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ORIGINAL RESEARCH article

Front. Immunol.

Sec. Alloimmunity and Transplantation

Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1644529

This article is part of the Research TopicFinding New Hope in Old Treatments: Repurposing Immunotherapy in TransplantationView all 7 articles

GlyT1 Inhibition by ALX-5407 Attenuates Allograft Rejection Through Suppression of Th1 Cell Differentiation

Provisionally accepted
Xiaohan  ZhangXiaohan Zhang1,2Weiqi  ZhangWeiqi Zhang1,2Shuai  JinShuai Jin2,3Zhen  WangZhen Wang2Hui  WangHui Wang2Gang  FengGang Feng2Jie  ZhaoJie Zhao2*
  • 1Research Institute of Transplant Medicine, School of Medicine, Tianjin First Central Hospital, Nankai University, Tianjin, China
  • 2Department of Renal Transplantation, Tianjin First Central Hospital, Nankai University, Tianjin, China
  • 3The First Central Clinical School, Tianjin Medical University, Tianjin, China

The final, formatted version of the article will be published soon.

Objective: 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.: RNA sequencing, flow cytometry, and qRT-PCR were employed to analyze GlyT1 expression in Th1-polarized CD4⁺T cells. ALX-5407 (0.5-500 nM) was tested in vitro 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. Results: GlyT1 expression was significantly upregulated in Th1 cells and rejection cohorts. ALX-5407 suppressed Th1 differentiation, reducing IFN-γ⁺CD4⁺T cells proportions (p < 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.Conclusions: 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.

Keywords: Solute carrier, GlyT1, ALX-5407, T-cell-mediated rejection, Organ Transplantation

Received: 10 Jun 2025; Accepted: 19 Aug 2025.

Copyright: © 2025 Zhang, Zhang, Jin, Wang, Wang, Feng and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Jie Zhao, Department of Renal Transplantation, Tianjin First Central Hospital, Nankai University, Tianjin, China

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