Spleens were aseptically harvested from euthanized male C57BL/6 mice (male, 6-8w) and mechanically dissociated into single-cell suspensions. CD4⁺T cells were isolated using magnetic bead-based negative selection kits (STEMCELL) and cultured in anti-CD3ϵ pre-coated 96-well plates (5μg/mL, BioLegend) under Th1-polarizing conditions: RPMI 1640 medium supplemented with recombinant IL-2 (100 IU/mL, PeproTech), IL-12p70 (10 ng/mL, PeproTech), anti-IL-4 (10μg/mL, BioLegend), and anti-CD28 (2μg/mL, BioLegend). ALX-5407 (MCE HY-10711A) was administered at 0.5–500 nM concentrations, with vehicle-treated cells serving as controls. Cells were harvested after 72-hour stimulation for flow cytometry or RNA sequencing.

Full-thickness tail skin grafts (1×1 cm) from donor BALB/c mice (male, 6-8w) were transplanted onto the dorsal region of anesthetized C57BL/6 recipients (male, 6-8w) using 6–0 Prolene sutures. Postoperatively, mice received daily intraperitoneal injections of ALX-5407 (100 mg/kg), rapamycin (50 mg/kg), or vehicle (n=5/group). Graft viability was monitored until Day 7, with >90% necrosis defined as rejection. Surviving grafts and secondary lymphoid organs were collected for histopathological and immunological analyses.

BALB/c mice (male, 6-8w) were anesthetized by inhalation and systemically heparinized via intraperitoneal injection. The thoracic cavity of the mouse was then opened. After the heart stopped beating, the superior vena cava and pulmonary veins of the mouse heart were ligated, and the aorta and pulmonary artery were disconnected. The donor heart was harvested and stored in ice-cold saline. C57BL/6 mice (male, 6-8w) were anesthetized by inhalation, and the skin on the right neck was incised to locate the right internal jugular vein and the right internal carotid artery. The right internal jugular vein and the right internal carotid artery were ligated, disconnected, and cannulated. The donor heart aorta was then sleeved onto the recipient’s internal carotid artery end, and the donor heart pulmonary artery was sleeved onto the recipient’s internal jugular vein end. After fixing and ligating the interfaces, blood flow was restored, and the heart filled and restarted beating. After the operation, the recipient mice were anesthetized by inhalation daily, and the beating of the donor heart was assessed by manual palpation to determine the heart beating situation, and a graft survival curve was plotted.

Single-cell suspensions from in vitro cultures or recipient spleens were stained with fluorochrome-conjugated antibodies against surface markers (Live/Dead, CD4, CD8α, CD25, CD44, CD69, NK1.1, CD19, CD11b, Ly6G, Ly6C, CD11c, F4/80), followed by intracellular fixation/permeabilization for IFN-γ, IL-17A, and Foxp3 detection. For cytokine profiling, cells were stimulated with PMA/ionomycin (1 μg/mL each, Invitrogen™) in the presence of brefeldin A (10 μg/mL, Invitrogen™) for 6 hours prior to staining. Data acquisition was performed on a BD LSR Fortessa X-20 cytometer and analyzed using FlowJo v10.8.

Total RNA was extracted from The CD4⁺T cells sorted from the spleen and subsequently cultured in vitro for 3 days using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA with PrimeScript RT Master Mix (TransGen). qPCR was performed on a LightCycler^® 96 system (Roche) using 2X SYBR Green Premix (TransGen) in 10 μL reactions. Primer pairs were designed via NCBI Primer-BLAST and validated by melting curve analysis. Relative gene expression was calculated using the 2^(-ΔΔCt) method with GAPDH as endogenous control. All reactions were performed in triplicate.

Transplanted skin grafts and transplanted heart grafts were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 μm thickness. Hematoxylin and eosin (H&E) staining was performed using standard protocols (Servicebio) for histopathological evaluation. For CD4 immunofluorescence, antigen retrieval was performed with citrate buffer (pH 6.0, 95°C, 15 min). Sections were blocked with 10% goat serum (Beyotime) and incubated with anti-CD4 primary antibody (1:100, Invitrogen) at 4°C overnight, followed by Alexa Fluor 488-conjugated secondary antibody (1:500, Invitrogen) for 1 h at room temperature. Nuclei were counterstained with DAPI (Servicebio). Images were captured using a Olympus upright fluorescence microscope. Negative controls omitted primary antibodies.

The CD4⁺T cells sorted from the spleen were labeled with 5 μM CFSE (Invitrogen) in PBS at 37°C for 10 min, followed by quenching with complete RPMI-1640 medium. Cells were seeded in 96-well plates and treated with ALX-5407 at concentrations of 0.5, 5, 50, or 500 nM, while control groups received vehicle (0.1% DMSO). After 72 h incubation (37°C, 5% CO₂), cells were harvested and analyzed by flow cytometry (BD LSR Fortessa 20X). Proliferation indices were calculated using FlowJo v10.8 software based on CFSE dilution.

The CD4⁺ T cells sorted from the spleens of C57BL/6 mice were labeled with CellTrace™ Violet (CTV) (20) and activated in vitro for 1 day using anti-mouse CD3 (5 ng/ml) and anti-mouse CD28 (2 ng/ml). They were then intravenously infused into C57BL/6 mice, which were randomly divided into an ALX-5407 group and a control group. According to the group, mice received intraperitoneal injections of ALX-5407 (50 mg/kg, once daily)(n=5). Three days later, spleens were collected from the mice for flow cytometry analysis to compare the proliferation of CD4⁺ CTV⁺ cells between the two groups by flow cytometry (BD LSR Fortessa 20X). Proliferation indices were calculated using FlowJo v10.8 software based on CTV dilution.

The CD4⁺T cells sorted from the spleen were polarized into Th0 and Th1 subsets under standard conditions for 3 days. For ALX-5407 treatment, cells were exposed to 500 nM ALX-5407 or vehicle (0.1% DMSO) for 24 h. Total RNA was extracted using TRIzol (Invitrogen), followed by ribosomal RNA depletion and library preparation with NEBNext Ultra II RNA Library Prep Kit (NEB). Differentially Expressed Genes (DEGs) were identified using DESeq2 (|log2FC| >1, adj. p <0.05). Gene Ontology (GO) and KEGG pathway analyses were conducted via clusterProfiler (v4.0).

The CD4⁺T cells sorted from the spleen and subsequently cultured in vitro for 3 days were lysed in RIPA buffer (Beyotime) containing protease inhibitors (Beyotime). Protein concentrations were quantified via BCA assay (Thermo Fisher). Equal amounts (20 μg/lane) were separated by 10% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk for 1 h, membranes were incubated overnight at 4°C with primary antibodies as loading control. HRP-conjugated secondary antibodies were applied for 1 h at RT. Signals were detected using ECL chemiluminescence and quantified by ImageJ v1.53.

Data were analyzed using GraphPad Prism v10.0 and expressed as mean ± SD. Multiple group comparisons employed two-way ANOVA with Bonferroni post hoc correction. Pairwise comparisons between specific groups used unpaired Student’s t-test. Skin graft survival rates were analyzed by Kaplan-Meier method with log-rank test for significance. p < 0.05 was considered to be statistically significant.

To investigate differences between Th0 and Th1 cells, CD4⁺T cells isolated from spleens of male C57BL/6 mice via magnetic bead sorting were cultured under Th0 or Th1 conditions for 3 days. RNA sequencing revealed distinct transcriptomic profiles between Th0 and Th1 cells ( Figure 1A ). Solute carrier (SLC) family proteins, as primary transporters of intracellular substances, profoundly influence cellular metabolic homeostasis through expression changes. Therefore, we prioritized analysis of differential SLC protein expression between the two experimental groups. Among differentially expressed genes, SLC6A9 (GlyT1), a cell-surface glycine transporter critical for maintaining intracellular/extracellular glycine homeostasis, exhibited significantly higher expression in Th1 cells compared to Th0 cells (p < 0.05, Figure 1B ). These findings suggest that GlyT1-mediated glycine regulation may influence Th1 proliferation and differentiation.

To investigate whether GlyT1 could influence CD4⁺ T cell proliferation and differentiation, we utilized ALX-5407, a specific inhibitor of GlyT1. According to literature, ALX-5407 selectively inhibits GlyT1 function by reducing glycine transmembrane flux and is currently under experimental investigation for treating neuropsychiatric disorders, with a recommended concentration of 5 nM for neuronal cells. To determine the impact of ALX-5407 on Th1 differentiation and identify effective doses, we treated CFSE-labeled CD4⁺T cells with a concentration gradient (0.5 nM, 5 nM, 50 nM, 500 nM) under Th1-polarizing conditions for 3 days. Flow cytometry results after 3 days showed that ALX-5407 at various concentrations had minimal effects on Th1 cell proliferation ( Figure 2A ). However, cell viability began to decline at 500 nM, prompting us to select this concentration for subsequent experiments. To further investigate the impact of ALX-5407 on CD4⁺T cells proliferation in vivo, we isolated CD4⁺T cells from the spleens of C57BL/6 mice, labeled them with CTV (20), and activated them in vitro. These cells were then injected into C57BL/6 mice. Flow cytometry analysis of splenocytes 3 days post-injection revealed that the proliferation of CD4⁺CTV⁺cells in the ALX-5407 treatment group appeared moderately reduced compared to the control group. However, this result showed no statistical significance, suggesting that, consistent with in vitro results, ALX-5407 exerts minimal impact on CD4⁺T cell proliferation in vivo ( Figure 2B ).

However, Flow cytometry analysis revealed that the proportion of CD4⁺IFN-γ⁺ T cells in the ALX-5407-treated group was significantly lower than in the control group, with IFN-γ MFI also markedly reduced ( Figure 2C ). Concurrently, MFI values of CD25 and CD69 in the ALX-5407 group decreased, indicating reduced T cell activation ( Figure 2D ). Further analysis of cytokine mRNA expression levels demonstrated consistent results: Ifng expression was significantly downregulated, while Il1β and Il10 were upregulated, with no change in Tgfb1 ( Figure 2E ). These results suggest that ALX-5407, a GlyT1 inhibitor, significantly suppresses Th1 differentiation and impairs Th1 cell activation and function.

To further validate our findings, we established a BALB/c (male, 6–8 weeks) to C57BL/6 (male, 6–8 weeks) murine skin allograft model. Starting from the day of surgery, four groups received daily intraperitoneal injections: control, ALX-5407 alone, rapamycin alone, and ALX-5407 combined with rapamycin. Graft survival analysis demonstrated that ALX-5407 monotherapy failed to prolong graft survival, while the combination treatment significantly extended survival compared to both single-agent groups (p=0.018, Figure 3A-B ). Histological evaluation on day 7 post-transplantation revealed better-preserved graft architecture with reduced inflammatory infiltration in the combination group ( Figure 3C ). Immunofluorescence analysis further confirmed significantly fewer CD4⁺T cells infiltrating the grafts in the combination group compared to other treatments ( Figure 3D and Supplementary Figure 2 ). These results indicate that ALX-5407 synergizes with rapamycin to exert significantly enhanced protective effects on allografts.

Based on the above skin transplantation results, to further clarify the impact of ALX-5407 on solid organ transplantation, we established a mouse allogeneic heart transplantation model for validation. Experimental data demonstrated that the survival time of the transplanted heart in the ALX-5407 treatment group was significantly prolonged compared with the control group, with a statistically significant difference ( Figure 3E , p=0.0020). Pathological analysis of the transplanted heart tissue collected on post-transplantation day 7 revealed that, compared with the control group, the structural integrity of the transplanted heart in the ALX-5407 treatment group was significantly improved, and the degree of inflammatory cell infiltration was notably reduced ( Figure 3E ).

To investigate the mechanisms underlying the protective effects of ALX-5407 combined with rapamycin on allografts, we performed flow cytometry analysis on splenocytes from skin-grafted mice at postoperative day 7 (gating strategy shown in Supplementary Figure 1 ). No significant differences were observed among groups in splenocyte viability, CD4⁺/CD8⁺ T cell ratios, or CD4⁺/CD8⁺ T cell proportions ( Figure 4A ). Notably, the proportion of CD4⁺IFN-γ⁺ T cells was reduced in all treatment groups compared to controls, with the most pronounced reduction in the combination group ( Figure 4B ). A parallel reduction was observed in CD8⁺IFN-γ⁺ T cell frequencies. Furthermore, combination therapy significantly reduced activation marker expression (CD25, CD44, CD69 MFI) in both CD4⁺ and CD8⁺ T cells ( Figures 4C, D ). These findings demonstrate that ALX-5407 and rapamycin synergistically suppress Th1 polarization while attenuating T cell activation.

To determine whether ALX-5407 impacts other T cell subsets, we analyzed regulatory T cells (Tregs) and Th17 cells, which showed no significant intergroup differences ( Supplementary Figure 3 ). Analysis of broader immune populations, including B cells, NK cells, macrophages, dendritic cells, and myeloid-derived suppressor cells, revealed no treatment-associated alterations. Collectively, these data indicate that the anti-rejection efficacy of ALX-5407 combined with rapamycin primarily stems from coordinated inhibition of Th1 differentiation and functional activity.

To investigate the mechanism by which ALX-5407 inhibits Th1 differentiation, we performed RNA sequencing (RNA-seq) on ALX-5407-treated Th1 cells and conducted KEGG/GO enrichment analyses compared to untreated controls ( Figures 5A, B ). The results revealed significant upregulation of the PI3K-AKT-mTOR pathway and downregulation of the MAPK pathway in ALX-5407-treated cells, which were further validated by Western blot analysis ( Figures 5C–E ). Given the critical roles of PI3K-AKT-mTOR and MAPK pathways in apoptosis regulation, we assessed apoptotic activity in ALX-5407-treated cells. Apoptosis was significantly increased in the ALX-5407 group ( Figure 5F ), with Western blot confirming elevated levels of cleaved caspase-3 and reduced expression of the anti-apoptotic protein BCL-2 ( Figure 5D ) (21, 22). These findings suggest that ALX-5407 reduces the Th1 cell population by inducing cellular apoptosis.

Notably, PI3K-AKT-mTOR pathway activation promotes T cell proliferation and differentiation, potentially explaining the limited efficacy of ALX-5407 monotherapy. Combining ALX-5407 with the mTOR inhibitor rapamycin partially counteracted mTOR pathway activation induced by ALX-5407, resulting in a synergistic enhancement of Th1 suppression (1 + 1>2 effect).