Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Chimeric antigen receptor (CAR)-T cell therapy has transformed treatment of relapsed/refractory DLBCL, yet resistance driven by regulatory T cells (Tregs) limits its efficacy. Here we identify Timosaponin AIII (TAIII), a clinical-stage natural product, as an effective modulator of CAR-T function that depletes CAR-Tregs while enhancing effector activity. Mechanistically, TAIII acts as an allosteric A2AR inhibitor by competing with cholesterol, suppressing CREB-dependent FoxP3 transcription and disrupting the A2AR-Treg axis. Ablation of A2AR or Tregs in vitro and in vivo abolishes TAIII activity, confirming specificity. Furthermore, TAIII reduces intratumoral Tregs, increases CD8⁺ T cells infiltration, and potentiates PD-1 blockade in solid tumor models. Importantly, TAIII promotes central memory T-cell formation and enhances CAR-T cytotoxic cytokine secretion. Combining or pretreating CAR-T cells with TAIII markedly improves antitumor efficacy and prevents later relapse across preclinical models. These findings establish TAIII as a combinatorial strategy to deplete CAR-Tregs, enhance CAR-T activity, and extend therapeutic durability.

Introduction
CD19 chimeric antigen receptor (CAR) T-cell therapy is an effective treatment for relapsed and refractory B-cell malignancies, but in practical clinical application, approximately 60% of patients eventually experience relapse or resistance1–5. Previous investigations suggest that immunosuppressive molecules and cell subsets are critical factors limiting CAR-T efficacy6. The immunosuppressive tumor microenvironment (ITME), including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs)), and molecules like hypoxia-driven adenosine and IL-107,8, is a major barrier to CAR-T cell efficacy by disrupting immune function.

Additionally, CAR-T cell dysfunction or reduced persistence, marked by diminished cytotoxicity and elevated inhibitory receptor expression, is another significant obstacle. Clinical trials evaluating the correlation between CAR-T cell expansion and therapeutic outcomes found that higher CAR-T cell levels post-infusion did not correspond with tumor burden, suggesting that immunosuppressive molecules or cell subsets may play a crucial role in mediating CAR-T therapy response9–11. Nevertheless, the precise mechanisms behind this ITME-mediated CAR-T cell dysfunction remain poorly understood12.

Tregs are crucial for maintaining self-tolerance and immune homeostasis, primarily through the high expression of the transcription factor FoxP3, which is essential for their function13–15. Tumor-infiltrating Treg cells (TITRs) are particularly immunosuppressive within the tumor microenvironment, impairing anti-tumor immune responses and thus promoting tumor immune evasion8. Two recent studies have highlighted the role of CAR-Treg cells in mediating CAR-T resistance through comprehensive single-cell profiling. Using single-cell proteomics and transcriptomics, Good et al. demonstrated that higher levels of Helios+FoxP3+ CAR-Treg cells are closely associated with poor clinical outcomes, providing insight into why CAR-T cell expansion in vivo does not always correlate with clinical response16,17. Based on single-cell transcriptome and TCR sequencing data, Haradhvala et al.18 analyzed samples from 32 patients treated with CAR-T cell infusion and found that high levels of CAR-Tregs led to clinical non-response, and the addition of just 5% of CAR-Tregs in CAR-T cell products was sufficient to reduce conventional CAR-T (CAR-Tconv) expansion and cause relapse in JeKo-1 lymphoma-engrafted NSG mice. These findings reveal the immunosuppressive impact of CAR-Tregs on CAR-T therapy and suggest that an effective Treg-depleting strategy could optimize CAR-T efficacy19.

Current strategies under development mainly include immune checkpoint inhibitors (ICI) and targeted drug combinations, such as CpG oligonucleotide combined with low-dose anti-OX40/CTLA-420 treatment, or blocking chemokine pathways like anti-CCR421. Additionally, antibodies targeting Treg cell surface receptors, such as anti-APRIL and anti-CD122, have been explored. These neutralizing monoclonal antibodies (mAbs), often combined with ICIs, have shown potential in restoring immune tolerance in animal models22,23. However, no clinically effective strategy for selectively depleting CAR-Tregs to enhance CAR-T efficacy has been developed. One significant limitation is that anti-Treg antibodies lack selective targeting, leading to unintended depletion of antitumor effector T-cell populations or adverse side effects24. Furthermore, certain strategies show modest or marginal effects due to the limitations of monotherapy or inadequate drug delivery approaches24. Therefore, novel approaches to improve the specificity of Treg targeting and enhance anti-tumor efficacy are still under development.

Natural products are characterized by their structural diversity and abundance of bioactive compounds, making them valuable sources for drug discovery and lead compound development in the treatment of major diseases, contributing significantly to human health. Compared to synthetic compounds, natural products often exhibit better bioavailability, biodegradability and safety profiles25. Many successful cases have shown the discovery of lead drugs from natural product extracts, which are subsequently optimized into more effective drugs. With the growing appreciation for functional assays and phenotypic screening, there has been a revitalized interest in natural product-based drug discovery26,27. The combination of CAR-T therapy with small-molecule immunomodulators offers advantages such as high efficiency, flexibility and personalization. Recent studies suggest that using small-molecule immunomodulators to eliminate Tregs and enhance CAR-T cytotoxicity against tumor cells may be a promising strategy to overcome therapy resistance.

To identify potential inhibitors of Treg function, we established a Treg functional assay system and conducted high-throughput screening of an in-house compound library containing over 3000 compounds. This led to the identification of a natural steroidal saponin, Timosaponin AIII (TAIII), which is currently in clinical trials as a safe and effective anti-wrinkle agent due to its anti-inflammatory properties (KCT0002223)28, which also demonstrated inhibitory effects on Tregs. TAIII is derived from Anemarrhena asphodeloides Bunge, a traditional Chinese medicinal herb29. Previous studies suggest that TAIII regulates multiple signal pathways30, including NF-κB, AKT/mTOR, and ERK1/2/MAPKs, exhibiting both anti-inflammatory and anti-tumor activities. Research has shown that TAIII can restore the balance between Treg and Th17 cells in mice with TNBS-induced colitis31. However, the precise mechanisms underlying TAIII’s effects on human T cells and supporting evidence remain unclear.

In this study, we found that TAIII allosterically targets adenosine receptor A2 (A2AR) in human T or CAR-T cells, blocking adenosine-mediated immunosuppressive signals by down-regulating key transcriptional markers, including FoxP3 and CTLA-4, and inhibiting Treg recruitment and cholesterol metabolism pathways, thereby reducing the CAR-Treg population. Genetic depletion of A2AR in both Treg and CAR-T cells, along with Treg depletion in CAR-T cells in vitro, and the use of a Treg-depletion CAR-T therapy model (Nalm6-Luc) in vivo, led to the loss of TAIII activity, further reinforcing its selective targeting of the A2AR-Treg axis. By disrupting the adenosine-induced immunosuppressive microenvironment, TAIII significantly suppresses tumor growth and enhances the efficacy of anti-PD-1 immunotherapy by reducing intratumoral Tregs. Importantly, pretreatment or combination with TAIII enhances CAR-T cell expansion, persistence, and anti-tumor activity, helping to overcome relapses across various tumor models. These findings establish TAIII as a potent CAR-T cell adjuvant and provide evidence that this natural product reduces Tregs, thereby improving CAR-T cell function and preventing therapy resistance.

Results
High-throughput screening identified Timosaponin AIII as a Treg inhibitor
Given that Treg percentage and function are crucial for CAR-T treatment outcomes, the transcription factor FoxP3a serves as the most important functional marker of Tregs by inhibiting the transcription of genes such as IL-213–15. To establish a high-throughput screening platform for Foxp3-mediated Il-2 inhibition, we introduced the human IL-2 promoter region (−326 to +145,473 bp) containing a FoxP3a (FoxP3) binding site into the Lenti-PGL4 reporter vector. This system was packaged into lentivirus, and the IL-2 promoter-firefly luciferase (Fluc) reporter with a puromycin resistance cassette was transduced into Jurkat T cells. After puromycin selection, monoclonal cells were isolated and tested for luciferase activity upon TCR stimulation. The best-performing clone was selected, and a lentiviral construct for FoxP3a overexpression, containing a BSD resistance cassette, was introduced. Following another round of TCR stimulation and selection, the final monoclonal cell line with optimal performance was chosen, allowing Treg function to be evaluated by Fluc fluorescence intensity (Fig. 1a).

To identify potential Treg inhibitors, we conducted high-throughput screening of an in-house natural product and FDA-approved drug library (containing over 3000 compounds). The Fluc intensity was normalized to cell viability after 16-hour treatment (Fig. 1a). During the initial screening, over 50 compounds, including Timosaponin AIII (TAIII), exhibited significant modulation of the FOXP3–IL-2 axis (Fig. 1b). To prioritize undisclosed candidates, previously reported compounds such as HDAC inhibitors were excluded through literature and patent database review. The top 10 candidates were subsequently subjected to secondary screening, including single-dose testing in human PBMCs (Fig. 1c, upper panel), with careful consideration of both potency and toxicity. Toxicity was assessed by evaluating the viability of human PBMCs as a surrogate for off-target cytotoxicity (Fig. 1c, lower panel). From this process, TAIII, a natural product derived from Anemarrhena asphodeloides Bunge and also a traditional Chinese medicine, emerged as the lead candidate (Fig. 1d), demonstrating robust dose-dependent efficacy and a favorable safety profile. TAIII significantly reversed FoxP3-mediated IL-2 transcription inhibition in a dose-dependent manner (Fig. 1e). TAIII, a steroidal saponin, has been primarily reported for its anti-cancer, anti-inflammatory, and anti-thrombotic properties30,31. In inflammation models, TAIII reduces cytokines such as IL-6, IL-1β, and TNF-α. However, its role in activating IL-2 or inhibiting human Tregs remains largely unexplored, as do the molecular mechanisms underlying Treg proportion and function.

To verify TAIII’s effect on human Tregs, we isolated human CD4+ naïve T cells and cultured them under iTreg-polarizing conditions. TAIII significantly impaired Treg differentiation and reduced expression of functional markers (FoxP3, CD25, CTLA4) in a dose-dependent manner (Fig. 1f). Similar results were observed when surveying the proportion of FoxP3+ iTreg cells during late differentiation stages (days 5–8) (Fig. 1g, h). These findings suggest that TAIII is a previously unrecognized natural product that inhibits Tregs and reduces their proportion.

TAIII enhances the antitumor efficacy of CAR-T cells by eliminating FoxP3+ Treg cells in vitro
To investigate whether TAIII improves the antitumor efficacy of CAR-T cells, we first generated the CD19 CAR-T by transducing T cells isolated from human peripheral blood mononuclear cells (PBMCs) with lentiviral CAR constructs. We then assessed the baseline T cell phenotypes at the time of CAR-T manufacturing. These included the proportions of CD8, CD4, Tnaive, Tcm, Teff, Temra, and Tregs, assessed by flow cytometry three days post-CAR transduction. T cell subsets were defined as follows: CD45RO⁻CD62L⁺ (Tnaive), CD45RO⁺CD62L⁺ (Tcm), CD45RO⁺CD62L⁻ (Teff), and CD45RO⁻CD62L⁻ (Temra), following established refs. 32,33 (Supplementary Fig. 1a). To evaluate phenotypic changes upon in vitro TAIII exposure, CD19-CAR-T cells were treated with either DMSO or TAIII for 72 hours, followed by flow cytometry analysis. The proportions of CD4⁺ T cells, CD8⁺ T cells and Teff remained largely unchanged, whereas the TAIII-treated group exhibited an increase in Temra cells and a corresponding decrease in Tnaive cells. Notably, TAIII treatment led to a significant reduction (~40%) in Treg proportions (Supplementary Fig. 1b, c, Fig. 2a), suggesting that TAIII may enhance CAR-T efficacy by attenuating immunosuppressive Treg populations.

To directly assess the effects of TAIII on untransduced T cells (UNT) and CAR-T cells in the absence of tumor antigen stimulation, we treated these cells with TAIII under non-stimulatory conditions and measured their proliferation, viability, and apoptosis. As shown in revised Fig. 2d–e, TAIII treatment for 24 and 72 h did not significantly affect the proliferation or viability of either cell type, indicating that TAIII’s effects are not due to nonspecific T cell stimulation, but rather are dependent on tumor antigen presence and Treg modulation (Fig. 2b, c).

Given these phenotypic alterations, we next evaluated the functional impact of TAIII on CAR-T cell-mediated tumor cell killing in a co-culture cytotoxicity assay. CAR-T cells were co-cultured with Raji or Nalm6 cells at effector-to-target (E:T) ratios of 1:2 and 1:4, with or without TAIII treatments. TAIII significantly enhanced CAR-T cell-mediated killing of Raji or Nalm6 cells in a dose-dependent manner compared to the CAR-T-only control (Fig. 2d, e). Specifically, TAIII increased the cytotoxicity of CAR-T cells, killing approximately 80% of target Nalm6 cells (Fig. 2e). Additionally, we observed a significant upregulation of T-cell effector cytokine secretion, including IFN-γ, IL-2, and TNF-α, in the TAIII-treated CAR-T group (Fig. 2d, e).

To further investigate the kinetics of TAIII’s effect, CAR-T-cell-mediated killing was monitored over time via immunofluorescence every 2 h for 36 h using the Incucyte® cytolight in a co-culture cytotoxicity assay (E:T=1:2 or 1:4). Similar results showed that TAIII significantly enhanced CD19 CAR-T activity against Raji cells in a time-dependent manner, with effects observed as early as 4 h (Fig. 2f).

Next, we investigated whether TAIII enhances CAR-T cell cytotoxicity by modulating Tregs. In co-cultures of CAR-T cells with or without TAIII post-activation with Raji (E:T=1:1), we observed that T-cell effector cytokine secretion, including IFN-γ and TNF-α, was significantly upregulated in the TAIII-treated CAR-T group (Fig. 2g). Moreover, the expression of FoxP3, the key Treg-associated transcription factor, was significantly downregulated (Fig. 2h), consistent with the reduction observed in TAIII-treated CAR-T cells cultured alone (Supplementary Fig. 1b, c, Fig. 2a). These results suggest that TAIII reduces the proportion of FoxP3+ CAR-Treg cells and enhances CAR-T cell cytotoxicity by targeting Tregs.

To specifically evaluate whether TAIII exerts its effect through Treg modulation, we generated Treg-depleted CAR-T (ΔTreg-CAR-T) cells by sorting CD4+CD25+CD127low cells from human PBMCs, reducing their frequency to 1.28% (Supplementary Fig. 2a, b). Notably, flow cytometric analysis was performed 72 h after CAR transduction to capture the dynamic expansion of CAR-T subsets over time, rather than immediately following sorting. Additionally, the Treg depletion strategy targeted <5% of CD4⁺CD25⁺CD127low cells and did not significantly alter the overall CD8/CD4 ratio, reflecting differential expansion post-depletion rather than a loss of CD4⁺ effector cells (Supplementary Fig. 2c). The resulting CAR-T cells were co-cultured with Nalm6-Luc cells for 24 h, and luciferase activity was measured to assess target cell killing. At various E:T ratios, Treg depletion enhanced the short-term killing effect of CAR-T cells, confirming that Tregs negatively influence CAR-T cell function (Supplementary Fig. 2d). This highlights the critical role of CAR-Tregs in modulating CAR-T-mediated cytotoxicity. Purified CD4+ or CD8+ ΔTreg-CAR-T cells were co-cultured with Raji-Luc cells for 24 h. As previously observed, CD8⁺ CAR-T cells were primarily responsible for short-term cytotoxicity11,34. Notably, TAIII treatment did not alter the killing capacity of ΔTreg-CAR-T cells, regardless of subset (Supplementary Fig. 2e–g), indicating that TAIII’s functional enhancement requires Treg presence.

This was further supported by parallel experiments using parental CAR-T cells (5.18% Tregs), in which TAIII significantly enhanced cytotoxicity (Supplementary Fig. 2e), suggesting that TAIII’s functional enhancement is dependent on the presence of Tregs. ELISA analysis revealed no significant differences in IL-2, IFNγ, or TNFα secretion between TAIII-treated and DMSO-treated ΔTreg- or CD4⁺ ΔTreg-CAR-T cells. While TAIII modestly increased cytokine secretion in CD8⁺ ΔTreg-CAR-T cells, this was insufficient to enhance cytotoxic activity (Supplementary Fig. 2g, h).

Collectively, these results demonstrate that TAIII potentiates CAR-T cell efficacy primarily by alleviating Treg-mediated suppression, rather than through direct effects on Teff cells. This targeted mechanism offers therapeutic advantages by preserving intrinsic T cell function while selectively countering immunosuppressive elements within the CAR-T population.

TAIII is a selective allosteric inhibitor of A2AR
To explore the intrinsic mechanism underlying TAIII-mediated Treg inhibition, we sought to identify the direct targets of TAIII using reverse screening methods. We designed and synthesized a biotinylated TAIII probe (Biotin-TAIII-P) with a photoactivatable group using click chemistry35 (Fig. 3a), incorporating a photosensitive fishhook structure for proximity labeling in combination with mass spectrometry (LC-MS) (Supplementary Fig. 3a). This led to the identification of adenosine A2A receptor (A2AR) as a key immunosuppressive target of TAIII (Supplementary Fig. 3b). To further validate this finding, we also used the SwissTargetPrediction online tool, which identified GPCRs as the most prominent class of predicted targets for TAIII. Notably, A2AR (ADORA2A) and A1AR (ADORA1) were identified as the top candidates, reinforcing A2AR as a promising target (Supplementary Fig. 3c–d). Adenosine receptors are members of the rhodopsin-like family of G protein-coupled receptors (GPCRs), which consist of four subtypes: A1R, A2AR, A2BR, and A3R. Each subtype is coupled to distinct intracellular small G proteins, mediating different signaling pathways. To further investigate TAIII’s selectivity, we conducted microscale thermophoresis assays using EGFP-fused adenosine receptor constructs. These assays compared the binding affinity of TAIII for A2AR with that for other adenosine receptor subtypes. The results revealed that TAIII displays high selectivity for A2AR, with negligible or no binding to other receptors and only a modest interaction with A2BR (Supplementary Fig. 3e). A2AR is a well-known immune checkpoint expressed on various immune cells, particularly T cells. Studies show that A2AR plays a critical role in the tumor immunosuppressive microenvironment and promotes tumor immune escape, making it a therapeutic target in immunotherapy. Although its immunosuppressive function via Treg has been established, the molecular mechanisms regulating the activity of Treg remains largely unknown. Moreover, it is unknown whether blocking this signaling pathway improves CAR-T cell manufacturing.

We then confirmed the binding of TAIII to A2AR. Since 293T cells express low levels of A2AR, we generated the A2AR-overexprssing 293T cells (293T-A2AR-OE) stably expressing A2AR-GFP. Using a cellular thermal shift assay (CETSA), we investigated the effect of TAIII on A2AR protein stability across different temperatures. As expected, TAIII robustly increased the thermal stability of A2AR (Supplementary Fig. 3f). Moreover, Microscale Thermophoresis assay further confirmed TAIII-A2AR binding. AZD4635, an A2AR inhibitor currently in phase II clinical trials, was used as a positive control (Supplementary Fig. 3g).

We then performed a Target engagement assay using biotin-streptavidin pull-down method and demonstrated a dose-dependent interaction between TAIII and A2AR in both overexpression system and primary human T cells (Fig. 3b, Supplementary Fig. 3h). To further confirm the target engagement of A2AR, we performed a competition assay with Biotin-TAIII-P after 4 h of TAIII treatment, and observed that 5 μM TAIII labeled ~50% of A2AR (Fig. 3c, d), confirming A2AR as a direct target of TAIII.

Given that A1R and A3R couple to Gi proteins, while A2AR and A2BR couple to Gs proteins. A2AR activation by ligand binding triggers Gs coupling, activating adenylate cyclase (AC) to produce cAMP and initiates the classic cAMP-PKA-CREB signaling pathway (Supplementary Fig. 4a), which mediates various immunosuppressive responses36,37. To evaluate the functional selectivity of TAIII among the adenosine receptor subtypes, we constructed expression vectors for A1R, A2AR, A2BR, and A3R and over-expressed them in 293T cells. Using the LANCE cAMP assay, we measured intracellular cAMP levels following A2AR activation with 0.1 μM of the adenosine agonist 5-(N-ethylcarboxamido)adenosine (NECA). The results demonstrated that TAIII exhibited high selectivity for A2AR, with minimal or no effects on the other subtypes and only a modest effect on A2BR (Supplementary Fig. 4b), consistent with previous findings on its binding selectivity (Supplementary Fig. 3e). Considering that A2BR has a substantially lower sensitivity to adenosine compared to A2AR, and its expression is particularly sparse on T cells, especially Tregs, A2BR was not prioritized as a key factor in our analysis. We then compared TAIII’s activity with the phase II A2AR antagonist AZD4635 using both CREB reporter and LANCE cAMP assays. TAIII displayed comparable potency to AZD4635 across various cell models, with cell viability maintained at all tested concentrations, suggesting that TAIII is likely non-toxic to T cells (Fig. 3e, f). To rule out off-target effects on the A2AR-cAMP/PKA/CREB pathway, we assessed TAIII sensitivity in A2AR-overexpressing cells (A2AR-OE) versus parental cells. TAIII exhibited greater sensitivity in A2AR-OE cells, with potency comparable to other A2AR antagonists (Fig. 3g). Taken together, these findings suggest that TAIII is a selective A2AR inhibitor.

To explore TAIII’s binding mode to A2AR, we utilized computer-aided structural analysis to investigate its interaction within the A2AR complex (PDB ID: 4EIY) (Fig. 3h). Studies show that the cholesterol-sensitive motifs (CCM) in the transmembrane region of A2AR are highly conserved. The cholesterol content in the cell membrane modulates the ligand-binding pocket of A2AR, thus affecting its function. Cholesterol depletion or CCM mutations disrupt A2AR structure, impairing orthosteric ligand binding and downstream signaling38,39. As a steroidal saponin, the core structure of TAIII resembles cholesterol and cholesterol replacement with TAIII in the liposome bilayer enhances drug delivery efficiency, highlighting their structural similarity40. Given that membrane cholesterol is crucial for A2AR activation38,39, we hypothesized that TAIII might compete with cholesterol to affect A2AR function. Structural modeling analysis showed the polyhydroxyl head of TAIII extends into a polar pocket formed by Q38, W32, Y43 and K122, allowing stable binding. The other hydrophobic moiety aligns with the non-polar region of the cell membrane along the transmembrane helix, consistent with the phospholipid bilayer model structure (Fig. 3h).

To validate this hypothesis, we introduced point mutations into three key CCM residues, which have been reported to bind cholesterol38, and investigated their contribution to the binding of A2AR and TAIII by CREB-luciferase reporter assay. Notably, the W129A mutation retained partial activity, which is an important consideration when interpreting the sensitivity of TAIII in these experiments. Compared to wild-type A2AR, all mutations decrease CREB activity and abolish sensitivity to even increasing doses of TAIII (Fig. 3i, j), indicating that TAIII affects A2AR function by competing with cholesterol and binding allosterically to these critical residues.

TAIII activates T cell responses through impairing Treg function in an A2AR-dependent manner
Adenosine suppresses immune cells within the tumor microenvironment by inhibiting T cell activation and cytotoxic signaling, which contributes to the immunosuppressive activity of Tregs44,45. To evaluate the efficacy of TAIII in enhancing T cell immune responses through A2AR signaling, we isolated PBMCs from healthy donors and cultured them under pan-T cell conditions with NECA (a potent adenosine agonist) or TAIII. Transcriptomic analysis (bulk RNA-seq) of T cells revealed that in the presence of NECA, TAIII treatment significantly upregulated immune activation genes, including TNF, GZMB, IFN-γ and IL-2, while downregulating immunosuppressive genes, such as FoxP3, IKZF2 (Helios), IKZF4 (Eos) and CCR8 (Fig. 4a). Additionally, it enriched immune suppression-related genes and pathways in Tregs, as shown by Reactome pathway enrichment analysis (Supplementary Fig. 5a). To validate these findings, we conducted ELISA and qRT-PCR, demonstrating that TAIII effectively counteracted NECA-mediated suppression of IL-2 and IFN-γ expression, enhancing T cell activation. Moreover, TAIII dose-dependently reduced FoxP3 transcription in T cells, showing greater potency than AZD4635 (Fig. 4b–c, Supplementary Fig. 5b, c). Similar results were observed when evaluating the proportion of FoxP3+ iTreg cells during differentiation. NECA (5 μM) upregulated Treg differentiation and functional markers (FoxP3, CTLA4, CD25, and TGF-β), which were significantly suppressed by TAIII treatment (Fig. 4d, Supplementary Fig. 5d).

Furthermore, we demonstrate that TAIII significantly prevented NECA-mediated immunosuppressive effects of Tregs on effector T cells (Teffs) (Fig. 4e, f, Supplementary Fig. 5e). To assess the direct effects of NECA and TAIII on Teff cell proliferation in the absence of Tregs, we performed CFSE dilution assays. CFSE-labeled Teff cells were cultured with or without NECA or TAIII, and proliferation was measured by CFSE dilution. As shown in Supplementary Fig. 5g, neither NECA nor TAIII significantly affected Teff proliferation under Treg-depleted conditions. These findings suggest that the effects of TAIII on Teff cells in the presence of Tregs are likely mediated through Treg modulation, rather than direct modulation of Teff proliferative capacity. Lentiviral delivery of A2AR-silencing shRNA into primary human iTregs markedly reduced FoxP3 protein levels and increased IL-2 transcription, while non-targeting controls showed no effect (Fig. 4g). To exclude off-target effects, we treated A2AR-depleted Tregs with TAIII and found that TAIII-mediated FoxP3 suppression and cAMP production were significantly attenuated by A2AR depletion, as confirmed by qRT-PCR and LANCE cAMP assays, suggesting that TAIII specifically targets A2AR to regulate FoxP3+ Tregs (Fig. 4h, Supplementary Fig. 5f).

To investigate the mechanism underlying TAIII-mediated FoxP3 transcriptional inhibition, a CUT & RUN assay revealed a robust reduction in CREB occupancy at the FoxP3-CNS-2 promoter region in human iTregs (Fig. 4i), confirming that TAIII inhibits FoxP3+ Tregs and enhances T cell activity through A2AR signaling blockade.

TAIII improves CAR-T cell antitumor activity through blocking A2AR in vitro
To determine whether TAIII improves CAR-T function through A2AR signaling, we performed co-culture cytotoxicity assay (E:T=1:2 or 1:4 in Fig. 5, E:T=1:1 to another donor in Supplementary Fig. 6a, b). NECA impaired CAR-T cytotoxicity by increasing of FoxP3+ CAR-Tregs, but TAIII restored cytotoxic function of NECA-treated CAR-T cells against Nalm-6 or Raji cells and upregulated effector cytokines, including IL-2, IFN-γ and TNF-α (Fig. 5a–c, Supplementary Fig. 6a, b). Given the importance of the proportion of CAR-Tregs in determining patient response and outcome to CAR-T therapy17,18, and TAIII’s ability to impair Treg function and differentiation, we then sought to investigate whether TAIII reduces FoxP3+ CAR-Tregs through A2AR signaling. In co-cultures of CAR-T cells and Raji cells in media with or without TAIII, the levels of FoxP3 and the frequency of FoxP3+ Tregs were significantly upregulated by NECA. Importantly, these effects were completely reversed by TAIII treatment without affecting the proportions of total CD4+ and cytotoxic CD8+ T cells (Fig. 5d, Supplementary Fig. 6c). This selective modulation, which aligns with our previous observations (Supplementary Fig. 1), demonstrates TAIII’s target specificity. These findings suggest that TAIII enhances the cytotoxicity of CAR-T cells by blocking A2AR signaling.

Given that central memory T cells (Tcm) not only exhibit anti-tumor activity but also prevent recurrence46,47, we further explored the impact of TAIII on Tcm populations (CD62L+CD45RO+) using a co-culture and FACS assay (E:T=1:1, 1:2 or 1:4). We observed that NECA stimulation robustly reduced the proportion of Tcm cells, while TAIII treatment completely restored Tcm levels across all conditions (Fig. 5e, Supplementary Fig. 6d).

To validate these findings, we conducted single-cell sequencing (scRNA-seq) using 10× Genomics on CAR-T only or co-cultures system, with or without TAIII treatment. By clustering and quantifying the proportions of B (Raji) and T (CAR-T) cells, we observed that TAIII robustly enhanced Raji cell (B cell subset) killing (E:T=1:1) (Supplementary Fig. 7a), consistent with our previous results. By re-clustering the combined T cell single-cell data, we isolated CD4+ T cells and further identified a FOXP3high CD4+ CTLA+ Treg subset (Supplementary Fig. 7b). In the co-culture condition, the proportion of this Treg subset within CD4⁺ T cells decreased by >40% upon TAIII treatment. Likewise, in the CAR-T–only group, the FOXP3high CD4⁺ CTLA4⁺ Treg cluster and A2AR⁺ CD4⁺ Treg cells were both markedly reduced (>40%) following TAIII treatment, underscoring the compound’s consistent suppressive effect on Treg populations (Supplementary Fig. 7c, d). These findings support the modulatory effect of TAIII on Treg populations in the context of NECA stimulation.

Notably, scRNA-seq analysis revealed a significant shift in cytotoxic CD8+ T cells. In CAR-T/Raji co-cultures, CD8+ CAR-T cells exhibited enhanced cytotoxicity after co-culture with tumor cells, which was further amplified in the TAIII-treated group. Differences in cytotoxicity were observed between key cytotoxic subsets, CD8+ GZMB+ Tex (Exhausted T cells) and CD8+ GZMA+ Tem. CellChat analysis revealed the strongest interactions between these subsets and Tregs (Supplementary Fig. 7e–f), suggesting that this increase is a secondary effect resulting from the reduction in Tregs. Additionally, TAIII treatment significantly upregulated the expression of IFNγ and TNFα (Supplementary Fig. 7g), reinforcing our hypothesis that TAIII enhances CAR-T cytotoxicity via Treg modulation, indirectly affecting CD8+ T cells.

Furthermore, bulk RNA-seq results showed that TAIII treatment counteracted NECA’s effects by modulating T cell proliferation and activation, cytotoxic cytokines and Treg immune suppression-related genes and pathways, as shown by Reactome Pathway enrichment analysis (Supplementary Fig. 7h). These findings align with our observations in T cells (Supplementary Fig. 5a). Importantly, TAIII treatment significantly counteracted the effects of NECA by upregulating TNF expression to enhance the T cell immune response, while downregulating the transcription of Treg functional markers, such as FoxP3, CTLA4, and CD25, in CAR-T cells, suggesting resistance to adenosine-mediated transcriptional changes (Supplementary Fig. 7i-j). These results were consistent with our previous T cell data. We also observed significant enrichment of fatty acid metabolism pathways, particularly cholesterol metabolism, following TAIII treatment (Supplementary Fig. 7k), consistent with TAIII’s role in regulating lipid metabolism40,48. Given that membrane cholesterol homeostasis regulates A2AR activity and is essential for Treg stability and function39,49, and considering TAIII’s ability to compete with cholesterol, these results suggest that TAIII modulates A2AR activity through both cholesterol metabolism regulation and allosteric binding to A2AR.

To better assess the specificity of TAIII, we replaced NECA with the high-affinity A2AR agonist CGS-21680. In functional assays (e.g., cytotoxicity and cytokine secretion), CGS-21680 induced significant inhibition, validating that our system responds robustly to a known A2AR agonist. This further ensures that the observed effects of TAIII are due to specific A2AR targeting, rather than non-specific receptor interactions (Supplementary Fig. 8a–b). Building on this, we next aimed to confirm that the effects of TAIII are A2AR-dependent and exclude potential off-target effects. To achieve this, we conducted additional experiments using A2AR knockdown (KD) in CAR-T cells. Briefly, CD19-CAR-T cells were transduced with A2AR-targeting shRNA (GFP+) and sorted by FACS. Knockdown efficiency was confirmed both at the protein level via Western blot and at the mRNA level using qRT-PCR (Supplementary Fig. 8c, Fig. 5f). First, we treated A2AR-KD CAR-T cells with TAIII and observed that TAIII no longer modulated A2AR-mediated cAMP production following A2AR knockdown, further confirming the specificity of TAIII for A2AR (Fig. 5g). A2AR-KD CAR-T cells were subsequently co-cultured with Nalm6-Luc cells, and the results demonstrated that A2AR knockdown enhanced CAR-T cell cytotoxicity compared to the SCR control (GFP-). Importantly, TAIII did not further enhance this cytotoxic effect, confirming that TAIII’s action is dependent on A2AR signaling (Fig. 5h). Furthermore, A2AR-KD CAR-T cells exhibited higher IFN-γ and IL-2 secretion but were unresponsive to both NECA and TAIII treatments, further supporting the A2AR dependence of TAIII’s effects (Fig. 5i, Supplementary Fig. 8d, e). These findings align with data from A2AR-KD Tregs, where TAIII had no impact on Treg activity (Fig. 4g, h, Supplementary Fig. 5f), providing strong evidence that A2AR is a critical mediator of TAIII’s effects on both Treg and CAR-T cell function.

Collectively, these results suggest that TAIII plays an important role in enhancing CAR-T cell function by modulating A2AR signaling and may help prevent relapse after CAR-T therapy.

TAIII suppresses tumor growth through reversing A2AR-FoxP3+ Treg mediated immunosuppression
To assess the antitumor efficacy of TAIII in vivo, we treated a syngeneic mouse model (C57BL/6J) inoculated with MC38 cells with 5 mg/kg TAIII administrated intraperitoneally for 22 days (Supplementary Fig. 9a). TAIII demonstrated a high tumor growth inhibition (TGI=55.9%) compared to the vehicle group, with no apparent adverse effects, as determined by monitoring body weight over time (Supplementary Fig. 9b–e). Importantly, TAIII treatment significantly increased the levels of CD8, CD4 and GZMB, while downregulating FoxP3 levels in tumor tissues (Supplementary Fig. 9f). Furthermore, TAIII significantly upregulated the expression of activation-associated cell surface molecules, such as CD69, on cytotoxic T lymphocytes (CTL) cells in TDLNs (Supplementary Fig. 9g). Additionally, the IHC staining of excised tumors revealed a significant decrease in FoxP3 protein levels and a marked increase in CD8+ T cells, indicating that blocking A2AR signaling with TAIII reduced infiltrating Tregs while simultaneously enhancing CD8+ T cell infiltration in the TAIII treatment group (Supplementary Fig. 9h). Next, we measured cytokine release in both tumor tissues and serum, finding that TAIII significantly increased the secretion of cytotoxic IFN-γ and IL-2 (Supplementary Fig. 9i).

Similar results were observed in a dose-escalation experiment, where doses ranged from 5 mg/kg to 20 mg/kg (once daily, QD) in the MC38 tumor model, with 50 mg/kg of AZD4635 (twice daily, BID) used as a positive control (Fig. 6a). TAIII exhibited dose-dependent tumor growth suppression, achieving a TGI=50.5% without noticeable body weight loss, and showed higher potency than AZD4635 (TGI=34.6%) (Fig. 6b–c, Supplementary Fig. 10a). Moreover, immunohistochemistry (IHC) staining of tumor-draining lymph nodes (TDLNs) revealed a robust decrease in FoxP3 protein levels and a reduction in infiltrated Treg cell numbers in a dose-dependent manner (Fig. 6d, Supplementary Fig. 10b), suggesting that TAIII suppresses tumor growth by impairing Foxp3+ Tregs in vivo. Furthermore, TAIII significantly enhanced the secretion of cytotoxic IFN-γ and IL-2 in both tumor tissues and serum (Fig. 6e). Collectively, these results suggest that TAIII suppresses tumor growth in vivo by blocking A2AR-mediated FoxP3+ Treg signaling.

A recent study demonstrated that PD-1 blockade expands Tregs in tumors, limiting the efficacy of immunotherapy50. To determine whether TAIII enhances the antitumor activity of PD-1 therapy, we treated MC38 tumor-bearing mice (C57BL/6J) with isotype control, anti-PD-1 (5 mg/kg, twice weekly, BIW), TAIII alone (10 mg/kg) administrated intraperitoneally, or the combination of TAIII and PD-1 therapy for 22 days (Fig. 6f). TAIII significantly enhanced the efficacy of anti-PD-1 therapy compared to monotherapy, without apparent adverse effects, as evidenced by body weight monitoring (Fig. 6g–i, Supplementary Fig. 10c, d). Moreover, TAIII significantly increased the production of antitumor cytokines IFN-γ and IL-2 in both tumor tissues and serum (Fig. 6j). Similarly, western blotting of excised tumors showed a marked reduction in A2AR-downstream signaling (pCREB) (Fig. 6k, Supplementary Fig. 10e), demonstrating that the observed therapeutic benefits were mediated through A2AR pathway blockade. Although CREB is relatively evenly expressed across different tissues, it is expressed at higher levels in immune cells, reflecting its key role in immune responses (The Human Protein Atlas database). We acknowledge the limitation that differential CREB expression across cell types in the tumor microenvironment could influence the observed changes in pCREB levels. Future studies will address this by analyzing cell-type-specific responses to TAIII and anti-PD-1 treatment.

Flow cytometric analysis of tumor-infiltrating lymphocytes (TILs) revealed a significant increase in the proportion of Tcm cells in the combination therapy group, relative to the anti-PD-1 monotherapy group (Fig. 6l), suggesting that TAIII + anti-PD-1 therapy potentiates T cell responses by promoting Tcm differentiation. Consistent with this, IHC staining demonstrated a marked reduction in FoxP3+ Tregs, along with an increase in CD8+ T cells within TILs, further corroborating the enhanced antitumor efficacy of the combination treatment (Fig. 6m–n).

Taken together, these results highlight the role of TAIII in enhancing the efficacy of PD-1 immunotherapy by reducing intratumoral Tregs through combination treatments.

TAIII prevents relapse after CAR-T therapy in various preclinical models by targeting A2AR-Treg signaling
Given the importance of FoxP3+ CAR-Treg levels in determining prognosis after CAR-T infusion17,18, we analyzed clinical samples from databases of patients who received CAR-T therapy. CD19 CAR-T products were collected pre-infusion from patients with B-lymphoblastic leukemia who experienced either long-term remission (R, over 8 years, n = 25) or no objective response to the therapy (NR, n = 6), and these were analyzed using scRNA-seq (GSE262072). The analysis revealed that the ratios of A2AR+ FOXP3+ cells within total CAR-T populations are significantly correlated with the percentage of cytotoxic cells, cytokine production and clinical response, consistent with findings from Good and Haradhvala et al.17,18 (Fig. 7a–b). Specifically, A2AR signaling and Tregs-related pathways, such as NF-κB1, TGF-β1, and the CD8+ T cell-related gene CRTAM (Class-I MHC-restricted T-cell associated molecule)51,52, are closely associated with tumor response following CAR-T infusion (Fig. 7b), positioning A2AR-FoxP3 as a potential biomarker for predicting therapeutic response and suggesting that A2AR blockade by TAIII may enhance CAR-T cell efficacy in patients.

Our findings further demonstrate that TAIII effectively reduces Treg proportions and function by blocking A2AR signaling, thereby potentially enhancing the efficacy of CAR-T cells in preventing tumor relapse. To test this hypothesis, we investigated the effect of TAIII on tumor relapse following CAR-T cell therapy in vivo. Raji-Luc cells were intravenously injected into NSG immunocompromised mice, followed by treatment with CAR-T or UNT cells two days later, with or without TAIII combination treatment (QD), starting at day 3 (post-Raji cells engraftment) through day 58 (Fig. 7c). Both CD19-CAR-T cells alone and the combination group effectively cleared Raji cells within a week. However, relapse occurred after remission in the CAR-T-only group two weeks later, whereas no relapse was observed in the combination group. In the CAR-T only group, 4 out of 5 mice died following the reappearance of lymphoma cells at 8 weeks (Fig. 7d, e, Supplementary Fig. 11a). The combination of TAIII and CAR-T significantly extended mouse survival compared to the vehicle and CAR-T-only groups (Fig. 7f). Moreover, the combination therapy significantly upregulated CAR-T and Tcm cell populations while downregulating FoxP3+ Tregs in both the bone marrow and blood, compared to all other conditions (Fig. 7g, h). These findings suggest that TAIII combination therapy is an effective and affordable therapeutic strategy to prevent relapse after CAR-T cell therapy in lymphoma.

To further validate our findings and demonstrate the broader applicability of TAIII in enhancing CAR-T cell potency, we assessed its effects in a Nalm6 intravenous xenograft model. NSG immunocompromised mice were intravenously injected with 3×10⁶ Nalm6 lymphoma cells (n=6–7), followed by infusion of 6×10⁶ CAR-T cells or UNT controls on day 3. TAIII (5 mg/kg, QD) was co-administered starting on day 3 post-inoculation and continued through day 16 (Fig. 7i). In this model, all control groups ultimately succumbed, whereas CAR-T treatment significantly prolonged survival. Importantly, the combination of CAR-T and TAIII therapy resulted in the longest survival time, with no mice in the CAR-T+TAIII group succumbing to the disease by the end of the experiment, while five out of seven mice (5/7) in the CAR-T only group had died (Fig. 7j). In contrast, TAIII did not prolonged the survival of UNT group, suggesting that its effects are antigen-specific rather than non specific. This combination therapy also enhanced CAR-T cell persistence and reduced Treg frequencies (Fig. 7k), with no significant effect on the CD8/CD4 ratio. These findings provide direct evidence of durable, antigen-specific tumor control and improved survival, without apparent toxicity, as indicated by stable body weight (Supplementary Fig. 11b, c).

We further extended our analysis to an immunocompetent MC38-mCD19 solid tumor model, where TAIII synergized with murine CD19-CAR-T therapy (Fig. 7l). C57BL/6J immunocompetent mice were subcutaneously injected with 1×10⁶ MC38-mCD19 cells (n=5). On day 6, mice were treated with 4.5 mg cyclophosphamide (CTX) and 0.75 mg/200 μL fludarabine (FLU) via intraperitoneal injection for lymphodepletion. On day 7, mice were treated with the optimal dose of CAR-T cells (4×10⁶), TAIII (10 mg/kg, daily), or a combination of both. Mice receiving the combination treatment showed more rapid tumor eradication, with a TGI of 90% (Fig. 7m). Tumor microenvironment profiling revealed a significant increase in intratumoral antigen-specific CD8⁺ T cells, accompanied by a reduction in Tregs, providing direct in vivo evidence that TAIII enhances CAR-T efficacy through immune modulation rather than nonspecific activation (Fig. 7n). In addition, we assessed cytokine release in mouse serum and found that TAIII significantly increased the secretion of cytotoxic TNF-α. Flow cytometry analysis also revealed an increase in CD8⁺ CD45.1⁺ T cells in the spleen (SP) and bone marrow (BM) of MC38-mCD19-bearing mice on day 6 post-engraftment (Supplementary Fig. 11d–e). These findings indicate that TAIII selectively targets Tregs in vivo, and modulates their population to enhance CAR-T cell expansion and persistence. This likely contributes to the improved antitumor efficacy and survival observed in the combination therapy group, and no apparent toxicity, as evidenced by stable body weight (Supplementary Fig. 11c, f). These results align with our observations in the Raji-Luc model, reinforcing TAIII’s role as a potent adjunct to CAR-T therapy and its potential to improve outcomes in both leukemia and solid tumors. Together, these findings support the generalizability of TAIII’s immunomodulatory effects.

To assess whether TAIII pretreatment enhances CAR-T cell antitumor activity in vivo, we treated Raji-Luc-bearing mice with CD19-CAR-T cells six days post-tumor engraftment (heavy tumor burden). Mice were treated CD19-CAR-T cells either with or without TAIII pretreatment (1 μM, 36 h) (Supplementary Fig. 12a). TAIII-pretreated group—which exhibited reduced FoxP3 levels both in vitro and in vivo (Supplementary Fig. 12b, c)—demonstrated significantly lower Raji-Luc cell percentages in vivo compared to CAR-T alone and the vehicle control groups. The pretreatment group exhibited greater potency and achieved earlier tumor clearance than the CAR-T only group (Supplementary Fig. 12d).

To further substantiate the potentiating effect of TAIII pretreatment on CAR-T cell-mediated cytotoxicity and its broader applicability, we employed the Nalm6-Luc subcutaneous tumor model. NSG mice were subcutaneously injected with 1×10⁶ Nalm6-Luc lymphoblastic leukemia cells, and six days later, 5×10⁶ CAR-T cells or TAIII-pretreated CAR-T cells (1 μM, 72 h) were infused. As anticipated, TAIII pretreatment significantly enhanced CAR-T cell antitumor activity in vivo. Compared to the CAR-T-only group, the TAIII-pretreated group exhibited a rapid reduction in tumor burden and achieved tumor eradication earlier, underscoring the potentiating effect of TAIII pretreatment on CAR-T cell-mediated cytotoxicity (Fig. 7o and Supplementary Fig. 12e). Subsequently, we tracked T cell frequencies and phenotypes in peripheral blood (on days 4, 7, and 14 post-CAR-T infusion), bone marrow and spleen (on day 14 post-CAR-T infusion) through flow cytometry. The results revealed that the TAIII pretreatment group exhibited a marked expansion of CAR-T cells and a significant reduction in CAR⁺ FoxP3+ Tregs in both peripheral blood (BL) and bone marrow (BM) compared to the CAR-T-only group (Fig. 7p). From day 4 to day 7, we observed an increase in the overall number of CD3+ T, CD4+ T, CD8+ T, Tcm, and effector memory T (Temra) cells in peripheral blood in the TAIII pretreatment group. Importantly, the CD4/CD8 ratio remained unchanged in the combination group compared to the CAR-T-only therapy group (Supplementary Fig. 12f–k). Notably, the sustained alterations in T cell populations suggest that TAIII’s modulation of Tregs provides long-term benefits, enhancing CAR-T cell persistence and functionality well beyond the initial treatment phases. These results are consistent with our findings in the Raji-Luc model and further underscore the generalizability of TAIII’s effects.

Additionally, we collected CD19 CAR-T products from 8 patients with B-lymphoblastic malignancies experiencing uncontrolled disease progression after CAR-T infusion (Table 1) and evaluated them with TAIII in co culture models. CAR-T cells from patients were co-cultured with Raji-Luc cells at E:T ratios of 1:1 to 1:4, with or without TAIII treatment (Fig. 7q). TAIII significantly enhanced CAR-T cell-mediated killing of Raji-Luc cells compared to the CAR-T-only group, with the killing efficiency in some patients increasing by several folds (Fig. 7r, Supplementary Fig. 13a, b). To determine whether TAIII enhances CAR-T cell cytotoxicity by targeting Tregs, we co-cultured Raji-activated CAR-T cells with or without TAIII (E:T=1:2). TAIII treatment significantly increased T cell-derived cytokine secretion (IL-2, IFN-γ) (Fig. 7s) and markedly decreased FoxP3 expression levels (Fig. 7t), indicating a reduction in FoxP3+ CAR-Tregs. To investigate whether TAIII improves patient-derived CAR-T function through A2AR signaling, we performed co-culture assay (E:T=1:2) and found that NECA treatments significantly impaired CAR-T cell cytotoxicity. However, TAIII restored the cytotoxic function of NECA-treated CAR-T cells, enabling effective Raji cell killing (Supplementary Fig. 13c, d). These findings demonstrate that TAIII pretreatment and combination with CAR-T cells enhances antitumor activity, accelerating tumor clearance and reducing recurrence in preclinical models compared to CAR-T monotherapy.

To test whether the immune-modulatory effects of TAIII are dependent on immune cells (e.g., T cells), Raji-Luc cells were intravenously injected into NSG immunocompromised mice, followed by treatment with or without TAIII. TAIII exhibited minimal effect on Raji cell growth in T cell-deficient mice (Supplementary Fig. 14a–c), with no significant survival benefit (Supplementary Fig. 14d). Additionally, no apparent body weight loss was observed, confirming that TAIII’s safety in immunocompromised mice (Supplementary Fig. 14e).

To further investigate the role of TAIII in modulating Tregs during CAR-T cell therapy in vivo, we performed additional experiments using the Nalm6-Luc xenograft model with ΔTreg-CAR-T cells (Tregs reduced to 1.28%). NSG immunodeficient mice were intravenously injected with Nalm6-Luc cells, followed by CAR-T cell infusion on day 2. TAIII treatment (QD) was initiated concurrently and continued through day 18 (Supplementary Fig. 15a). We ensured the inclusion of appropriate controls, such as the UNT cell control, in the study design. Consistent with our in vitro findings (Supplementary Fig. 2), depletion of Tregs abrogated the therapeutic benefit of TAIII. While CAR-T therapy alone induced partial tumor regression, the addition of TAIII to ΔTreg-CAR-T cells conferred no further tumor suppression (Supplementary Fig. 15b, c) and did not significantly improve survival (Supplementary Fig. 15d). In the Treg-depleted CAR-T+TAIII group, the apparent loss of therapeutic benefit after day 19 is not due to a late-onset effect, but rather reflects the absence of TAIII’s primary target, Tregs. Notably, TAIII did not enhance CAR-T efficacy at any timepoint in the absence of Tregs, highlighting the essential role of Tregs in mediating TAIII’s therapeutic effects. Flow cytometry analysis of peripheral blood, bone marrow, and spleen on day 7 post-CAR-T infusion revealed that TAIII had no significant effect on CAR-T expansion or persistence in the Treg-depleted model, with no changes observed in the Tcm population or overall CAR-T contraction (Supplementary Fig. 15e–f). These findings underscore that TAIII enhances CAR-T efficacy primarily through Treg modulation, rather than by directly promoting CAR-T persistence. By selectively modulating Treg activity without impairing other immune components, TAIII improves CAR-T function while preserving systemic immune balance, thereby improving therapeutic outcomes.

Collectively, these findings reveal that TAIII, through Treg-targeting mechanisms, augments CAR-T cell efficacy and reduces relapse when used as a pretreatment or in combination therapy, offering a promising approach to optimize CAR-T therapeutic outcomes and production.

Methods
Ethics statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Sciences (Approval Nos: DWLL(p)-2023-23, DWLL(E)-2024-34, DWLL(E)-2025-35). The maximal permitted tumor burden was 2000 mm³ in volume or 2 cm in diameter per animal, as specified by the committee guidelines. Tumors were measured every 2 days, and animals were closely monitored for signs of distress. If tumor volume approached or exceeded the approved limit at the time of measurement, animals were immediately euthanized humanely. We confirm that no animal was allowed to maintain a tumor exceeding the permitted burden for any extended period. All procedures complied with the NIH Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines.

For human samples, this study was approved by the Ethics Committee of Tongji Hospital (Approval Nos: TJ1537, NCT02537977) for the collection and use of clinical samples, and by the Ethics Committee of the Chinese Academy of Sciences (Approval No: YXLL-2023-17) for the isolation and use of PBMCs and naive T cells from healthy donors. Written informed consent was obtained from all participants prior to sample collection. The study was conducted in compliance with the principles of the Declaration of Helsinki and all relevant institutional guidelines.

Cell lines and cell culture
The HEK-293T and Jurkat T cell lines were obtained from the ATCC (Manassas, VA, USA) Cell Line Bank. Jurkat T cells used in this study were cultured with 5% CO2 at 37°C, at a concentration of 2×105 to 5×105 in RPMI (Gibco, Thermofisher), with 10% FBS (VivaCell, C04002) and supplemented with 1% penicillin/streptomycin. Human T or CAR-T cells were cultured in X-VIVO (Lonza, 04-418Q) with 10% FBS (Gibco, 10100-147), 1% GlutaMAX (Gibco, 35050061), 1% NEAA (Gibco, 11140050), 1% Antibiotic-Antimycotic (Gibco, 15240062), 100 IU IL-2 (R&D Systems, 202-IL) and Dynabeads Human T-activator CD3/CD28 (Gibco, 1132D). HEK-293T cells were cultured in DMEM with 10% FBS (VivaCell, C04001) and 1% penicillin/streptomycin. Human cell lines were authenticated within 6 months of manuscript preparation through cell line short tandem repeat (STR) profiling conducted by a certified Contract Research Organization (CRO), SHANGHAI BIOWING BIOTECHNOLOGY Co. LTD. All cell lines tested matched >85% with the STR profiles of their corresponding lines listed in the ATCC or DSMZ Cell Line Bank and were confirmed to be virus- and mycoplasma-free.

Biological reagents
For immunoblotting, the following primary antibodies were used: β-Actin (Yeason, 30101ES60, 1:5000), CREB (Cell Signaling Technology, 9197T, 1:1000), phospho-CREB (Ser133; Cell Signaling Technology, 9198T, 1:1000), A2AR (Santa Cruz Biotechnology, sc-32261, 1:1000), and GAPDH (Cell Signaling Technology, #2118, 1:5000). The secondary antibodies (Goat anti-Rabbit lgG antibody, M21002L) and (Goat anti-Mouse lgG HRP, M21001L) were obtained from Abmart. Streptavidin beads magnetic beads were purchased from MedChemExpress (MCE, Shanghai). The cell viability detection kit (Cell Counting-Lite 2.0 Luminescent Cell Viability Assay) was obtained from Vazyme (DD1101-02). The Bright-Glo™ Assay Reagent was obtained from Promega (E2620). The immobilon western HRP substrate was purchased from Millipore (WBKLS0500). The LANCE™ cAMP 384 Kit was purchased from PerkinElmer (AD0262E) and One Step qRT-PCR SYBR Green Kit was purchased from Yeasen (11143ES80). The chemical compound library comprised both in-house synthesized compounds and commercially acquired compounds from MCE and TargetMol (Shanghai). NECA was purchased from MCE (Shanghai). AZD4635, CPI-444 and Timosaponin AIII were purchased from TargetMol (Shanghai). Inhibitors were dissolved in DMSO to obtain 10 mM stock solutions. Serial dilutions were then made to obtain final dilutions for cellular assays with a final concentration of DMSO between 0.2% and 0.5%. All buffer solutions were prepared using pH adjustment calculations performed with Mettler Toledo (FE28-Standard).

TAIII probe synthesis
To a mixture of Timosaponin AIII (74 mg, 0.1 mmol) and 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl) propanoic acid (17 mg, 0.1 mmol) in 2 mL DMF were added HATU (38 mg, 0.1 mmol) and DIEA (26 mg, 0.2 mmol). The mixture was stirred for 2 h at room temperature in the dark. Subsequently, the reaction was quenched by water (10 mL), and the mixture was extracted by CH2Cl2. The organic phases were combined together and dried over anhydrous Na2SO4. Upon solvent evaporation, the residue was purified by flash column chromatography to yield TAIII probe (7 mg, 8%) as a white solid.

Click Chemistry and proximity labeling proteomics
293T cells were cultured in a 10 cm dish until 80–90% confluence was reached. The cells were washed with PBS and lysed by 1 ml of NP-40 lysis buffer. Centrifugation (13,000g; 10 min; 4°C) was applied to remove the insoluble fraction from the cell lysate. DMSO or TAIII-P was added into cellular lysates, followed by incubation at 4°C for 2 h and all samples were UV (365 nM)-irradiated for 30 min using a UV crosslinker, then biotin-azide (10 μM), TCEP (1 mM), TBTA (100 μM), and CuSO4 (1 mM) were added sequentially to the lysate, followed by 1 h incubation at room temperature, protected from light. The proximity labeled proteins were then acetone-precipitated and air-dried, then resolubilized in a 1.5% SDS solution. The sample was incubated with avidin-agarose beads at room temperature for 2 hours before centrifugation, the supernatant was removed and the beads were washed with PBS three times, finally subjected to LC-MS/MS based proteomics analysis or western blotting. The raw mass spectrometry data generated has been deposited in the PRIDE public repository under accession code: PXD060766.

Cellular thermal shift assays
A2AR OE 293T cells were harvested and freeze-thawed for three cycles, centrifuged to remove the precipitates (13,000g; 10 min; 4°C). TAIII or DMSO was added to cell lysates in a dose-dependent manner and incubated for 1 h. Samples were then divided into 100 μL aliquots in 0.2 ml PCR tubes and transiently heated in a PCR machine for 3 min at indicated temperatures, followed by 3 min of cooling at RT. Samples were then centrifuged (13,000g; 10 min; 4°C) to separate the soluble proteins from the cell debris and aggregates. The supernatant was transferred to new tubes and analyzed by western blotting.

LANCE cAMP assay
cAMP levels were measured by LANCE™ cAMP 384 kit (PerkinElmer, AD0262) according to the manufacturer protocol. Briefly, cells were harvested and resuspended in Stimulation Buffer (PBS with 0.1% BSA and 0.5 mmol/L IBMX) at a concentration of 6×105 cells per mL, and then transferred 5 μL of cell suspension to each well of a 384-well plate. Then stimulated cells with TAIII dilutions for 30 min in the presence of 0.1 μM NECA. After that add Detection Mix and incubate for 60 min at room temperature in the dark. Then, reading 340/665 nm and 340/615 nm fluorescence intensity, the ratio of 665 nm/615 nm is inversely proportional to cAMP concentration.

Pan-T cells activation and expansion
PBMC were isolated from fresh human peripheral blood by density gradient centrifugation. For inducing PBMC into pan-T cells, PBMC were cultured in a suitable tissue culture plate or dish at a concentration of 1×106/ml, and added Dynabeads Human T-Activator CD3/CD28 (Gibco, 11131D) at a bead-to-cell ratio of 1:1, 100 IU/ml IL-2 was also added into the culture medium. Dynabeads were removed on day 5, and after 48 h of removal of the beads, the pan-T cells were harvested for further analysis.

For assessed cytokine secretion and mRNA expression, PBMC or T cells were cultured at a concentration of 1×105 cells per mL in 96-well or 6-well flat-bottom plates, cells were activated by 1:1 Dynabeads and treated with indicated TAIII or AZD4635 in the present of 5 μM NECA. After 16 h treatment, PBMC were collected for qRT-PCR analysis (Yeason), and the secretion levels of IFN-γ and IL-2 were analyzed by an ELISA kit (MultiSciences) after 48 h of culture.

Treg induction
Human PBMCs (n=3) were obtained from blood samples of healthy donors (Shanghai Blood Center) with ethical approval from the Shanghai Blood Center Ethics Committee. Monocytes were first isolated from fresh umbilical cord blood (n=3) by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare), and then isolated CD4+ Naïve T cells by using Human Naïve CD4+ T Cell Isolation Kit II (Miltenyi Biotec, 130-094-131). Subsequently, naïve T cells were activated by Dynabeads Human T-Activator CD3/CD28 (Gibco, 11131D) and cultured under Treg-induced conditions as previously described: IL-2 (100 IU/ml), TGF-β1 (5 ng/ml), indicated drug treatments were also administered. 5 days later, removing the TGF-β1 from the culture medium, differentiation efficiency was detected by FACS before subsequent experiments. The PBMCs and naive T cells used in our experiments were sourced from different healthy donors (Ethics approval number: YXLL-2023-17), ensuring that the data reflects variability across individuals and is representative of the general population. This approach enhances the robustness and generalizability of our findings.

Treg suppression assay
Human CD4+CD25highCD127lowCD45RA- Teff cells were sorted from healthy adult PBMC by BD FACS Aria III flow cytometer. Teff cells were incubated with 2.5 μM CFSE (Invitrogen, C34570) at 37°C for 20 min, and then added five times the original staining volume of complete culture medium and incubated for 5 min. After that, remove the supernatant by centrifugation and resuspend cells in pre-warmed culture medium. These stained cells were stimulated by CD3/CD28 Dynabeads (bead-to-cell ratio is 1:4) and then co-cultured with Treg cells at the indicated ratio in 200 μL medium in a round bottom 96-well plate (20,000 Teff cells per well). After 96 h of co-culture, the proliferating peaks were analyzed by FACS.

Flow cytometry (FACS)
For surface protein staining, cells were harvested and incubated with the fluorochrome-conjugated antibodies for 30 min at room temperature and protected from light.

The following fluorescent dye-conjugated antibodies were used for flow cytometric analysis: For human T cell staining, the abundance of distinct cell populations and the corresponding flow cytometry gating strategies were applied to delineate CD4⁺ T (CD4-FITC, BD222346; CD4-Alexa Fluor 700, BD561030), CD8⁺ T (CD8-PC5.5, BD560662), CD3⁺ T (CD3-PE, BD561803), CAR⁺ T (CAR-Alexa Fluor 647, BIOSWAN, 200102), regulatory T (Treg), naïve T (Tnaive), central memory T (Tcm), effector T (Teff), and terminally differentiated effector memory T (Temra) cells. CAR-T cells were gated on the basis of CD3 or CD4 in combination with CD19-CAR (CD19-BV421, BD562440) expression. Tregs were defined as CD4⁺FoxP3⁺ cells. For intranuclear FoxP3 staining, cells were fixed and permeabilized after surface staining, by using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions and then stained with anti-FoxP3-PE-Cy7 (eBioscien, 25-4776-41) or FoxP3-PE (BD Pharmingen™, 560082). Human T cell subsets were classified according to surface markers as follows: CD45RO⁻CD62L⁺ (Tnaive), CD45RO⁺CD62L⁺ (Tcm), CD45RO⁺CD62L⁻ (Teff), and CD45RO⁻CD62L⁻ (Temra). For Treg depletion experiments, natural Tregs (nTregs) within PBMCs were identified as CD4⁺CD25⁺CD127low cells. For mouse T cell subsets, we used CD44 as a substitute for CD45RO. CD45RO-PE (BD555493), CD62L-BV421 (BD 563862), and CD44-BV421 (BD 563970) were employed. Cell proliferation was tracked using CFSE (Invitrogen, C34554), and viability was assessed with Fixable Viability Stain 660 (BD564405) or Fixable Viability Stain 520 (BD564407). For mouse T cell staining, anti-mouse CD45-FITC (BD553080), CD3-APC-Cy7 (BD 557596), CD4-APC (BD 553051), CD8-PC5.5 (BD 551162), FoxP3-PE (BD 560408), CD44-BV421 (BD 563970), and CD62L-Alexa Fluor 700 (BD 560517) were employed. Data were collected by Beckman CytoFLEX and analyzed with FlowJo V10.8.1 software.

Microscale Thermophoresis assay
MST assay was done as previously described65, A2AR OE 293T cells were lysed by RIPA buffer added PMSF, centrifuged to remove the precipitates (13,000g; 10 min; 4°C), and then 10 μL cellular lysates were incubated with 10 μL serial dilution of TAIII or AZD4635 at room temperature for 30 min. Then the mixture was loaded into capillaries supplied by Nano-Temper, and thermophoresis was scanned by Monolith (Nano-Temper).

Human samples for scRNA-seq & bulk RNA-seq
Human CAR-T products were obtained under the approval of the Tongji Hospital Review Board (Clinical Trial No: NCT02537977, Sponsor: Shanghai Tongji Hospital, Tongji University School of Medicine) and written informed consent was obtained from all participants or their legally authorized representatives. Clinical characteristics of B-lymphoblastic malignancy patients can be found in Table 1. The scRNA-seq and bulk RNA-seq were performed and carried out by Novogene Co., Ltd. following the manufacturer’s instructions. The raw data generated in this study have been deposited in the GEO database under accession numbers GSE289388 and GSE302847.

Cut&Run assay
The Cut&Run assays were performed as previously described66, carried out and analyzed by Novogene Co., Ltd. following the manufacturer’s instructions. Details provided in Supplementary and methods.

Molecular modeling
The crystal structure of A2AR was obtained from Protein Data Bank (PDB ID: 4EIY). Then, water molecules and original ligands were manually removed by using PyMol software. This docking job to predict the binding pose of Timosaponin AIII was carried out by Autodock (version 4.2.6). To the beginning, prepare_ligand4.py and prepare_recptor4.py scripts from AutoDockTools 1.5.6 were used to prepare the initial files, including adding charges and hydrogen atoms. Next, a grid box of 60×60×60 with a spacing of 0.375 Å was set to enclose the whole binding site38. The Lamarckian genetic algorithm (LGA) was adopted to search for the best binding poses. The specific docking settings were as follows: trials of 100 dockings, 300 individuals per population with a crossover rate of 0.8, and the local search rate was set to 0.06. Other parameters were set as default during the docking.

High-throughput screening platform for Foxp3-mediated IL-2 inhibition
To generate a high-throughput screening platform for Foxp3-mediated IL-2 inhibition, the human IL-2 promoter region (−326 to +145,473 bp), which contains a FoxP3 binding site, was introduced into the Lenti-PGL4 delivery vector. In this approach, lentivirus was packaged by Hanbio, and the IL-2 promoter-Fluc reporter was introduced into the Jurkat T cells followed the lentivirus transduction protocol in the presence of 5–8 μg/ml Polybrene (Hanbio) for 24 h, and then fresh medium was added. 72 h post-infection, the cells were selected with 0.3 μg/ml puromycin for 48 h. Following puromycin selection, monoclonal cells were isolated. These clones were tested for luciferase activity upon TCR stimulation, and the best-performing monoclonal clone was selected. Subsequently, a lentiviral construct for FoxP3a overexpression, containing a BSD resistance cassette, was introduced into the selected clone. After another round of TCR stimulation and selection, the final monoclonal cell line was chosen based on optimal performance. Treg function was evaluated by measuring the fluorescence intensity of Fluc.

Animal models
(1) Syngeneic transplant models:
Six-week-old male C57BL/6J mice (Gem Pharmatech, Nanjing, China) were housed in IVC cages in SPF facility and used according to the animal care regulations of Hefei Institutes of Physical Science, Chinese Academy of Sciences (Hefei, China). After a week of adaptive feeding, 1×106 MC38 cells in PBS harvested during exponential growth were injected into the subcutaneous space on the right flank of mice. Daily oral administration was initiated when the tumor volume had reached about 50 mm3. TAIII was delivered intraperitoneally daily in 5% DMSO/0.5% Methocel/0.4% Tween 80 solution at 5 mg/kg, 10 mg/kg, and 20 mg/kg, or anti-PD-1 (5 mg/kg in saline, BIW), and its vehicle was administered as control. The reference compound AZD4635 was delivered orally twice a day in 5% DMSO corn oil at 50 mg/kg. Body weight and tumor growth were measured every two days. Tumor volume (mm3)=[(W2×L)/2], in which the width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements. The harvested tumor-draining lymph nodes or the tumor tissues were further evaluated by the IHC assay using Foxp3 and CD8 staining according to the instructions. Briefly, after fixing in 4% neutral buffered formalin and processing in paraffin, the tumor-draining lymph nodes or the tumor tissues were sectioned into 4 μm sections, and then stained with FoxP3 or CD8 antibody.

(2) Non-invasive in vivo bioluminescence model:
5.5-week-old NSG mice were injected intravenously with 3×106 Raji-Luc or Nalm6-Luc cells (Shanghai Model Organisms Center, Inc., NM-B07-1). Two or six days later, 6×106 of CAR-T cells manufactured from day 6-10 were given intravenously. Effect of CAR-T only or TAIII combination treatment (5 mg/kg, IP, HKI) on Raji-Luc+ or Nalm6-Luc+ cell growth in a non-invasive in vivo bioluminescence model of lymphoma (n=5 or 6). For a pretreatment model, the CD19-CAR-T cells with or without TAIII pretreatment (1 μM, 36 h) were infused post-6 days of injection of Raji-Luc to mice. Correlation between luciferase-positive lymphoma burden as measured by Bright-Glo assay and luminoskan every 3 days or every week and bioluminescent images of representative mice with matched starting lymphoma burden were taken. Survival curves were compared using the logrank Mantel–Cox test. The percentage of Luc (CD19) and CAR-T cells, along with other T cell subsets, was measured by flow cytometry using the indicated antibodies in blood or bone marrow or spleen samples from transplanted mice (n=5 or 6). The body weights of mice transplanted were measured every two days.

In the MC38-mCD19 subcutaneous tumor model, C57BL/6J immunocompetent mice were subcutaneously injected with 1×10⁶ MC38-mCD19 cells (n=5). On day 6, mice were treated with 4.5 mg cyclophosphamide (CTX) and 0.75 mg/200 μL fludarabine (FLU) via intraperitoneal injection for lymphodepletion. On day 7, mice were treated with the optimal dose of CAR-T cells (4×10⁶), TAIII (10 mg/kg, daily), or a combination of both. Tumor size was measured after treatment. Subsequent assays, including tumor growth inhibition (TGI) calculations, tumor tissue staining, flow cytometry, and ELISA, were performed following procedures similar to those used in the syngeneic transplant models. TGI=((MTVcontrol-MTVtreated)/MTVcontrol)×100%. MTV: Mean Tumor Volume.

For the Nalm6-Luc subcutaneous tumor model with pretreatment, NSG mice (n=3) were subcutaneously injected with 1×10⁶ Nalm6-Luc lymphoblastic leukemia cells. Six days later, 5×10⁶ CAR-T cells or TAIII-pretreated CAR-T cells (1 μM, 72 h) were infused. Correlation analysis was performed between the luciferase-positive leukemia burden, measured by the Bright-Glo assay and Luminoskan. Total flux was quantified and plotted. T-cell frequencies and phenotypes were assessed in peripheral blood (on days 4, 7, and 14 post-CAR-T infusion), bone marrow and spleen (on day 14 post-CAR-T infusion) via flow cytometry.

Mice were sacrificed according to the experimental protocols or when they met prespecified endpoints defined by the IACUC. All animal studies were performed by blinded personnel in specific-pathogen-free, Helicobacter-free facilities in Tongji University and the Chinese Academy of Sciences Animal Resource Center following national, state, and institutional guidelines, including the ARRIVE guidelines, and according to protocols approved by the Institutional Animal Care and Use Committee of Tongji University and the Chinese Academy of Sciences (DWLL(p)-2023-23, DWLL(E)-2024-34, DWLL(E)-2025-35).

ELISAs on tumor samples
ELISAs on tumor samples were performed as described before67,68. Briefly, the procedure involved two main steps:
(1) Tumor Tissue Preparation: Tumor tissues were excised immediately post-sacrifice and placed on ice to minimize protein degradation. The tissues were minced and homogenized in a tissue lysis buffer optimized for protein extraction. After homogenization, samples were centrifuged at 4°C to collect the supernatant containing soluble proteins.
(2) ELISA Procedure: The supernatant was analyzed using commercially available ELISA kits (MultiSciences), following the manufacturer’s instructions, to quantify IL-2, IFN-γ, and other cytokines of interest in both tumor homogenates and serum samples.

Statistical analysis
Each experiment was conducted independently three times, with three technical replicates per experiment. Biological replicates are defined as independent experiments performed on different days using distinct cell passages or animals. For in vitro experiments, each replicate represents cells from a separate culture batch; for in vivo studies, each replicate represents an individual mouse. Technical replicates (e.g., multiple wells from the same culture) were not used for statistical analysis. Standard deviation (SD) was used for all data unless otherwise specified. Normality of the data was assessed using the Shapiro-Wilk test. Parametric tests (Student’s t-test or ANOVA) were applied only to data that passed the normality test. For data that did not follow a normal distribution, non-parametric tests, such as the Mann-Whitney U test or Kruskal-Wallis test, were used as appropriate. Statistical significance was determined accordingly, with exact P values reported in the figures for clarity. All analyses were conducted using GraphPad Prism (Version 9).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Hou and colleagues have submitted a manuscript where they identify a novel compound that would inhibit Treg function to enhance effector T cell function in general and of CAR T cells in particular. The paper claims that this would function via Adenosin receptor signaling. The impact of A2AR or other adenosine receptors as well as on CAR T cells and T cells is well known and established and so are highly potent inhibitors of the pathway. A multitude of A2AR antagonists or inhibitors have been proposed for cancer treatment up to clinical trials, also in the context of CAR T cells and in fact A2AR known antagonist perform similar if not better then the new compound. Altogether this does not provide a conceptual advance and limited novelty beyond the specific compound. Impact could only come from superior activity to other antagonists but this is not investigated. I have identified further conceptual problems and limititations that further prevent evaluation of the paper:\n1) while the screening concept is understood, it is totally unclear why the authors focused on TAIII in spite of numerous others performing similarly well in the screen. Experimental evidence is required here\n2) overall the concentrations of TAIII are very high microM range. Good inhibitors act in the two digit nM range, which challenges specificity here\n2) the authors back the paper on the claim of the relevance of CARTreg for CAR activity but I wonder how much a problem this really is. How is performance of either CD8 or CD4 CAR T cell population when using killing as a surrogate? What is the prevalence of Foxp3 Treg in the cultures? Are these cells specifically suppressive? This is aggravated by the fact that this reduction is mostly not shown by assay.\n3) Overall the reduction in Foxp3 can be quite modest and it is questionable if this suffices differences in some the killing assays than look quite massive.\n4) essential controls are missing throughout: diluent and irrelevant CAR should always be included as controls with or without TAIII.\n5) It is totally unclear how the authors ended up with A2AR. This is neither explained nor justified.\n6) MC38 is not a xenograft and again key controls are missing, such as TAIII only or with isotype versus TAIII with PD1, this will likely change the conclusions\n7) same for the xenograft model in NSG, controls are missing (see above)\n8) it is very unclear how many healthy donors were used for each experiment and subexepriment\n9) for each and every figure and subfigure it must be made very clear how often experiments were done, if these were pooled and how\n10) how was cell line identity ascertained?", "reviewer_2": "In this study, Hou and coworkers characterize the immunomodulatory effects of Timosaponin III (TAIII) and find that it may help resolve a crucial issue: the contamination of CAR-T cell preparations by CAR-Tregs, which are responsible for resistance to therapy. The significance of the author’s finding is huge. In addition, the authors use a variety of assays and several in vitro and in vivo models to make the point that TAIII works as an A2AR inhibitor, decreasing cAMP, PKA, and CREB signaling to counteract FOXP3 expression. TAIII works as a standalone therapy against solid tumors, and also enhances the efficacy of PD-1 blockade. In a xenograft model of B cell lymphoma, TAIII improves the efficacy of CAR-T cell therapy. There is also some evidence of better efficacy of CAR-T cells pretreated with TAIII. In all, I am enthusiastic about the significance, scientific soundness, and potential impact of this work.\nWhat I am less enthusiastic about, though, is the writing. English needs to be corrected across the paper to convey scientific messages properly. There are imprecisions, contradictions, and outright errors in the interpretation of the experiments (while the experiments themselves are correct!). Some references are called out wrongly, and some figures are difficult to follow because they are mislabeled or with panels arranged in non-alphabetical order. Thus, while the experimental part to correct is quite straightforward, I urge the authors to rewrite the paper to improve its readability and, ultimately, its high potential scientific impact.\nExperimental issues:\nThe main experimental gap is that it is impossible to conclude that TAIII works by impacting Tregs only without ruling out its effect on Teff cells. None of the experiments included in the paper does so, as the improved function of Teff cells is always read out when Tregs are concomitantly inhibited (Figs. 5, 6, 7, S3, S4, S5, S6). Thus, I recommend to run an in vitro experiment in which Teff are purified and devoid of Tregs. It might be easier to do it with mouse effector T cells rather than in the CAR-T setting, but it would be OK as the authors proved the efficacy of TAIII even in the context of boosting the endogenous T cell response (Fig. …). So, would NECA and TAIII modulate IFN , TNF, and IL-2 production from Teff when Tregs are completely absent? If not, the authors can conclude that TAIII has an exclusive effect on Tregs.\nFig. 3b. the input lane is oversaturated and impossible to interpret. Please substitute it. The authors may include GAPDH as a further control.\nFig. 3e. Was NECA used in the assay? If not, please show a positive control of NECA treatment increasing CREB luciferase signal.\nFig. 6i. An experiment including a group of TAIII treated mice would be necessary to test whether combo immunotherapy is more effective than TAIII single therapy.\nDiscussion issues:\n(lines 484-486) TAIII does not cause “an additional increase in the secretion of inflammatory cytokines.” I disagree, as Fig. 2d, 4b, 5a, 6g, 7m indicate otherwise. Please reword more cautiously.\nDiscuss potential usefulness of TAIII + anti-PD-1 for solid tumors.\nCompare TAIII to currently available inhibitors of A2AR. Why it is better, beyond being a natural product?\nPaper readability issues:\nGeneral:\n1. Please revisit for English correctness.\n2. Some references are inaccurately cited. For instance, ref. 16 (line 77) does not evaluate CAR-Tregs as a mechanism of resistance to therapy. Moreover, on lines 378 and 384, refs. 16 and 17 are called out, while it should be refs. 17 and 18. These examples are non-exhaustive, please check references all over the paper.\n3. Please arrange figure panels in alphabetical order (e.g., Fig. 7 is not).\nSpecific:\nFig. 1a. Better describe the screening procedure by showing the transduction with the IL2-Luc reporter AND with the FOXP3a-encoding plasmid. Was T cell stimulation through the TCR necessary to induce luciferase expression? Show the high-throughput readout for screening (Light production before-after TAIII stimulation, one non-changing and one changing sample) to give an idea of the robustness / statistical significance of the assay.\nFig. 1b. How was the cutoff limit selected? Explain why TAIII was selected despite not having the best IL-2 induction activity in the assay.\nFig. 1c. (line 144) text callout to assay is wrong – the figure shows the chemical formula of TAIII.\nFig. 1g. Move Y axes to 0 please. Also please check this throughout the paper.\nFig. S1b. How was A2AR selected over other TAIII-binding candidates (CCT2, GFUS, HSPA1B, EEF2)? Even if the downstream analysis was convincing in attributing immunomodulatory effects to A2Ar inhibition, what was the original rationale for selecting A2AR over the other candidates?\nFig. S1c. Change the panel organization to make clear that the line plot quantifies thermal stability (and is not related to panel e).\nFig. 3f. It is very confusing to see a cAMP readout increasing (rather than decreasing) upon administration of A2AR antagonists. I understand this is because the 655/615nm ratio is inversely proportional to cAMP, but the point needs to be clarified in the main text as well as in the figure, to help the reader follow the logic of the experiment. Alternatively, can the 615/655 ratio be shown? This consideration also applies to Fig. S2b. Also, values for 655/615 ratio in Fig. 3f, 3g are very different from these shown in Fig. S2b. Is it because relative values are shown in Fig. S2b? Please make it uniform with Fig. 3f and g.\nFig. 3i. It should be noted that even the W129A mutation does not abolish CREB activity. This aspect is crucial because sensitivity to TAIII cannot be tested if A2AR activation by cholesterol is completely abolished. Using a logarithmic Y axis instead of a broken scale may help clarify. Also, was NECA used in the assay?\n(line 222) The effects of TAIII on A2BR do not seem minimal (Fig. S2b). Please rephrase.\n(line 226). To rule out off-target effects of TAIII, the most important controls are cells not expressing A2AR and not responsive to TAIII and other A2AR inhibitors. These are shown in Fig. S2b. It would also be appropriate to precisely say “to rule out off-target effects on cAMP/PKA/CREB pathway triggering”, as other off-target effects cannot be excluded.\n(line 249) The sentence is unclear. It should read “All mutations decrease CREB activity and abolish sensitivity to even increasing doses of TAIII”.\nFig. 4a. Please say which assay was used. Bulk RNA seq?\nFig. 4c. What is the difference with Fig. S3a? Also, there is a mislabeling in the figures: treatment with NECA and TAIII should not increase Foxp3, nor decrease IL-2 and IFN .\nFig. 4e. Please indicate the Teff to Treg ratio, and show CFSE dilution plots for no CD3/CD28 and no Tregs conditions. Setting the scale of the X axis from the fluorescence value of non-proliferated Teff to the lowest value of CFSE fluorescence may help show suppression more convincingly.\nFig. 4f. How were the suppression values calculated?\nFig. 5a. Please show Raji cells in the absence of CAR-T cells and indicate how the percentage of killing was calculated.\n(lines 298 to 303). I disagree that the described experiments allow us to make the point that TAIII works by blocking A2AR signaling – this point is convincingly settled by the shRNA experiments in Fig. 4g, h and S3d. Rather, the experiments described in the paragraph try to convince the reader that the effect of TAIII was Treg specific. As previously discussed, additional experiments are needed to make this point.\nFig. S4.a. Can the authors exclude that the B/CD4 hybrid population evident in the first panel (and disappearing due to the CAR-T effect) is not an artifact created by cell doublets? Also, it seems that TAIII leads to the killing of only one population of B cells, while a separate B cell cluster is spared. What is special about these B cells? Third, can you please show the Treg cluster? Finally, how do the “unknown” cells look like? They are interesting as they appear only after TAIII treatment.\nFig. S4b and S4c. It is counterintuitive that the treatment with NECA and TAIII increases immune suppression genes – they should be decreased, especially considering the reduction in Foxp3, CTLA-4, and CD25 shown in panel d. Please explain.\nFig. S4d. Please label the “TAIII” group as “NECA + TAIII”. Also note that the first NECA + TAIII replicate does not match with the other two (it appears TAIII did not work well). Can this replicate be eliminated? Also indicate if IFN  is also upregulated in the NECA + TAIII group.\nFig. S4e. The p value for the enrichment of cholesterol metabolism genes after TAIII treatment should be indicated.\nFig. 6. General: please indicate how many experiments were conducted and how many mice were analyzed in total. Also applies to Fig. S5.\nFig. 6a. MC38 is not a xenograft – that term is reserved to graft procedures across species barrier (e.g., human Raji tumors in NSG mice). Please also correct panel h, Fig. S5a, and several instances in the manuscript text.\n(line 339) How was TAIII administered? Line 339 says intratumorally, lines 726 and 898 intraperiotoneally.\nFig. 6c. It seems that tumors are normalized to the volume on day 0. This is incorrect, as day 0 volume is largely due to the liquid in which tumor cells are resuspended. It would be better to represent tumor growth as non-normalized volumes. This consideration also applies to Fig. S5c.\nFig. 6g. ELISAs on tumor samples are difficult. Please explain the procedure in detail. The same applies to Fig. S5i, especially for IL-2.\nFig. 6l. The Western blot for pCREB needs to be shown, including lanes for total CREB and housekeeping genes. Is CREB equally expressed by all cell types in the tumor environment? If not, less pCREB can also be secondary to the modulation of cells after administration of anti-PD-1 and TAIII.\nFig. S5g. Report the percentage of Tregs (especially CD44+CD62L- activated Tregs). Is it decreased? How does this compare with CTL activation? This comparison may further help making the point of about the specific action (or lack thereof) of TAIII on Tregs.\nFig. 7b. (line 385-386). It is confusing (and possibly inaccurate) to state that NF-kB signaling is downstream of A2AR or specifically important for Tregs – it is also key for Teff cells. Also, the figure shows NF-kB signaling increases in the CAR-T cell product administered to responders. Please restate.\n(line 432) It is good to test whether TAIII inhibits tumor growth in the absence of T cells. However, the rationale indicated in line 432 is wrong: the experiments test whether TAIII has a direct antitumor effect, rather than testing off-target effects on CAT-T cells (which are absent in these experiments).", "reviewer_3": "In this interesting article, Hou et al. describe the use of Timosaponin AIII (TAIII) in the context of cancer therapy, either as monotherapy or in combination with PD-1 axis blockade or with CAR-T cells, and they argue that the TAIII improves the potency of T-cell mediated therapies via A2AR-blockade-mediated Treg suppression. Contrary to the title, the manuscript broadly and nicely shows TAIII’s A2AR-axis-mediated anti-cancer activities using orthogonal approaches. The data and story have translational relevance and would be of great interest to the field. However, on the other hand, the presented data in the current form are somewhat limited surrounding the concept of if/how TAIII in fact augments CAR-T potency, and if so, whether it is through CAR-Treg suppression.\n1. Throughout the T-cell functional experiments, both in vitro and in vivo, it is critical to add non-specific T cell control, such as CAR-T cells targeting an irrelevant antigen or at least untransduced T cells. It is unclear whether the TAIII has the role in augmenting antigen-specific T cell responses, or whether it is non-specific. In particular, in the NSG xenografts (such as Fig 7 and Extended Fig 6) known to be confounded by xeno reactivities, without negative control T cells with or without TAIII, the data don’t support the therapeutic utility of combining CAR-T and TAIII. Please also provide whether xeno-GVHD-related symptoms changed when animals were co-exposed to both T cells and TAIII compared to T cells alone.\n2. Please provide T cell phenotypic data with frequencies, including CD8/CD4 ratio, Tnaive/Tcm/Teff/Temra composition, and T reg frequencies 1) at the time of cell manufacturing, 2) before and after in vitro TAIII exposure (for the experiments with 36-hr “pretreatment” of CAR-T cells with TAIII before infusion), and 3) longitudinal changes in the in vivo frequencies (persistence) and phenotype of T cells with and without TAIII exposure at select timepoints. Does exposure to TAIII change these parameters? Does T-cell phenotypic or functional changes (if any) induced by “pretreatment” TAIII exposure retained in vivo after infusion or were they transient? Fig 7h and 7i may partially address this, but the timepoint at which the data was obtained is not described in the Legend, and the critical controls are missing as described in #1.\n3. Alternatively or additionally, is it possible to do selective Foxp3+ T cell depletion experiments or do studies like A2AR-ko CAR-T +/- TAIII, or other genetic validation to describe mechanistic association between CAR-T reg suppression and TAIII?\n4. CAR-T experiments were done only with one model (Raji). Generalizability of the finding should be shown in a second xenograft model (especially if the intended main message remain to be the effect of TAIII in the context of CAR-T therapy or more broadly adoptive T cell therapy).\n5. Does Raji express A2AR, and what are the A2AR expression levels relative to other leukemia and lymphoma cell lines? Does A2AR expression on T cells and tumors change over time in in vivo models?\n6. In Extended Fig. 6 (Fig S6) in which CAR-T “pretreated” with TAIII were used to treat xenografts, what was the timepoint at which spleens were harvested to generate the Figure S6b? What was the parental population for the %FoxP3+ in the y-axis? (was the %FoxP3+ in the whole splenocytes or was it gated specifically on CAR-T cells, or on CD4+ CAR-T cells?). Fig S6c, which shows the luminescence data from day9, please show day 0 baseline values (or pre-treatment values) for all treatment groups.\n7. Ideally, it would be better to use immunocompetent adoptive T cell therapy models (B6 murine CD19 CAR x E2aPBx, or pmel, OT-1, etc.) to actually study the T cell biology (Treg differentiation) and concepts of disease relapses. If difficult, showing in vivo T cell phenotypic data with frequencies (CD8/CD4 ratio, Tnaive/Tcm/Teff/Temra composition, and T reg frequencies) from Fig6. immunocompetent B6/MC38 model might help strengthening your case.\n8. Have the authors studied the effect of TAIII on other T cell subsets (e.g. CD8+)? Is the anti-tumor effects attributable solely to either CD4+ Treg induction/function, or does TAIII have broader effects on T cells’ anti-tumor functions? Further showing the effects of TAIII on T cells are desired before concluding the effects to be “through impairing CAR-T regs”.\n9. What is the benefit of using the TAIII compared to other A2AR-targeting technologies, for example existing agents such as AZD4635 or genetic silencing of A2AR as others have shown in the field? Or can similar effects be achieved by depleting Treg from infusion products?\n10. Figure 6. TAIII-only group is missing in the in vivo experiments. Was combining PD-1 blockade and TAIII more effective than TAIII alone? The data currently show the comparison of only anti-PD1 and combination, but that alone is not sufficient to support the statement that TAIII augmented the effect of anti-PD-1 therapy.\n11. Because there are other nice in vitro cytotoxicity data (such as Fig 2c), I assume the overall conclusion is valid, but I don’t think the method of FACS-based in vitro cytotoxicity data presented in the Fig 2a/2b and Fig 5a (and the way in which “precent killing” were calculated as described in the methods section) are technically consistent with the valid and well-accepted FACS-based T-cell cytotoxicity evaluation method in our field (for example, Hermans et al, J Immunol Methods, 2004. PMID: 14871532). The issue with the presented FACS-based cytotoxicity data is that it does not account for (or control for) the effect of non-specific killing. Additionally, another issue is that the residual leukemia seems to be defined by cell-surface CD19 expression (assessing from the fact that the presented FACS dot plots gate on CD19-BV421+ population). CD19 cell-surface expression rapidly downregulates after anti-CD19CAR (or antibody) engagement, and the percentage of CD19+ population is not synonymous to the residual live leukemia fraction in this type of antigen-specific T-cell killing experiments (in another word, depending on the experimental condition, antigen-negative or antigen-low “live” leukemia can be there). These should be resolved if these figures are going to be included in the manuscript.\nMinor points:\n12. In Fig 7j, there are four culture conditions presented. In the subsequent figures, some of these conditions appear to be missing. Could all conditions be presented in the subsequent figures? (It is possible that I’m getting confused by the order in which figures are presented: this is a minor suggestion but I think it is easier to understand if the figures are presented in a linear fashion. For example, the way in which Figure 7g, h, i, j, k, l are ordered are quite difficult for readers to follow as we have to go back and forth trying to visually identify an appropriate subheading corresponding to the legend/text. Similar issues are seen with Figure 6e/f and 6l).\n13. C57BL/6 and MC38 model is a syngeneic murine model, and it is not a “xenograft” model as described throughout in the manuscript, figures and legends.\n14. Are there any existing data on the safety of systemically dosing TAIII in humans? The existing clinical experience seems using TAIII as an anti-wrinkle dermatology treatment, but if authors could discuss about the expected translational promise of TAIII (including the existing safety data and/or lack thereof), that would be great.\n15. Line 154: there are no T cell functional data provided in the Fig 1\n16. Fig 6h in vivo treatment schema would benefit from clarification. It currently says “anti-PD1 w/o TAIII”, but there should be other treatment groups?\n17. Line 225, the reference to “dose-limiting toxicity” is confusing when the data is solely about in vitro data. Perhaps, you might want to consider saying something like, “cell viability was preserved across all TAIII concentrations tested in the assay, suggesting that TAIII is likely non-toxic for T-cell viability”.\n18. Line 485-486: “without causing an additional increase in the secretion of inflammatory cytokines”. This statement directly contradicts your data (for example, Fig 2d, 5c, 7m, etc.)\n19. I’m afraid I’m having a difficult time understanding Figure 3i, y-axis. In Figure 3e, CREB-Flu reporter activity is reduced in a drug dose-dependent manner. On the other hand, in Figure 3i, the CREB-Flu activity is reduced in conditions of binding-site mutations (e.g. K122A, W129). Isn’t the binding-site mutation expected to abrogate the reduction in CREB-Flu reporter readout (as shown in Fig 3j)? Or is Fig 3i using a different readout? If the authors could please clarify and specify which luciferase-based reporter assays in Fig 3 are measuring which effects, that would be greatly appreciated.\n20. Methods, line 674, please specifically describe the CD19 CAR construct used in the project, either referencing the original article describing the exact construct you used, and/or providing the exact sequences of the construct you designed in the supplementary materials.\n21. Figure 4e. CFSE dilution assay. The histograms shown are gated on which population? (CFSE dilution of “all T cells” in the culture, or Treg, or Teff?)\n22. Figure 4e and 5e – please provide gating strategy in supplementary.\n23. Minor editorial suggestion: It is advisable to use consistent color scheme in Fig 4b-f. For example, in Figure 4b, “green” bar is used for “NECA+, TAIII 1 uM”, but in the same “green” color means “NECA+ AZD4635” in Figure 4C and 4F. Similarly the definition of “blue”, “purple” and “pink” switches from figure to figure, which reduces the readability of the data.", "reviewer_4": "The paper discusses Car-T potential therapy. It is well written. Timosaponin AIII enhances CAR-T cell potency.High-throughput screening identified Timosaponin AIII as a Tregs inhibitor, TAIII enhances the antitumor efficacy of CAR-T cells by eliminating FoxP3+ Treg cells in vitro and TAIII activates T cell responses through impairing Treg function in an A2AR-dependent manner TAIII suppresses tumor growth through reversing A2AR-FoxP3+ Treg mediated immunosuppression methods are given as well. The study was done on animal model (Xenograft) and 6 high resolution figures are given. The methods includes statistical and biological ones. Major finding, found that TAIII specifically blocks A2AR signaling in CAR-T cells by acting as a novel allosteric inhibitor of A2AR, thereby reducing the proportion of CAR-Tregs without concurrently depleting cytotoxic T cells."}, "author_response": "We’d like to thank the reviewers for their careful and insightful review of our manuscript. We believe that in addressing the points raised, we have improved the quality of our manuscript. A point-by-point response to reviewer comments and questions is below.\nNCOMMS-25-06683-T\nPoint-by-Point Responses:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nHou and colleagues have submitted a manuscript where they identify a novel compound that would inhibit Treg function to enhance effector T cell function in general and of CAR T cells in particular. The paper claims that this would function via Adenosin receptor signaling. The impact of A2AR or other adenosine receptors as well as on CAR T cells and T cells is well known and established and so are highly potent inhibitors of the pathway. A multitude of A2AR antagonists or inhibitors have been proposed for cancer treatment up to clinical trials, also in the context of CAR T cells and in fact A2AR known antagonist perform similar if not better then the new compound. Altogether this does not provide a conceptual advance and limited novelty beyond the specific compound. Impact could only come from superior activity to other antagonists but this is not investigated. I have identified further conceptual problems and limititations that further prevent evaluation of the paper:\n1) while the screening concept is understood, it is totally unclear why the authors focused on TAIII in spite of numerous others performing similarly well in the screen. Experimental evidence is required here\nAuthor response: We sincerely thank the reviewer for their thoughtful comment and apologize for any confusion caused. Figure 1b presents data from our initial screening, where over 50 compounds, including TAIII, demonstrated significant effects on the FOXP3-IL-2 axis. In order to prioritize novel compounds, we conducted a thorough review of the literature and patent databases, ensuring that previously reported compounds, such as HDAC inhibitors, were excluded.\nThe top 10 candidates were then subjected to secondary screening, which involved dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system and single-dose testing in human PBMCs, with toxicity and potency carefully assessed. From this screening, TAIII emerged as the lead compound, exhibiting strong dose-dependent efficacy and a favorable safety profile (see Fig. 1a, b for Reviewer).\nTo further clarify the rationale for our focus on TAIII, we have updated the manuscript to include this explanation and provide additional data in the revised Figure 1c (see Figure 1 for Reviewer). “During the initial screening, over 50 compounds, including Timosaponin AIII (TAIII), exhibited significant modulation of the FOXP3–IL-2 axis (Fig. 1b). To prioritize novel candidates, previously reported compounds such as HDAC inhibitors were excluded through literature and patent database review. The top 10 candidates were subsequently subjected to secondary screening, which included dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system (data not shown) and single-dose testing in human PBMCs (Fig. 1c), with careful consideration of toxicity and potency. From this process, TAIII, a natural product derived from Anemarrhena asphodeloides and also a traditional Chinese medicine, emerged as the lead candidate (Fig. 1d), demonstrating robust dose-dependent efficacy and a favorable safety profile.” We hope this resolves the reviewer’s concern and offers a clear justification for our prioritization of TAIII in this study.\n2) overall the concentrations of TAIII are very high microM range. Good inhibitors act in the two digit nM range, which challenges specificity here\nAuthor Response: We sincerely appreciate the reviewer’s insightful comment regarding the concentration of TAIII and its specificity. We agree with the Reviewer that being a natural product, TAIII operates at micromolar concentrations (300-600 nM in cells), it is crucial to emphasize that its efficacy, although lower compared to some synthetic small-molecule inhibitors, is accompanied by a significantly lower toxicity profile. This makes TAIII a promising candidate for further development, especially considering its favorable safety profile, which is often a key limitation for many synthetic compounds.\nIn our studies, TAIII demonstrated superior in vitro and in vivo efficacy compared to synthetic small-molecule inhibitors, including the clinical phase II positive control drug AZD4635, AB928 and CPI444 (revised Figures 4 and 6, Extended Data Fig. 5 and Fig. 5, Figure 2 for Reviewer). This demonstrates the therapeutic potential of TAIII, even at higher concentrations, and suggests that its safety and availability make it an ideal candidate for continued investigation.\nRegarding specificity, we have taken several additional steps to validate the selectivity of TAIII. Through a biotin-pull-down assay followed by mass spectrometry, we identified A2AR as a novel target of TAIII, which we believe mediates its selective action. This discovery was further corroborated using target engagement assays and competitive assays, which further support the specificity of TAIII towards its identified targets (Figure 3, Extended Data Fig. 3). In addition, we also conducted a luciferase-reporter assay to evaluate TAIII’s effect on the adenosine receptor family (Extended Data Fig. 4). These results provide strong evidence that A2AR is the key target of TAIII’s action.\nAdditionally, we acknowledge that TAIII’s pharmacokinetics (PK) profile limits its bioavailability. As reported in previous studies, TAIII (MW: 740.92 g/mol) exhibits poor permeability and solubility, with a bioavailability of approximately 10% (Wang et al., RSC Adv. 2023, PMID: 37077258; Wang et al., Current Pharmaceutical Biotechnology, 2020, ISSN: 1873-4316). The reported T1/2 is 1.82 ± 0.47 h, with Cmax of 3.67 ± 0.51 μg/L and Tmax of 4.67 ± 0.52 h, indicating relatively low absorption. However, based on our experience and the literature, we found that intraperitoneal injection of TAIII improves its Cmax and T1/2, enhancing its bioavailability. While the concentrations used in our study (5mg/kg) remain below 1 µM in blood, these improved pharmacokinetic parameters align with the observed efficacy in preclinical models. Given these considerations, we believe that optimizing TAIII’s formulation or delivery methods will be crucial for maximizing its therapeutic potential.\nFuture efforts should focus on developing advanced drug delivery systems to enhance its bioavailability and overall clinical efficacy.\nWe hope that these additional data and clarifications effectively address the reviewer’s concerns about the concentration and specificity of TAIII, providing a clearer understanding of its potential as a therapeutic agent."}, {"round": 2, "reviewer_comments": {"reviewer_1": "For the avoidance of doubts, I will be exclusively commenting on previous reviewer 1 and the associated comments and concerns. Hou and colleagues have submitted a revised manuscript where they aimed at alleviating key concerns of the previous review, which were the narrative, novelty, specificity, validity of conclusions and scientific rigor. While they provided a credible effort, including substantial novel data majority of concerns remain and are even strengthened by some of the data and argumentation provided. It is acknowledged that with the betterment of the narrative and results of the secondary screen and rationale, clarity has been improved relevantly. Specific comments:\n1) Novelty: the study does still not provide a fundamental advance, and even if the conclusions would hold (see below), would still revolve around known mechanisms of suppression. Added value or advancement could only come from substantial therapeutic benefit, which to this point remains unclear and probably borderline (see below).\n2) Specificity: the specificity of the compound remains unclear. Authors now provide western blot and signaling data as well as functional data, but the amounts required to reach potential effects remain excessive: while the data may be compatible with A2AR, they do not prove it. For example the western blot dot meet quality standards in 2025 (single image, no quantification, cropped blots, see also below) and importantly only show a potential band at very high concentrations, which hints towards lack of specificity and not unlikely would have pulled down more proteins but this is unclear. Similarly, while the use of NECA as a proxy of adenosine signaling is standard, the overall impact of NECA in the assays is weak to borderline, challenging the relevance of the assay. Similarly the fact that NECA does not antagonize the effect of the compound does not mean that the compounds prevents A2AR effect. Lastly the signaling molecules analyzed are not A2AR specific but broadly used by similarly structured (often suppressive) receptors, again taking away the specificity claim. This are just some example substantiating the concern on specificity.\n3) Validity of conclusions: both for A2AR as well as for Treg, the results may to a certain level be compatible with these elements being influenced but may simply be otherwise explained (see above for A2AR). In the case of Treg at best only weak and indirect evidence is provided. Authors deplete a Treg “enriched” population from PBMC prior to transduction to back up their claim, which does not answer the question of the actual role of Treg here. For example, a more likely explanation backed up by the authors own data is that in their hands CD4+CART hardly kill. Thus reducing CD4+, as done here, automatically will enhance killing as shown. This unrelated to Treg. This is further strengthened by the fact that even the evidence that Treg are truly present in these CAR cultures is weak in the present data set. The lineage defining marker Foxp3 hardly stains (just compare to the authors own “purer” Treg populations) and only on a handful of cells. Lastly even functionally only indirect evidence or literature is provided. Even at the level of activity strengthening of CAR T cells in vivo, the evidence is weak at best. In the data provided (for example figure 7j), even untransduced T cells perform equally well than the presumed better combination treatment, challenging the data presented and its conclusions. Overall the follow up is either very short or devoid of survival data, which anyways limit data interpretability and relevance.", "reviewer_2": "While the authors fully addressed my experimental concerns and I keep a high enthusiasm towards the significance of the paper, I keep having problems with the representation of statistical analyses, one item of discussion, and one piece of data to be moved from the reviewer’s figures to the extended data. All other suggestions are minimal.\nMajor:\nThe representation of many statistics keeps being incorrect. First, many figure legends say that “representative results” are shown, yet SEM is used as a dispersion value. Instead, it should be clarified throughout all legends whether the shown data are from one representative experiment with individual points representing technical replicates, or one pooled graph including all samples used (typical for mice). In these cases, SD should be used instead of SEM. SEM is to be used only when the graph plots the averages from individual, identical experiments.\nSecond, the boilerplate statement found in the statistical analysis section of the materials and methods says that each experiment has been repeated three times with three technical replicates. The number of replicates, however, appears wildly variable across groups, and individual values by experiment should be reported instead.\nFinally, the actual test used (Student’s t, Mann-Whitney, Kruskall-Wallis as per the Materials and Methods section) should be specified for every experiment, not just at the end of each figure legend. Was normality of the distribution verified in all cases a parametric test was used? Possibly not, as it is difficult for groups with just two replicates (e.g., TNF in Fig. 2e) to pass a normality test, and thus be analyzed using a Student’s t.\nFig. 7m,n. Please add information on whether TAIII-pretreated CAR-T cells prevent tumor relapse. If not, it just means that for long-term benefits, TAIII should be administered in vivo as well. Just comment on these issues.\nFig. S2: It is impossible to conclude that the moderate cytokine production observed after TAIII treatment (Fig. S2e) does not correlate with enhanced killing without showing the killing assay on the same cell line used for Fig. S2e (Raji). Please move this control from Reviewer Fig. 10e to the text, and ensure proper statistics (now only one sample is included). Similarly, a direct comparison between Fig. S2d (on Nalm6-luc) and Fig. S2e (on Raji), as per lines 229-231, is incorrect.\nMinor:\nFig S1b and lines 186-188. Can the authors explain why cultured T cells lose CD45RO expression? On the same figure, labeling of Foxp3 PE-CY7 on the x axis of flow plot is incorrect. Finally, it is unclear at which points Raji cells were added to the coculture.\nPlease show the computational analysis that helped remove CCT2, GFUS, HSPA1B, and EEF2 as candidates for TAIII binding (line 249).\nLine 133,“eliminates” is too strong. Replace with “reduces” or “inhibits”.\nLine 140, replace “Treg functional system” with “high-throughput screening platform for Foxp3-mediated Il-2 inhibition”.\nLine 159, explain how toxicity was evaluated in the secondary screening. PBMC viability?\nLines 276-278. Please clarify that the assay was performed with NECA with or without titrated amounts of TAIII.\nFig. 4e, f and S5e. It would be appropriate to show CFSE dilution plots in the absence of Tregs. Does NECA and TAIII modulate Teff proliferation?\nLine 353. It remains unknown whether the increased cytokine release after exposure to TAIII is directly responsible for the restoration of cytotoxic function of CAR-T cells. An increase in contact-dependent cytotoxicity is equally possible. I would suggest changing the words “by upregulating” to “and upregulated”.\nFig. 5b (top panel) and 5c seem to report the same results twice.\nIn several instances, “TEMRA” cells are mislableled as “terma”.\nLine 392, substitute “enriching” with “modulating”.\nIt looks like lines 395-398 and 405-408 repeat identical concepts.\nLine 445. Please spell out “QD” and “BID”.\nLine 459. Please spell out “BIW”.\nFig. 6f and 7i Substitute w/o (“without”) with ±.", "reviewer_4": "The revised paper does not include 'track changes' option. The paper content is impressive and important. It looks like the authors addressed the majority of reviewer comments.\ni did not find addressing of the following comment - The following comment - he concentrations of TAIII are very high microM range. Good inhibitors act in the two digit nM range, which challenges specificity here.\nspecifically, experimental evidence was given to the TAIII uissue - it increases the proportion of central memory T cells (Tcm) and enhances\n49 overcoming late relapses in vivo. They also note that TAIII as a potent adjuvant for CAR-T\n50 cell function and manufacturing by reducing CAR-Tregs proportion. non targeting controls were detected non-targeting\ncontrols. N=3 healthy donors. Tregs were\n876 defined as CD4⁺FoxP3⁺ cells. For intranuclear FoxP3 staining. Data from three independent experiments (each with\nthree technical replicates) were\n1159 pooled and analyzed to provide a total of nine data points for each condition. 2 cell lines (HEK-293T and Jurkat T cell\nlines) were included.\nreviewer 2: paper language looks corrected. Figures and legends are clearer. FoxP3+\nTreg cells in vitro. a, CD19 CAR-1103 T cells were generated from human T cells. Human\npan-T cells (CD3+ 1104 T) were isolated from PBMC with a human pan-T isolation kit and incubated\n1105 with CD3/CD28 cross‐linking antibodies beads, CAR lentiviral constructs were delivered into\nIn the discussion, cytokines are still mentioned - Moreover, TAIII eliminates Tregs from CAR-T cells while simultaneously\nactivating. IL2-Luc screening is not described. the IL-2 promoter-firefly luciferase (Fluc) reporter. the W129A mutation\nretained\n308 partial activity, which is an important consideration when interpreting the sensitivity of TAIII\n309 in these experiments. Effect of TAIII on A2BR is not discussed.\n667 immune responses, without causing excessive inflammatory cytokine secretion, which could\n668 lead to toxicity. This suggests that TAIII carries a low risk of triggering cytokine release\n669 syndrome (CRS), a common side effect associated with CAR-T cell therapy. Importantly, TAIII\n670 increases the percentage of Tcm and Temra cells upon tumor antigen stimulation in vitro and in\n671 vivo, enhancing T-cell expansion and persistence. Inhibitor comparison - Given its immunoregulatory properties and\nability to downregulate CTLA4 expression\n685 in T cells, TAIII holds promise as an adjuvant to immune checkpoint inhibitors (ICIs) such as\n686 anti-PD-1 antibodies. Notably, PD-1 blockade alone has been shown to paradoxically expand\nintratumoral Tregs, thereby limiting therapeutic efficacy. References list was revised. Line 249 was not corrected. Bulk\nRNASeq was used. Tregs significance is mentioned (but not ratio). Suppression essays are mentioned. P val for Choesterol\nis not given. The following sentance is included To assess the antitumor efficacy of TAIII in vivo, we treated a syngeneic\nmouse model\n430 (C57BL/6J) inoculated with MC38 cells with 5 mg/kg TAIII administrated intraperitoneally for\n431 32 days (Extended Data Fig. 9a). The authos tracked tracked T cell frequencies\n553 and phenotypes in peripheral blood. Cells were examined with or without A2AR. To determine whether TAIII improves\nCAR-T function through A2AR signaling, the authors performed co-culture cytotoxicity assay. western blotting of excised\ntumors showed a marked\n465 reduction in A2AR-downstream signaling (pCREB) (Fig. 6k, Extended Data Fig. 10e),\n466 demonstrating that the observed therapeutic benefits were mediated through A2AR pathway\n467 blockade. Although CREB is relatively evenly expressed across different tissues, it is expressed\n468 at higher levels in immune cells, reflecting its key role in immune responses (The Human\nfurther supported by parallel experiments using parental CAR-T cells (5.18%\n227 that TAIII’s functional enhancement is dependent on the presence of Tregs. Line 674 details added. I did not note added\ngating information for figures 4e and 5e. Figure 4 color bars were improved for better visibility. Figure axis titles were added.\nCell population is Treg.\nI think the paper can be accepted for publication.", "reviewer_5": "Hou et al presented an improved revised manuscript where they identified a Timosaponin III (TAIII) as an allosteric inhibitor of A2AR that improve tumor control by impairing T reg differentiation and function. The significance of this finding is that is a traditionally remedy less toxic that current inhibitors of A2AR, could be used in combination with immunotherapy to improve therapeutic responses.\nSignificant changes include the addition of necessary controls, specifically, no transduced T cells and TAIII effect alone for the in vitro and in vivo models. Further characterization of T cell phenotype after TAIII exposure was including, evidencing that the TAIII therapy increase the Tcm phenotype and expansion to the TAIII therapy was added. The effect of TAIII combined with CAR-T cells was evaluated in a second leukemia cell line and killing efficiency was validated with orthogonal assays, resolving the concern of using flow cytometry analysis to evaluate killing effect when CD19 molecule could be downregulated.\nMajor concerns:\n1. Authors claim that Treg depletion enhanced short-term killing of CAR t cells when co-culture with Nalm6-luc (Figure Ext data Fig2c). The killing capacity of CAR-T cell is increased when t reg population is depleted. They claimed that TAIII effect is lost if the T reg are not present validating that the effect is specific because drug effect on Treg, (Figure Ext data Fig2d) but in this experiment the DMSO controls with CAR-T cells and depleted Treg-CAR-T cells showed no therapeutic benefit. I would like to know how the relative luciferase activity is measured, and why they are not measuring luciferase activity as in c. This is not explained in the figure legend.\n2. Although cytotoxic effect is assessed in combination with the drugs tested, how does the drug tested directly affect untraduced T cell and CAR-T cell proliferation and viability? Is TAIII increase proliferation in vitro? There is no data addressing this.\n3. Figure 2B and C the ELISA is the CAR-T cells alone after treatment, It is confusing that the amount of protein measure by ELISA are extremally different between experiments. Figure 5h is measuring IL2 levels of CAR-T cells measure after co-culture in a 1:1 ratio and the levels are significantly lowers that the levels shown in figure 5a even when the E:T ratio is lower, (more CAR-T cell per tumor cells). It requires clarification.\n4. Immunocompetent model testing mouse CAR-T cells in MC38 model in combination with TAIII will respond the relevance of combining TAIII with CAR-T cell, since the TAIII administration alone reduce tumor burden, it will answer if there is real relevance in the combination in vivo. Specially because it will provide a more comprehensive understanding of antitumor effect when the interplay of CAR-T cells /endogenous immune cells is also present, especially endogenous CD8+ and Tregs in the TME.\n5. There are several concerns regarding the NSG models. To start, Raji model lacks the untransduced T cell (UNT) control with and without TAIII, what are necessary to exclude xenoreactivities to T cells. However, the UNT control was included in the Nalm6 model in vivo. Although the authors state that TAIII does not enhance the UNT effect, the data raise questions about antigen-specific efficacy of the CAR-T cells used, especially since the UNT group outperformed CAR-T cell in controlling tumor (Figure 7J-K and Supplementary Fig 11 b) compared with CD19-CAR group. In fact, 4 out of 6 mice treated with untransduced T cells showed reduced tumor burden that the mice treated with CAR-T + TAIII. Additional experiments are urgently needed to demonstrate that tumor control is not driven by nonspecific T-cell activity. Specifically, the study should include side-by-side comparisons of UNT versus CD19-CAR T cells in both Nalm6 and Raj models, followed by evaluation of the combination with TAIII.\n6. Figure 7i, Nalm6 model was used subcutaneously. Nalm-6 is a leukemia model, TAIII effect should be addressed in the standard i.v. disseminated Nalm-6 models with and bioluminescence is used to measure tumor. If the reason is to evaluate the effect in a solid tumor, a different models should be used (colon, osteosarcoma…) and the efficacy should measure alone and in combination.\n7. Extended data Fig 15, the adequate control will be to use untransduce T cells with T reg depleted on them, with and without TAIII. Please comment on why the Treg-CAR-T cell plus TAII arm shows a marked loss of benefit after day 19. Does this reflect a decrease of T cell persistence? How would the survival curves change with extended follow-up?\n8. Extended data fig 15b, why conventional CAR-T cell alone and in combination with TAIII is not presented in this model?\nMinor concerns:\n1. Figure 5: there is a typo in the figure b in the main figure the or in the figure legend b panel has CAR-T + Raji in the first panel of ELISA results, it is confusing. It requires clarification.\n2. Supplementary figure 11a has no legends.\n3. Extended figure 11a has no legends\n4. Figure 7 is missing the statistical asterisks"}, "author_response": "We’d like to thank the reviewers for their careful and insightful review of our manuscript. We believe that in addressing the points raised, we have improved the quality of our manuscript. A point-by-point response to reviewer comments and questions is below.\nNCOMMS-25-06683-T\nPoint-by-Point Responses:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nHou and colleagues have submitted a manuscript where they identify a novel compound that would inhibit Treg function to enhance effector T cell function in general and of CAR T cells in particular. The paper claims that this would function via Adenosin receptor signaling. The impact of A2AR or other adenosine receptors as well as on CAR T cells and T cells is well known and established and so are highly potent inhibitors of the pathway. A multitude of A2AR antagonists or inhibitors have been proposed for cancer treatment up to clinical trials, also in the context of CAR T cells and in fact A2AR known antagonist perform similar if not better then the new compound. Altogether this does not provide a conceptual advance and limited novelty beyond the specific compound. Impact could only come from superior activity to other antagonists but this is not investigated. I have identified further conceptual problems and limititations that further prevent evaluation of the paper:\n1) while the screening concept is understood, it is totally unclear why the authors focused on TAIII in spite of numerous others performing similarly well in the screen. Experimental evidence is required here\nAuthor response: We sincerely thank the reviewer for their thoughtful comment and apologize for any confusion caused. Figure 1b presents data from our initial screening, where over 50 compounds, including TAIII, demonstrated significant effects on the FOXP3-IL-2 axis. In order to prioritize novel compounds, we conducted a thorough review of the literature and patent databases, ensuring that previously reported compounds, such as HDAC inhibitors, were excluded.\nThe top 10 candidates were then subjected to secondary screening, which involved dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system and single-dose testing in human PBMCs, with toxicity and potency carefully assessed. From this screening, TAIII emerged as the lead compound, exhibiting strong dose-dependent efficacy and a favorable safety profile (see Fig. 1a, b for Reviewer).\nTo further clarify the rationale for our focus on TAIII, we have updated the manuscript to include this explanation and provide additional data in the revised Figure 1c (see Figure 1 for Reviewer). “During the initial screening, over 50 compounds, including Timosaponin AIII (TAIII), exhibited significant modulation of the FOXP3–IL-2 axis (Fig. 1b). To prioritize novel candidates, previously reported compounds such as HDAC inhibitors were excluded through literature and patent database review. The top 10 candidates were subsequently subjected to secondary screening, which included dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system (data not shown) and single-dose testing in human PBMCs (Fig. 1c), with careful consideration of toxicity and potency. From this process, TAIII, a natural product derived from Anemarrhena asphodeloides and also a traditional Chinese medicine, emerged as the lead candidate (Fig. 1d), demonstrating robust dose-dependent efficacy and a favorable safety profile.” We hope this resolves the reviewer’s concern and offers a clear justification for our prioritization of TAIII in this study.\n20\n15\n10\n5\n3\n2\n1\n0\n285 286 287 288 446 447 498 591 634 1142-TAIII\nFigure 1 for Reviewer: Secondary screening in Jurkat-FoxP3a-IL-2-Fluc reporter system and human PBMCs.\na. Secondary screening of the top 10 candidates selected from the initial screening, conducted in IL-2-Fluc-FoxP3a reporter cells. Compounds were tested at concentrations of 0, 0.1, 0.3, and 1 μM. b. Human PBMCs were activated through TCR stimulation and treated with 1 μM of the indicated compounds for 48 hours. IL-2 secretion levels were measured by ELISA.\n2) overall the concentrations of TAIII are very high microM range. Good inhibitors act in the two digit nM range, which challenges specificity here\nAuthor Response: We sincerely appreciate the reviewer’s insightful comment regarding the concentration of TAIII and its specificity. We agree with the Reviewer that being a natural product, TAIII operates at micromolar concentrations (300-600 nM in cells), it is crucial to emphasize that its efficacy, although lower compared to some synthetic small-molecule inhibitors, is accompanied by a significantly lower toxicity profile. This makes TAIII a promising candidate for further development, especially considering its favorable safety profile, which is often a key limitation for many synthetic compounds.\nIn our studies, TAIII demonstrated superior in vitro and in vivo efficacy compared to synthetic small-molecule inhibitors, including the clinical phase II positive control drug AZD4635, AB928 and CPI444 (revised Figures 4 and 6, Extended Data Fig. 5 and Fig. 5, Figure 2 for Reviewer). This demonstrates the therapeutic potential of TAIII, even at higher concentrations, and suggests that its safety and availability make it an ideal candidate for continued investigation.\nRegarding specificity, we have taken several additional steps to validate the selectivity of TAIII. Through a biotin-pull-down assay followed by mass spectrometry, we identified A2AR as a novel target of TAIII, which we believe mediates its selective action. This discovery was further corroborated using target engagement assays and competitive assays, which further support the specificity of TAIII towards its identified targets (Figure 3, Extended Data Fig. 3). In addition, we also conducted a luciferase-reporter assay to evaluate TAIII’s effect on the adenosine receptor family (Extended Data Fig. 4). These results provide strong evidence that A2AR is the key target of TAIII’s action.\nAdditionally, we acknowledge that TAIII’s pharmacokinetics (PK) profile limits its bioavailability. As reported in previous studies, TAIII (MW: 740.92 g/mol) exhibits poor permeability and solubility, with a bioavailability of approximately 10% (Wang et al., RSC Adv. 2023, PMID: 37077258; Wang et al., Current Pharmaceutical Biotechnology, 2020, ISSN: 1873-4316). The reported T1/2 is 1.82 ± 0.47 h, with Cmax of 3.67 ± 0.51 μg/L and Tmax of 4.67 ± 0.52 h, indicating relatively low absorption. However, based on our experience and the literature, we found that intraperitoneal injection of TAIII improves its Cmax and T1/2, enhancing its bioavailability. While the concentrations used in our study (5mg/kg) remain below 1 µM in blood, these improved pharmacokinetic parameters align with the observed efficacy in preclinical models. Given these considerations, we believe that optimizing TAIII’s formulation or delivery methods will be crucial for maximizing its therapeutic potential.\nFuture efforts should focus on developing advanced drug delivery systems to enhance its bioavailability and overall clinical efficacy.\nWe hope that these additional data and clarifications effectively address the reviewer’s concerns about the concentration and specificity of TAIII, providing a clearer understanding of its potential as a therapeutic agent."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors answered to all my comments thoroughly. The only comment I have left is to clarify the statements on lines 316-319. On line 316, it is stated that overexpression assays were conducted in the absence of NECA. However, line 319 says that 0.1uM NECA was used. Just clarify further. I now recommend acceptance.", "reviewer_4": "The paper was corrected and can be accepted for publication", "reviewer_5": "Hou et al presented an improved revised manuscript where they identified a Timosaponin III (TAIII) as an allosteric inhibitor of A2AR that improve tumor control by impairing T reg functionality. The significance of this finding is that is a traditionally remedy less toxic that current inhibitors of A2AR, could be used in combination with immunotherapy to improve therapeutic responses.\nSignificant revisions to the manuscript include the addition of experiments assessing the impact of TAIII on CAR-T cell proliferation and viability following co-culture, demonstrating that TAIII does not alter T-cell biology in the absence of antigen stimulation (Fig. 2B) In addition, the expanded methodological description improves data interpretation of the experiments.\nNew data provided, with the repetition of the Biotin-pulldown assay, binding affinity assay and the use of the CGS-21680, further validate the mechanism of action of TAIII as an allosteric inhibitor of A2AR.\nThe authors also incorporated systemic Nalm-6 model (Fig. 7) and added key controls (e.g., vehicle and untransduced T cells), which substantially strengthen the findings and address concerns regarding potential xenoreactivity driven by T cells as well as the lack of antigen specific efficacy observed in the subcutaneous Nalm-6 model. Importantly, the inclusion of the immunocompetent MC38 model enables a more thorough evaluation of TAIII in combination with CAR-T cells or immune checkpoint blockade, increasing the clinical relevance of these approaches by interrogating TAIII’s mechanism of action in a complex, immune-intact setting. Overall, the data support the conclusion that TAIII enhances antitumor activity by modulating the tumor immune microenvironment, via A2AR-Treg inhibition, highlighting its potential as a combination partner for immunotherapies. Overall, these new data further support the proposed mechanism of TAIII, showing enhanced CAR-T responses mediated through immune modulation, addressing the reviewer’s concerns.\nMinor changes appreciated by the reviewer including annotation labels and including some of the stats that were missing from the figures.\nSome minor comments to be considered prior to publication:\n1. Some statistical analyses are annotated with * or #. Using both symbols in the same graph requires clearer explanation in the figure legend (e.g., to indicate different comparisons, statistical tests, or experiments). If all comparisons come from the same analysis, the notation should be unified and applied consistently across the figure.\n2. To facilitate readability, it would be helpful to use consistent colors for the same treatments across all the panels in the same figure. For example, in Figure 5 panels A–B, TAIII is shown in yellow, NECA in green, and the combination in purple, whereas in panel C the combination appears in yellow. Similarly, in Figure 6 panels H, I, and L, PD-1 treatment is shown in red, but in panel K it switches to blue. Maintaining a consistent color scheme throughout would improve clarity and reduce the risk of misinterpretation.\n3. Figure 6K and l, I would consider including the statistic comparisons for the remaining groups.\n4. Including the statistics in the extended data fig 9d."}, "author_response": ""}]

Decision letter
Jing Yang, Ph.D.
Professor
Department of Hematology,
Tongji Hospital,
School of Life Science and
Technology, Tongji University
Shanghai, P.R. China 200092
Tel: (+86)13761232316
jingy@tongji.edu.cn
We’d like to thank the reviewers for their careful and insightful review of our manuscript. We believe that in addressing the points raised, we have improved the quality of our manuscript. A point-by-point response to reviewer comments and questions is below.
NCOMMS-25-06683-T
Point-by-Point Responses:
REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author):
Hou and colleagues have submitted a manuscript where they identify a novel compound that would inhibit Treg function to enhance effector T cell function in general and of CAR T cells in particular. The paper claims that this would function via Adenosin receptor signaling. The impact of A2AR or other adenosine receptors as well as on CAR T cells and T cells is well known and established and so are highly potent inhibitors of the pathway. A multitude of A2AR antagonists or inhibitors have been proposed for cancer treatment up to clinical trials, also in the context of CAR T cells and in fact A2AR known antagonist perform similar if not better then the new compound. Altogether this does not provide a conceptual advance and limited novelty beyond the specific compound. Impact could only come from superior activity to other antagonists but this is not investigated. I have identified further conceptual problems and limititations that further prevent evaluation of the paper:
1) while the screening concept is understood, it is totally unclear why the authors focused on TAIII in spite of numerous others performing similarly well in the screen. Experimental evidence is required here
Author response: We sincerely thank the reviewer for their thoughtful comment and apologize for any confusion caused. Figure 1b presents data from our initial screening, where over 50 compounds, including TAIII, demonstrated significant effects on the FOXP3-IL-2 axis. In order to prioritize novel compounds, we conducted a thorough review of the literature and patent databases, ensuring that previously reported compounds, such as HDAC inhibitors, were excluded.
The top 10 candidates were then subjected to secondary screening, which involved dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system and single-dose testing in human PBMCs, with toxicity and potency carefully assessed. From this screening, TAIII emerged as the lead compound, exhibiting strong dose-dependent efficacy and a favorable safety profile (see Fig. 1a, b for Reviewer).
To further clarify the rationale for our focus on TAIII, we have updated the manuscript to include this explanation and provide additional data in the revised Figure 1c (see Figure 1 for Reviewer). “During the initial screening, over 50 compounds, including Timosaponin AIII (TAIII), exhibited significant modulation of the FOXP3–IL-2 axis (Fig. 1b). To prioritize novel candidates, previously reported compounds such as HDAC inhibitors were excluded through literature and patent database review. The top 10 candidates were subsequently subjected to secondary screening, which included dose-dependent evaluation in the Jurkat-FOXP3a-IL-2-Fluc system (data not shown) and single-dose testing in human PBMCs (Fig. 1c), with careful consideration of toxicity and potency. From this process, TAIII, a natural product derived from Anemarrhena asphodeloides and also a traditional Chinese medicine, emerged as the lead candidate (Fig. 1d), demonstrating robust dose-dependent efficacy and a favorable safety profile.” We hope this resolves the reviewer’s concern and offers a clear justification for our prioritization of TAIII in this study.