determined by cryo-EM and retaining the IL-21R–IL-21R interface observed in the crystal structure (EMDB: EMD-28278). (G) Close-up views of key contacts in the IL-21R–IL-21R site III interface.

Figure 2

Structural basis for common-gamma family receptor sharing (A) Structural alignment of the IL-21 ternary complex with IL-2 shown in beige (PDB: 2B5I), IL-4 in purple (3BPL), and IL-15 in light green (4GS7). (B) Surface representation of γc (pink, right), comparing the site IIa interaction with IL-2 (beige), IL-4 (purple), IL-15 (light green), and IL-21 (dark green). (C) Close-up view comparing helix D of IL-2 (beige) and helix D of IL21 (green), represented as cylinders to show the structural overlap of the site IIa binding residues. (D) Structure-based sequence alignment between IL-2, IL-4, IL-7, IL-9, and IL-15. Residues contacting γc are highlighted in pink. Residues contacting IL-21R are highlighted in blue. (E) Close-up views comparing the helix D interaction with γc. A hydrogen bond “hotspot” interaction is highlighted between P207^γc (shown in pink) and Q116^IL-21 (dark green), Q126^IL-2 (beige), R121^IL-4 (purple), and Q108^IL-15 (light green). (F) Close-up views comparing the helix B interaction with γc. Hydrogen bonding is highlighted between T105^γc (shown in pink) and D37^IL-21 (dark green) or D30^IL-15 (light green). There is no hydrogen bonding between γc and helix B for either IL-2 (beige) or IL-4 (purple).

Figure 3

Engineering the IL-21–γc interface modulates downstream signaling pathways (A) Schematic of the signaling pathways activated by WT IL-21 and the design of IL-21 variants with substitutions along the IL-21–γc interface. (B) Dose-response curves for phopsho-STAT3 (top) and phospho-STAT1 (bottom) in YT-1 cells stimulated with WT IL-21 or the indicated variants for 20 min and analyzed by flow cytometry. Data are mean ± SD for two replicates, shown as percentage of maximal WT IL-21 MFI. (C) Normalized E_max values for phopsho-STAT3, calculated from the dose-response curves shown in (B). Data are mean ± SD for two replicates. (D) Maximum MFI of phospho-STAT3 or phospho-STAT1 in YT-1 cells treated with saturating concentrations of IL-21 or variant. Data are mean ± SD for two replicates. The y axis is scaled to begin at the unstimulated background. (E) Dose-response curves for phospho-STAT3 in human CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and CD56⁺ NK cells. Cells were stimulated with WT IL-21 or the indicated variants for 20 min and analyzed by flow cytometry. Data are mean ± SD for two replicates, shown as percentage of maximal WT IL-21 MFI. (F) Normalized E_max values for phospho-STAT3 in CD4⁺, CD8⁺, CD19⁺, and CD56⁺ human PBMCs calculated from dose-response curves shown in (E). Data are mean ± SD for two replicates.

Figure 4

IL-21 variants modulate B cell activation and antibody production (A) Schematic depicting the T-dependent B cell interaction between a T follicular helper cell (Tfh) and B cell. (B) Activation of pERK in human B cells stimulated with anti-CD40, BCR crosslinking, and WT IL-21 or indicated variant. Data shown are the MFI analyzed by flow cytometry. (C) Activation of pS6 in human B cells stimulated with anti-CD40, BCR crosslinking, and WT IL-21 or indicated variant. Data shown are the MFI analyzed by flow cytometry. (D) Representative histograms of γc and IL-21R surface expression of naive, pre-germinal center (pre-GC), germinal center (GC), memory, and plasmablast (PB) B cells from unstimulated human tonsil organoids. (E) Activation of intracellular pS6 analyzed by flow cytometry in B cell subsets in tonsil organoids cultured with or without 100 nM WT IL-21. Data are mean ± SD for five human donors. (F) Frequency of plasmablasts from tonsil organoids cultured for 7 days in the presence of 100 nM IL-21 or variant, normalized to unstimulated tonsils. Data are mean ± SD for five human donors. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01 by one-way ANOVA. (G and H) Flu-specific IgG quantified by ELISA from the tonsil organoids vaccinated with LAIV and indicated IL-21 variant on day 7 post stimulation with vaccine. Data were fit by non-linear regression, and area under the curve (AUC) and IC₅₀s were calculated in Prism. Data are mean AUC ± SD (G) or log(1/IC₅₀) (H) for five human donors. ^∗p ≤ 0.05 by one-way ANOVA; ns, not significant. (I) Flu-specific IgG detected by ELISA on day 7 post vaccination from tonsil organoids cultured with or without an IL-21R blocking scFv. Data are mean ± SD for two human donors.