A Cytokine Receptor Signaling Atlas Reveals How STAT Mosaics Fine-Tune T Cell Function
Pingdong Tao, Ruchir RastogiiD, Hua JiangiD, Yang ZhaoiD, Leon SuiD, Kevin M. JudeiD, Anshul Bharat KundajeiD, K. Christopher GarcíaiD
Abstract
The extent to which JAK/STAT cytokine signaling is functionally redundant or selective remains debated. Here we engineered a double orthogonal IL-2/IL-2R{beta}/{gamma}c ternary system enabling programmable, interference-free activation of each of the 36 mammalian cytokine receptors, and their downstream six STATs, in T cells. At the membrane-proximal level, comprehensive phospho-signaling profiling revealed that while each receptor activates a dominant STAT, unique STAT activation fingerprints derived from combinatorial biases fine-tune nuanced T cell fates. At the membrane-distal level, single-cell transcriptomic atlas of all cytokine receptors confirmed that these STAT mosaics sensitively specify non-redundant transcriptional programs. STAT5-dominant receptors drove proliferative expansion at the expense of stemness; STAT3-driven programs instructed a continuum from stem cell memory to terminal effector states with preserved cytotoxic capacity and mediated superior curative antitumor responses; while other STATs specified highly restricted phenotypes. These findings decode a STAT signaling vocabulary that defines the intrinsic functional bandwidth of natural cytokines.
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In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (24 Aug 2026) · CC BY 4.0
A fully orthogonal IL-2 receptor platform isolates 36 cytokine receptors in T cells; receptor-specific STAT combinations specify distinct fates, and STAT3-dominant programs give curative antitumor responses.
Whether the 36 cytokine receptors are functionally redundant when they share only six STATs has been unresolved, partly because earlier systems were confounded by endogenous receptors and cytokines. This work gives complete synthetic control over both receptor chains, allowing single-intracellular-domain readouts of signaling, transcriptional state and antitumor function.
Key results
ShownEach cytokine receptor intracellular domain produces a quantitatively distinct STAT fingerprint. STAT1, STAT3, STAT4 and STAT5 are broadly engaged, while STAT2 and STAT6 are highly restricted.
30 decoupled receptor ICDs profiled by phospho-flow. STAT2 was confined to IFNAR2 and IFNLR1; STAT6 was largely IL-4R, with modest signal from IL-20R and IFNAR2. IL-23R favored STAT3 while IL-12Rβ2 favored STAT4. · Isolating individual cytokine receptor signaling…
ShownA single-cell atlas of CD8+ T cells under each receptor shows that STAT bias organizes cell states. STAT5-dominant receptors favor naive/central memory clusters, multi-STAT receptors favor stem cell memory clusters, and four receptor-specific clusters appear.
35 samples, 63,669 cells after filtering (1,101 to 2,370 per sample), 11 clusters. Separate clusters were specific to IL-12Rβ2 (C8), IL-4R (C9), IFNGR1 (C10) and IFNAR2/IFNLR1 (C11). The legend of Fig. 3 gives 58,624 cells. · Transcriptomic atlas of T cells across all…; Figure legends
ShownSix pseudotime trajectories align with dominant STAT programs. STAT3 drives the strongest progression to a terminal effector state with low TOX, STAT5 preserves stemness, and this was supported by repeated-stimulation assays.
Slingshot on non-cycling cells rooted at C1. STAT3- and STAT4-dominant receptors gave the highest Tex_term frequencies; STAT5-dominant receptors kept Tex_prog. STAT3-programmed cells kept TNFα, IFNγ and granzyme B production, and TOX stayed low even after repeated stimulation. · STAT-dependent T cell differentiation…; Analysis of single cell RNA-sequencing data
ShownProliferation and stem-like memory preservation trade off across receptors. Within a single receptor, attenuating STAT1/3/4 or the Shc-dependent MAPK/PI3K arm shifts the balance.
3-day DoIL-2 culture; STAT5-dominant IL-2Rβ, βc, IL-7R, EpoR and TSLPR expanded most with fewest CD44-CD62L+ cells (n = 4). IL-22RdC, which keeps STAT5 but loses most STAT1/3/4, expanded more. IL-2Rβ(Y341F) and OSMR(Y854F) reduced expansion. · Interwoven cytokine receptor signaling balances…; Figure legends
ShownIn B16F10 melanoma, the four individually tested STAT5-dominant receptors expanded and infiltrated tumors but achieved no complete regression in any animal. Five STAT3-dominant combinatorial receptors gave durable, often curative responses.
A pooled barcoded screen preceded individual tests. IL-2Rβ, βc, EpoR and TSLPR gave no complete regression in any animal; IL-9R, IL-31R, IL-21R, IL-22R and IL-23R gave durable, often curative control. All receptors improved control vs the Box control. · Systematic in vivo profiling of T cell antitumor…; Discussion
Methods, data and reproducibility
The authors selected an IL-2 Q126R mutein by saturation mutagenesis and evolved a compensatory orthogonal γc by yeast display, then swapped receptor intracellular domains into the system. They read out phospho-signaling by flow cytometry and transcriptional states by split-pool scRNA-seq, then ran trajectory, regulon and in vivo barcoded screening analyses.
- Mouse primary T cells expressing 31 reconstructed natural receptor pairs and 30 decoupled single-ICD receptors, phospho-flow · mouse · n = 31 pairs; 30 decoupled ICDs; duplicate
- scRNA-seq (Parse split-pool) of Pmel-1 CD8+ T cells, 24 h DoIL-2 stimulation, 30 natural receptors, 3 engineered receptors, 2 controls · mouse · n = 35 samples; 63,669 cells
- Yeast-displayed γc library (five degenerate positions) selected against IL-2 Q126R fusion · human γc displayed in S. cerevisiae · n = about 1 × 10^8 transformants
- YT STAT5 reporter cells and primary human T cells for orthogonality tests · human
- Pooled barcoded in vivo screen: 30 receptors plus mock in Pmel-1 T cells, B16F10 in Rag1-/- mice · mouse · n = 1× 10^7 T cells transferred; 5 × 10^5 tumor cells
- Individual receptor therapy tests in B16F10-bearing C57BL/6J mice with Pmel-1 T cells · mouse · n = 5 × 10^6 Pmel-1 T cells per mouse
- Methods & tools
- Yeast surface display, Phospho-flow cytometry, Parse Evercode / Trailmaker, Scanpy, scVI (scvi-tools v1.4.0), Leiden clustering (resolution 0.6), Slingshot, UCell, pySCENIC v0.12.1 (GRNBoost2, RcisTarget, AUCell), CollecTRI with univariate linear model, DESeq2, GraphPad Prism v10
- Reagents & environment
- scvi-tools v1.4.0; pySCENIC v0.12.1; GraphPad Prism v10; Seurat v5; mouse genome GRCm39 with Ensembl release 109; RcisTarget mm10 databases.
- Reproducibility gaps
- scRNA-seq data and analysis code are promised for GEO and public release only upon publication; other information is available from the lead contact on request.
Limitations (5)
- Transcriptomic states were mapped without concurrent TCR stimulation, so states in tumor-infiltrating T cells under chronic antigen exposure remain uncharacterized. · stated by the authors
- Direct gene regulatory programs of individual STAT factors remain undefined; chromatin accessibility profiling such as ATAC-seq could resolve STAT-centric cis-regulatory networks. · stated by the authors
- It is unknown whether receptor-specific signaling profiles and transcriptional programs are conserved between mouse and human systems. · stated by the authors
- The text and the Fig. 3 legend report different atlas sizes (63,669 vs 58,624 cells). The scaffold also fixes the IL-2Rβ transmembrane and Box regions and uses human STAT2 as a pSTAT2 surrogate, so native receptor behavior may differ. · noted in this summary
- Curative claims rest on one melanoma model with a transgenic Pmel-1 TCR and a single IL-2 mutein; per-group animal numbers are not given in the text, and robustness to other tumors is untested. · noted in this summary
Who should read:Read this if you design cytokine receptor-based T cell therapies or study how STAT signaling combinations map to T cell fate in single-cell data.
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