{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,24]],"date-time":"2025-09-24T00:06:09Z","timestamp":1758672369545,"version":"3.44.0"},"reference-count":42,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T00:00:00Z","timestamp":1758585600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Objective<\/jats:title><jats:p>Transplant rejection driven by Th1 cell-mediated immune responses remains a critical challenge. This study aimed to investigate the role of glycine transporter 1 (GlyT1\/SLC6A9) in Th1 differentiation and evaluate the therapeutic potential of its inhibitor, ALX-5407, in attenuating allograft rejection.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>RNA sequencing, flow cytometry, and qRT-PCR were employed to analyze GlyT1 expression in Th1-polarized CD4<jats:sup>+<\/jats:sup>T cells. ALX-5407 (0.5\u2013500 nM) was tested <jats:italic>in vitro<\/jats:italic> under Th1-polarizing conditions. A murine skin allograft model (BALB\/c to C57BL\/6) was established to assess graft survival and immune responses. Combination therapy with rapamycin and ALX-5407 was evaluated through histopathology, immunofluorescence, and splenocyte profiling. Mechanistic insights were derived from RNA-seq, KEGG\/GO enrichment, and Western blotting.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>GlyT1 expression was significantly upregulated in Th1 cells and rejection cohorts. ALX-5407 suppressed Th1 differentiation, reducing IFN-\u03b3<jats:sup>+<\/jats:sup>CD4<jats:sup>+<\/jats:sup>T cells proportions (p &amp;lt; 0.05) and activation markers (CD25, CD69), while inducing apoptosis via caspase-3 activation and BCL-2 downregulation. Although ALX-5407 monotherapy failed to prolong graft survival, combination with rapamycin synergistically enhanced efficacy (p = 0.018), reduced inflammatory infiltration, and attenuated splenic Th1 polarization. Mechanistically, ALX-5407 inhibited MAPK signaling but activated the PI3K-AKT-mTOR pathway, which rapamycin counteracted to amplify suppression.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>GlyT1 serves as a metabolic checkpoint in Th1 differentiation, and its inhibition by ALX-5407 attenuates allograft rejection through dual suppression of Th1 function and apoptosis induction. Synergy with rapamycin highlights a novel combinatorial strategy to mitigate rejection with reduced toxicity. These findings position GlyT1 targeting as a promising approach for clinical translation in transplantation immunotherapy.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1644529","type":"journal-article","created":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T04:14:03Z","timestamp":1758600843000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["GlyT1 inhibition by ALX-5407 attenuates allograft rejection through suppression of Th1 cell 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