Age-dependent Zap70 expression in thymocytes regulates selection of the neonatal regulatory T cell repertoire

nature immunology Article https://doi.org/10.1038/s41590-025-02292-7 Age-dependent Zap70 expression in thymocytes regulates selection of the neonatal regulatory T cell repertoire Received: 26 February 2025 Accepted: 27 August 2025 Published online: xx xx xxxx Check for updates Brian D. Stadinski 1,10, Elizabeth A. Mills 2,10, Preston A. Humphries 2, Sarah B. Cleveland 1, Parker Dow3, Koura Murakami4, Yue Ru Li5, Masaaki Murakami4, Masahiro Ono 6, Byron B. Au-Yeung 7, Gerald P. Morris3, Juan Carlos Zúñiga-Pflücker 5, Robert A. Campbell 8, Eric R. Griffiths9, Eric S. Huseby 1 & Wan-Lin Lo 2 The Foxp3⁺ regulatory T (Treg) cell repertoire carries age-dependent biases, with neonatal subsets enriched for highly self-reactive clones. However, the thymocyte features distinguishing neonatal from adult Treg selection remain unclear. Here, we show that neonatal double-positive mouse thymocytes, unlike their adult counterparts, fail to upregulate Zap70 during thymic selection, creating a calcium signaling bottleneck. This attenuated Zap70-dependent signaling limits negative selection, allowing highly self-reactive clones to evade deletion. Modulating Zap70 expression alters this balance; reducing Zap70 in adults rescues development of these clones, whereas increasing Zap70 in neonates enforces their deletion. Similarly, enhancing neonatal calcium signaling via increased LAT Y136-mediated PLCγ1 activation promotes clonal deletion. Analysis of pediatric human thymi reveals that ZAP70 expression remains low during the first year of life, aligning with the peak window for thymic Treg cell development. These findings suggest that age-dependent Zap70 expression regulates negative selection and thymic Treg cell development. Early life necessitates rapid immune development while maintaining tolerance to benign stimuli. Neonatal T cell microrepertoires, featuring unique T cell antigen receptor (TCR) clonotypes compared to those in adults, help establish immune homeostasis via their effector functions and tissue localization1–4. Among these microrepertoires, thymic-derived Foxp3+ regulatory T (Treg) cells and CD8αα intraepithelial lymphocytes (IELs) contribute to peripheral and mucosal tolerance, respectively. A defining feature of neonatal Treg cells and CD8αα IEL precursors is their enrichment for self-reactive clones generated via agonist selection during a tightly controlled developmental window5. While thymocyte-extrinsic factors (for example, antigen-processing machinery and thymic epithelial and antigen-presenting cells) are suggested to shape the conventional T (Tconv) cell and Treg cell repertoires6–10, A full list of affiliations appears at the end of the paper. Nature Immunology the extent to which neonatal thymocytes use cell-intrinsic pathways to adjust negative selection and diversify into regulatory subsets remains unclear. In the adult thymus, CD4⁺CD8⁺ double-positive (DP) thymocytes undergo positive selection by ligands for which they have very weak affinity, whereas strong TCR signals typically induce negative selection5. Critically, ligands that drive positive selection and many that induce negative selection generally do not support mature T cell expansion or effector differentiation5. These observations highlight two principles shaping the mature T cell repertoire: (1) thymocyte maturation is accompanied by a programmed reduction in TCR sensitivity, and (2) the TCR can precisely distinguish the strength and abundance of peptide bound to major histocompatibility complex (MHC) ligands, e-mail: ; Article https://doi.org/10.1038/s41590-025-02292-7 translating this information into distinct functional outcomes11–13. Thus, TCR sensitivity and function are progressively refined during T-lineage cell maturation. ζ-Associated protein of 70 kDa (Zap70), a cytoplasmic tyrosine kinase14, is pivotal in this process, mediating TCR signaling and ensuring the generation of a repertoire that balances immune responsiveness with self-tolerance. Zap70 expression begins after the double-negative (DN) 3 stage and is essential for supporting DP thymocytes through thymic selection15–17. Complete Zap70 deficiency causes severe immunodeficiency14, and both hypermorphic and hypomorphic mutations are linked to autoimmunity, emphasizing the necessity of precise Zap70 regulation in maintaining tolerance. In mature T cells, Lck-mediated phosphorylation of the ζ-chain facilitates Zap70 binding, functioning as a signaling rheostat: elevated self-reactivity increases ζ-chain phosphorylation, thereby enhancing TCR sensitivity12,18,19. After antigen recognition, Lck-activated Zap70 phosphorylates LAT and SLP76, assembling the signalosomes that trigger multiple pathways, including ERK and NFAT20,21. Interestingly, Zap70 phosphorylates LAT and SLP76 with varying efficiency20. Phosphorylation at LAT Y136, which recruits PLCγ1, is relatively inefficient compared to phosphorylation at other sites that recruit GADS and GRB2. Consequently, ERK signaling can be induced by weaker stimuli, whereas PLCγ1-dependent calcium and NFAT activation demand stronger signal inputs20,22,23. Thus, the LAT signalosome functions not just as a scaffold but as an active integrator of signal strength and ligand quality during TCR recognition of peptide bound to MHC12,13,20,22,23. A critical yet unresolved question is whether these signaling thresholds are inherently fixed or can adapt with the host’s age. If LAT-dependent signaling is recalibrated in an age-dependent manner, this could profoundly shape neonate thymocyte fate, biasing the repertoire toward greater tolerance or increased self-reactivity. Here, our findings reveal that thymic negative selection is limited during the neonatal stage, correlating with a modest reduction in Zap70 expression in both mouse and human thymocytes. Diminished neonatal Zap70 expression selectively dampens calcium signaling while maintaining ERK activation. Consequently, transcriptional activation is skewed: NFAT, IRF4, NR4A3 and EGR2 show reduced activation in neonatal DP thymocytes, whereas ERK-dependent factors, including NR4A1, Fos and EGR1, along with NF-κB p65, remain largely unaffected. Experimental modulation of Zap70 expression within physiological levels is sufficient to reprogram selection thresholds (Zap70 haploinsufficiency in adults recapitulates neonatal-like selection, and transgenic expression eliminates neonatal-biased clones). Thus, age-dependent Zap70 expression and its effects on calcium signaling serve as a developmental switch, allowing highly self-reactive thymocytes to escape clonal deletion and contribute to the neonatal Treg cell repertoire. (Fig. 1a and Extended Data Fig. 1c). Following lineage commitment, similar reductions in neonates were observed in agonist-selected populations, including CD25+ CD4-single-positive (CD4SP) Treg cell precursors, Foxp3+ Treg cells and IEL precursors (Fig. 1b and Extended Data Fig. 1d), suggesting that these alterations s (...truncated)