IL7-TBRII, a Dual Cytokine Modulator Targeting IL-7 and TGF-β Pathways, Inhibits Tumor Progression and Metastasis

Abstract

Tumor-infiltrating CD8⁺ T cells are a key determinant of anti-tumor efficacy in immunotherapy. IL-7 has been explored as a cytokine therapy to expand CD8⁺ T cells, showing promising anti-tumor effects in preclinical models. However, clinical outcomes remain limited, likely due to the immunosuppressive tumor microenvironment. To enhance the efficacy of IL-7 therapy, we reanalyzed publicly available single-cell RNA-sequencing (scRNA-seq) data of tumors treated with IL-7, identifying elevated TGF-β signaling in CD8⁺ T cells following treatment. As TGF-β impairs CD8⁺ T cell function and antagonizes IL-7 signaling, we developed a bifunctional fusion protein, recombinant human IL-7 (rhIL-7)-hyFc-sTBRII (IL7-TBRII), by fusing a TGF-β trap (Fc-TBRII) to rhIL-7-hyFc (IL7-Fc). We evaluated the binding affinities and functionalities of each domain in vitro and in vivo, and assessed anti-tumor effects in the MC38 colon cancer model. IL7-TBRII demonstrated superior anti-tumor efficacy compared to IL7-Fc or Fc-TBRII alone, primarily through increased infiltration of cytotoxic CD8⁺ T cells into tumors. Also, IL7-TBRII expanded the number of activated CD44⁺ CD8⁺ T cells. Furthermore, IL7-TBRII reduced metastasis in the 4T1 breast cancer model by reshaping the immune cell composition, and demonstrated synergistic efficacy when combined with radiotherapy or anti-CTLA-4 therapy in the EMT6 breast tumor model. These findings suggest that dual modulation of the IL-7 and TGF-β pathways by IL7-TBRII effectively reprograms the immune microenvironment in both primary and metastatic tumors, particularly by promoting CD8⁺ T cell activation and infiltration, thus offering a promising strategy to improve clinical responses to immunotherapy.

INTRODUCTION

Tumor-reactive CD8⁺ T cells play a pivotal role in tumor eradication. However, their efficacy is often compromised by immune checkpoint proteins such as PD-1 and CTLA-4, leading to reduced cytolytic activity and T cell exhaustion (1,2,3). While immune-checkpoint blockade (ICB) therapy has rejuvenated the anti-tumor potential of these cells, its benefits are limited to a subset of patients, often tied to the presence of T cells in the peripheral blood (PB) and tumor (4,5,6,7). This challenge underscores the need for strategies to expand T cell populations and improve ICB therapy outcomes.

To increase the number of T cells in tumors, IL-2 family cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) have been explored as tumor immunotherapies. These cytokines play critical roles in T cell survival, proliferation, differentiation, and functionality, with several currently undergoing clinical research (8). Among them, IL-7, essential for T cell survival and proliferation, has emerged as a promising immunotherapeutic agent (9,10,11). Our engineered Fc-fused recombinant human IL-7, recombinant human IL-7 (rhIL-7)-hyFc (efineptakin alfa; IL7-Fc), features improved stability and a longer half-life, displaying strong anti-tumor activity in preclinical models and enhanced efficacy in combination therapies (12). Currently undergoing clinical trials for various cancer types, both as a monotherapy and in combination with ICBs, IL7-Fc exemplifies the potential advancements in cancer immunotherapy. Despite these advancements, the efficacy of IL7-Fc in immunosuppressive tumor environments remains limited, prompting the development of a new bispecific fusion protein, rhIL-7-hyFc-sTBRII (IL7-TBRII). This innovative approach targets the significant role of TGF-β1, an immunosuppressive cytokine prevalent in ‘immune-cold’ tumors that also impairs T cell activation capabilities of IL-7 (13,14). IL7-TBRII not only promotes CD8⁺ T cell expansion but also neutralizes TGF-β1, thereby maximizing the therapeutic potential of IL-7. Additionally, it enhances the differentiation and tumor infiltration of cytotoxic CD8⁺ T cells, representing a significant advancement in IL-7-based cancer therapies and underscoring the importance of targeting TGF-β1 to optimize therapeutic outcomes.

MATERIALS AND METHODS

Expression and purification of bifunctional fused proteins

IL7-TBRII was expressed in mammalian cell culture using Chinese hamster ovary (CHO; ATCC, Manassas, VA, USA) cells transfected with a pAD-15 expression vector. The cells were cultured in fed-batch process for 17 days, and the cell culture harvests were purified using three-step chromatography process. In the first step, Eshmuno A (Merck, Rahway, NJ, USA) chromatography was used to capture IL7-TBRII. This was followed by Capto adhere (Cytiva, Marlborough, MA, USA) chromatography and Fractogel EMD COO (Merck) chromatography to remove impurities.

Cell lines

The murine colon carcinoma cell line, MC38, were kindly provided by Dr YC Sung (Genexine, Inc., Seoul, Korea). MC38 cells were cultured in DMEM (Welgene, Gyeongsan, Korea) supplemented with 10% FBS (HyClone, Bengaluru, India), 1× antibiotic-antimycotic (anti-anti; Gibco, Waltham, MA, USA). EMT6 cells were cultured in Waymouth MB752/1 Medium (Waymouth; Welgene) supplemented with 15% FBS, 1× Glutamax (Gibco), and 1× anti-anti. 4T1 cells were cultured in RPMI 1640 (Welgene) supplemented with 10% FBS and 1× anti-anti. All cells were cultured at 37°C in a humidified 5% CO₂ atmosphere.

2E8 cell line proliferation assay

2E8 cells (ATCC) were cultured in Iscove’s Modified Dulbecco’s Medium (ATCC) supplemented with 20% FBS (Gibco), 0.05 mM 2-mercaptoethanol (BME; Gibco) and 5 ng/mL of mouse IL-7 (mIL-7; Cell Signaling Technology, Danvers, MA, USA). On day 1, cultured 2E8 cells were washed out with growth medium and induced mIL-7 starvation. Following starvation, the cells were treated with each sample (e.g., IL7-Fc, IL7-TBRII) and incubated for 72 h at 37°C in a humidified 5% CO₂ atmosphere. After 72 h, MTS (Promega, Madison, WI, USA) was added to the 2E8 cells treated with each sample and incubated for 4 h at 37°C in a humidified 5% CO₂ atmosphere. After incubation, the absorbance was measured at 490 nm and the data were analyzed using a 4-parameter curve.

Mice

We used 6-wk-old female C57BL/6 mice purchased from Orient Bio Inc. (Seongnam, Korea) and 6-wk-old female in-house bread BALB/c mice. Seven- to 13-wk-old female Jh (C57BL/6) mice provided by A. macpherson and K. McCoy were used for experiments (15).

Ethics approval

All mouse strains were maintained under specific pathogen-free conditions in an approved animal facility at POSTECH Biotech Center. All animal experiments were conducted according to the protocols approved by the Institutional Animal Care and Use Committee of POSTECH (POSTECH-2022-0028, POSTECH-2023-0116, POSTECH-2024-0113).

Tumor models and treatments

IL7-Fc, Fc-TBRII, and IL7-TBRII were supplied by Genexine, Inc. C57BL/6 mice were subcutaneously (s.c.) inoculated with 1 × 10⁵ MC38. On day 6, mice were treated s.c. or intravenously (i.v.) with IL7-Fc, Fc-TBRII, IL7-TBRII, or an equivalent volume of PBS as a control. We monitored the tumor growth with digital caliper measurements 3 times a week. The tumor volume was calculated with the following formula: 0.5 × Length × Width × Width, where length is the largest diameter and width is the smallest in mm. Mice were euthanized when tumor volume reached 2,000 mm³, in accordance with animal ethics guidelines. For the immunological analysis in tumor-bearing mice, we collected tumor tissue and PB after treatment. For detection of TGF-β1 in serum, serum was collected 2, 6, 24, 48, 72, and 192 h after treatment, and the level of TGF-β1 in serum was measured with a Duoset ELISA kit (Mouse TGF-β1; R&D Systems, Minneapolis, MN, USA). For the multiple treatment study, Jh (C57BL/6) mice were inoculated s.c. with 1×10⁵ MC38 and treated i.v. with IL7-Fc, Fc-TBRII, or IL7-TBRII 3 times at weekly intervals 6-day after tumor inoculation. We measured size of tumors on 2-day after the first treatment of drugs. We harvested tumors on 1-day after the last treatment for flow cytometry analysis. For the memory response study, mice that had completely eliminated MC38 tumors, along with wild-type C57BL/6 controls, were inoculated subcutaneously with 1×10⁵ MC38 cells, and tumor growth was subsequently monitored. For the study of combination therapy, BALB/c mice were inoculated with 5×10⁴ EMT6 into the 4th mammary fat pad and then treated s.c. with IL7-TBRII on day 10 or 11 after inoculation for experiments with anti-CTLA-4 therapy and radiotherapy, respectively. Anti-CTLA-4 therapy (5 mg/kg) was injected intraperitoneally (i.p.) 4 times at 3-day intervals. Tumor irradiation was performed using an X-RAD 320 irradiator (Precision X-Ray Inc., North Branford, CT, USA) operated at 320 KV with a 1 mm cooper filter delivering a 3.5 Gy/min. Mice were anesthetized using i.p. injection of ketamine/xylazine. Mice were placed in lead shielding jigs and exposed tumors received 6 Gy irradiation. For the metastatic study, BALB/c mice were inoculated with 2×10⁴ 4T1 into the 4th mammary fat pad and treated s.c. twice at 3-day intervals with IL7-TBRII on day 9 after inoculation. Lung tissues were collected on 15-day after the last treatment and fixed with Bouin’s solution overnight for the quantification of visible metastatic tumor nodules.

For pharmacokinetics, 8-wk-old male Sprague-Dawley rats were treated s.c. or i.v. with IL7-TBRII, and the concentration of IL7-TBRII was measured by ELISA.

In vitro functional assay for CD8⁺ T cells

Cells isolated from inguinal lymph nodes of C57BL/6 mice were labeled with Cell Tracing Violet (Invitrogen, Waltham, MA, USA) and cultured with 1 μg/ml α-CD3e mAb (InVivoMab™), 10 μg/ml α-CD28 mAb (Tonbo Biosciences, San Diego, CA, USA), 1 ng/ml TGF-β1 (eBioscience, Santa Clara, CA, USA), and 1 nM drugs (PBS, IL7-Fc, Fc-TBRII, and IL7-TBRII). After 72 h, we collected culture supernatant and cells for measurement of IFN-γ with Mouse IFN-gamma DuoSet ELISA (R&D Systems) and flow cytometric analysis, respectively.

Preparation of single cells from tumor, lung, and bone marrow tissues

Tumor and lung tissues were collected, chopped, and digested with 400 Mandl units/ml collagenase D (Roche, Basel, Switzerland) and 2 U/ml DNase I (Roche) for 30 min at 37°C. Digested tumor and lung tissues were dissociated into a single-cell suspension using a 70 μm cell strainer. Tumors were weighed before digestion. Bone marrow cells were harvested by flushing femur and tibia with RPMI 1640 medium 40 (Welgene) containing newborn calf serum (Gibco).

Flow cytometry

Single-cell suspensions were stained with Ghost Dye Violet 510 (Tonbo Biosciences) in PBS to exclude dead cells. Cells were subsequently stained with anti-mouse CD16/32 and fluorescence-conjugated Abs. For intracellular protein staining, cells were fixed and permeabilized with eBioscience™ Fixation/Permeabilization solutions (Invitrogen). For detection of intracellular cytokines, cells were re-stimulated with eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors; Invitrogen) for 3 h at 37°C followed by surface marker staining. Cells were then fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA, USA) for cytokine staining. Stained cells were run through a CytoFLEX LX (Beckman Coulter, Brea, CA, USA), and data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Abs (clone) used for surface staining include CD45 (30-F11), TCRβ (H57-597), CD8α (53-6.7), CD4 (RM4-5), CD62L (MEL-14), CD44 (IM7), B220 (RA3-6B2), Nkp46 (29A1.4), PD-1 (RMP1-30), Tim-3 (RMT3-23), CD11b (M1/70), CD11c (N418), Siglec-F (E50-2440), Ly6G (1A8), Ly6C (HK1.4), c-kit (2B8), Sca-1 (D7), CD127 (A7R34), CD16/32 (93), CD34 (RAM34), and TER-119 (TER-119). Abs (clone) used for the intracellular staining include Foxp3 (FJK-16s), granzyme B (GzmB; GB11), TCF-1 (C63D9), Ki-67 (SolA15), TNFα (MP6-XT22), and IFN-γ (XMG1.2).

RNA extraction and RNA-sequencing analysis

Lung tissues were mechanically homogenized in TRIzol Reagent (Invitrogen). RNA was isolated using TRIzol-chloroform extraction according to the manufacturer’s protocol and shipped to GENINUS Inc. (Seoul, Korea). After quantity and quality check, the library was constructed using a Stranded mRNA prep kit (Illumina, San Diego, CA, USA), paired-end transcriptome sequencing was conducted using the NextSeq550 platform (Illumina), and then FASTQ data were aligned using a STAR (2.7.10a version) by GENINUS Inc. The read count data were processed based on fragments per kb per million reads (FPKM) + Geometric normalization method using EdgeR in R (R Development Core Team, Vienna, Austria, 2020). FPKM values were estimated using Cufflinks (16). Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc., Korea) by Ebiogen Inc.

TGF-β1 signaling luciferase reporter assay

HEK293 cells expressing TGFβ/SMAD Signaling Pathway SBE Reporter (60653; BPS Bioscience, San Jose, CA, USA) were cultured in the manufacturer’s growth medium (79531; BPS Bioscience) supplemented with 400 μg/ml of geneticin (11811031; Invitrogen). On day 1, HEK293 cells (ATCC) were seeded at 96 well plate. On day 2, cells were treated each sample (e.g., IL7-Fc, Fc-TBRII, and IL7-TBRII) and incubated for 4 to 5 h at 37°C in a humidified atmosphere containing 5% CO₂. Following this, human TGF-β1 was added, and the cells incubated for 15 to 18 h under the same conditions. On day 3, luciferase reagent (60690; BPS Bioscience) was added to the cells and incubated for 15 to 30 min at 37°C in a humidified 5% CO₂ atmosphere. Luminescence was then measured, and the results were analyzed using a 4-parameter curve.

Bio-layer interferometry (BLI) assay

The BLI assay was carried out using an Octet K2 instrument (Sartorius, Göttingen, Germany) at 30°C with shaking at 1,000 rpm, utilizing an amine-reactive 2nd generation (AR2G) biosensors (18-5092; Sartorius). The AR2G biosensor were prepared using a buffer including 1 M ethanolamine (pH 8.5), 10 mM acetate buffer (pH 5.0), and 1× kinetics buffer from the AR2G Reagent Kit (18-5095; Sartorius). IL-7Rα (4 µg/ml) or TGF-β (2 µg/ml) was immobilized as the ligand during loading step to measure single binding affinity. TGF-β (2 µg/ml) was immobilized as the ligand in the loading step, and IL-7Rα (1, 2, 4, and 8 µg/ml) were used in association 2 step for double binding affinity measurements. The AR2G biosensor on plate 1 was rehydrated in deionized water for at least 10 min, and loading samples were prepared on plate 2. The loading plate was applied to the procedure of Baseline 1 (distilled water [DW], 60 s), Activation (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide, 300 s), Loading (Ligand, 300 s), Quenching (1 M ethanolamine, 300s), Baseline 2 (1× kinetics buffer, 120 s), Association 1 (GX-72, 300 s) and Dissociation 1 (DW, 300 s) or Association 2 (IL-7Rα, 300 s) following to manufacturer’ instructions (Sartorius). The dissociation constants (K_D) value for binding affinity was analyzed using Octet BLI systems software (ForteBio Data Acquisition and Analysis 9.0; ForteBio, Fremont, CA, USA).

Data collection and scRNA-seq data processing

Public scRNA-seq datasets from Gene Expression Omnibus were re-analyzed to examine TGF-β signaling pathway of tumor-infiltrating CD8⁺ T cells. Seurat (v.5.1.0) R package was used for preprocessing and visualization. For CD8⁺ T cells in MC38 tumors from rhIL7-Fc treated mice, gene expression matrix data were downloaded from GSE237266 (17). For total cells in CT26 tumors from recombinant IL-7 (rIL-7) treated mice, gene expression matrix data were downloaded from GSE205307 (18). We excluded samples based on a previously conducted quality control analysis. Next, these samples were normalized, clustered, and annotated. We calculated IL-7 signaling pathway (BIOCARTA_IL7_PATHWAY, downloaded from https://data.broadinstitute.org/gsea-msigdb/msigdb/biocarta/mouse/m_il7Pathway.gif) and TGF-β signaling pathway (BIOCARTA_TGFB_PATHWAY, downloaded from https://data.broadinstitute.org/gsea-msigdb/msigdb/biocarta/mouse/m_tgfbPathway.gif) of CD8⁺ T cells by using the AddModuleScore function of the Seurat package. Gene Set Enrichment Analysis (GSEA) was conducted fgsea function in R.

Statistical analysis

The statistical analysis was assessed using Prism 9 software (GraphPad Software, San Diego, CA, USA). Data were presented as means ± SEM.

RESULTS

Dual effects of IL-7 cytokine therapy on CD8⁺ tumor-infiltrating lymphocytes (TILs): activation of IL-7 signaling while inducing TGF-β pathway

Through a series of previous studies, we have demonstrated that the antitumor activity of the rhIL-7-hyFc protein (epineptakin-alfa; IL7-Fc) is attributed to the enhancement and activation of CD8⁺ TILs (12). To identify strategies that could further augment the antitumor efficacy of IL7-Fc, we reanalyzed our previous scRNA-seq data from CD8⁺ TILs isolated after IL7-Fc treatment (Supplementary Fig. 1A) (17). In the MC38 colorectal cancer model in C57BL/6 mice, IL7-Fc treatment, compared to buffer-treated controls, led to increased expression of gene sets associated with the IL-7 signaling pathway in CD8⁺ TILs (Supplementary Fig. 1B). Unexpectedly, we also observed an increase in gene sets associated with the TGF-β signaling pathway in CD8⁺ TILs following IL7-Fc treatment, which appeared to be driven by upregulation of TGF-β receptor II (Tgfbr2) (Supplementary Fig. 1C and D). To validate this finding, we reanalyzed scRNA-seq data from another study using rIL-7 in the CT26 colorectal cancer model in BALB/c mice (18). In this dataset, total tumor cells were clustered into cancer cells, stromal cells, and immune cell populations, and transcriptomic changes in tumor-infiltrating T cells were investigated (Supplementary Fig. 1E). Notably, we observed a significant increase in Tgfbr2 expression exclusively in CD8⁺ T cells, compared CD4⁺ T cells (Supplementary Fig. 1F). Consistent with our initial findings, rIL-7 treatment also upregulated gene sets related to both the IL-7 and TGF-β signaling pathways in CD8⁺ TILs in this model (Supplementary Fig. 1G and H).

These results collectively suggest that IL-7 cytokine therapy enhances antitumor immunity by activating the IL-7 signaling pathway in CD8⁺ TILs but concurrently induces the immune-suppressive TGF-β pathway (13,14,19,20). This dual effect raises the possibility that while IL7-Fc promotes CD8⁺ TIL activation, the concurrent upregulation of the TGF-β pathway may counteract this activation by suppressing robust CD8⁺ T cell functionality.

Development and characterization of IL7-TBRII as a dual-function cytokine drug

Considering that TGF-β pathway activation is associated with CD8⁺ TIL dysfunction in tumors from ICB non-responders, our findings highlight the potential of combining IL-7 cytokine therapy with strategies to block the TGF-β pathway to improve antitumor efficacy (21). To address this, we developed IL7-TBRII, a novel bifunctional fusion protein engineered by incorporating a TGF-β trap (Fc-TBRII), extracellular domain of human TGF-βRII, at the C-terminus of the IL7-Fc construct (Fig. 1A; Patent AU2020383176A1). Binding affinity assays demonstrated that IL7-TBRII exhibits high specificity and strong interaction with its target proteins, CD127 (IL-7Rα) and TGF-β1, with sub-nanomolar K_D of 6.911 nM and 0.145 nM, respectively (Fig. 1B and C). Notably, IL7-TBRII retained the ability to simultaneously engage both targets (Fig. 1D), underscoring its dual functional potential. Functional characterization revealed that IL7-TBRII effectively promotes lymphocyte proliferation in B cell lymphoma cultures, though with approximately a two-fold decrease in potency compared to IL7-Fc (Fig. 1E). Additionally, IL7-TBRII robustly inhibited TGF-β signaling, as confirmed by a luciferase reporter assay in HEK293 cells transfected with a SMAD-binding element-driven luciferase gene. The inhibition achieved by IL7-TBRII was comparable to that of the Fc-fused TGF-β trap (Fc-TBRII) construct, further illustrating its capacity to counteract TGF-β1-induced signaling (Fig. 1F).

Figure 1. Binding affinity and functional characteristics of IL7-TBRII. (A) The schematic diagrams of structure of bifunctional fusion protein. (B-D) BLI in the presence of (B) CD127, (C) TGF-β1, or (D) CD127 and TGF-β1. The analytes are IL7-Fc, Fc-TBRII, or IL7-TBRII. (E) Proliferation assay of 2E8 B cell lymphoma in the presence of IL7-Fc or IL7-TBRII. (F) Inhibition of TGF-β1 signaling was measured using luciferase reporter assay in the presence of IL7-Fc, Fc-TBRII, or IL7-TBRII. (G) Number of immune cells in the PB of C57BL/6 mice after treatment of IL7-Fc or IL7-TBRII (n=3–4/group). Each dot indicates the number of cells in each mouse, and bar graph shows mean ± SD. One-way ANOVA with Bonferroni posttests between each group. (H) TGF-β1 levels in the serum of MC38-bearing mice treated with IL7-Fc, Fc-TBRII, or IL7-TBRII (n=3/group). Data are shown as mean ± SD and representative of 2 independent experiments. (I) Pharmacokinetics of IL7-TBRII in serum from Sprague-Dawley rats treated with IL7-TBRII.

EC₅₀, half-maximal effective concentration; IC₅₀, half-maximal inhibitory concentration.

^*p<0.05, ^**p<0.01.

Next, we assessed the pharmacodynamic effects of IL7-TBRII in C57BL/6 mice by administering equimolar doses of IL7-Fc and IL7-TBRII and evaluating changes in immune cell populations in PB 7 days post-treatment. IL7-TBRII effectively expanded T cell subsets (CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, and Tregs) to a comparable extent as IL7-Fc, demonstrating its potential to promote T cell proliferation. In contrast, neither treatment significantly increased NK cells or CD11b⁺ myeloid cells, although IL7-TBRII induced a slight expansion of B cells (Fig. 1G). To evaluate the efficacy of IL7-TBRII in regulating TGF-β1 levels, we administered the IL7-TBRII to MC38 tumor-bearing mice and measured serum TGF-β1 concentrations. Comparative treatments with IL7-Fc and Fc-TBRII were also included. As anticipated, IL7-Fc failed to neutralize TGF-β1, whereas IL7-TBRII successfully reduced TGF-β1 levels, maintaining this effect for up to 120 h post-administration. However, Fc-TBRII exhibited superior neutralizing capability, sustaining efficacy for up to 240 h (Fig. 1H). The pharmacokinetics of IL7-TBRII were characterized by measuring serum concentrations over time following i.v. and s.c. administration in Sprague-Dawley rats. The half-life of IL7-TBRII varied depending on the dose and route of administration, measuring 51.6, 50.0, and 25.6 h for i.v. doses of 1, 3, and 10 mg/kg, respectively, and 23.5 h for a s.c. dose of 10 mg/kg (Fig. 1I, Supplementary Table 1).

We also developed another bifunctional fusion protein, sTBRII-hyFc-rhIL-7 (TBRII-IL7), which features a TGF-β trap at the N-terminus of hyFc and rhIL-7 at the C-terminus (Supplementary Fig. 2A). Comparative analysis of the two bifunctional fusion proteins revealed that TBRII-IL7 promoted B cell proliferation to a similar extent as IL7-TBRII but was 4.5 times less effective in suppressing TGF-β1 signaling (Supplementary Fig. 2B and C). In C57BL/6 mice, both proteins enhanced CD8⁺ T cell expansion; however, IL7-TBRII induced a significantly greater increase in CD8⁺ T cell numbers (Supplementary Fig. 2D). Furthermore, in MC38 tumor-bearing mice, IL7-TBRII maintained lower serum TGF-β1 levels for a slightly longer duration compared to TBRII-IL7 (Supplementary Fig. 2E). Based on these findings, IL7-TBRII was prioritized for further experiments due to its superior functional performance.

IL7-TBRII restores CD8⁺ T cell function and enhances anti-tumor efficacy through TGF-β modulation

Because TGF-β signaling suppresses cytolytic activity, proliferation, and differentiation of CD8⁺ T cells into effector states upon stimulation, we evaluated whether IL7-TBRII could counteract the inhibitory effects of TGF-β1 and restore CD8⁺ T cell functionality (22,23). CD8⁺ T cells were stimulated with anti-CD3/CD28 in the presence of recombinant TGF-β1 and treated with IL7-Fc, Fc-TBRII, or IL7-TBRII. As expected, IL7-Fc failed to reverse the robust inhibition of CD8⁺ T cell proliferation caused by TGF-β1. In contrast, both IL7-TBRII and Fc-TBRII effectively restored CD8⁺ T cell proliferation by blocking TGF-β signaling (Fig. 2A and B). Furthermore, the suppression of key effector molecules in cytotoxic CD8⁺ T cells, including IFN-γ and GzmB, by TGF-β1 was reversed by both IL7-TBRII and Fc-TBRII. Notably, IL7-TBRII significantly enhanced IFN-γ secretion to a greater extent than Fc-TBRII (Fig. 2C and D). These results demonstrate that IL7-TBRII effectively counteracts the inhibitory effects of TGF-β1, restoring CD8⁺ T cell proliferation, enhancing effector differentiation, and upregulating the secretion of effector molecules.

Figure 2. IL7-TBRII counteracted TGF-β1 effect on CD8⁺ T cells and delayed tumor growth. (A-D) Functional assays for CD8⁺ T cells in the presence of IL7-Fc, Fc-TBRII, or IL7-TBRII. (A) Proliferation of gated CD8⁺ T cells in culture was shown as representative histograms of CTV dilution profiles. (B) Frequency of proliferating CD8⁺ T cells (left) and proliferation index (right) calculated with FlowJo software. Each dot indicates the value of triplicates. (C) Level of IFN-γ in the culture supernatant measured by ELISA in triplicates. (D) Frequency of CD8⁺ T cells expressing GzmB measured by flow cytometry in triplicates. (E) MC38-bearing C57BL/6 mice were treated with IL7-Fc, Fc-TBRII, or IL7-TBRII. Average tumor size (n=9/group). (F-G) MC38-bearing Jh (C57BL/6) mice were repetitively treated with IL7-Fc, Fc-TBRII, and IL7-TBRII once a week for 3 weeks. (F) Average tumor growth curve, and (G) survival curve (n=8-9/group). (H) Long-term memory response. Each dot indicates an individual mouse. Two-way ANOVA (E, F) or one-way ANOVA (B-D) with Bonferroni posttests or or simple survival analysis (G) between each group. Data are shown as mean ± SD and representative of 2 independent experiments.

CTV, CellTrace Violet.

^*p<0.05, ^**p<0.01, ^***p<0.001, and ^****p<0.0001.

To evaluate the anti-tumor efficacy of IL7-TBRII, we administered the drug to MC38 tumor-bearing mice when tumors became palpable. A single dose of IL7-TBRII significantly reduced tumor growth compared to PBS but did not show superior efficacy over IL7-Fc or Fc-TBRII (Fig. 2E). To enhance its therapeutic potential, we explored a multiple-dosing regimen. Given that serum TGF-β1 levels returned to baseline 7 days after a single IL7-TBRII treatment (Fig. 1H), we administered IL7-TBRII three times at 7-day intervals. As IL7-TBRII contains human protein sequences, it has the potential to induce anti-drug Ab formation in mice, which could impair its efficacy upon repeated dosing. To address this, treatments were conducted in Jh-deficient C57BL/6 mice, which lack Ab-producing B cells. We observed delayed tumor growth in the multiple-dosing group compared to PBS and Fc-TBRII (Fig. 2F). Notably, IL7-TBRII outperformed IL7-Fc in extending survival, achieving complete tumor regression in 5 out of 9 mice (55%) (Fig. 2G). To assess long-term immunity, MC38 cells were reintroduced into mice that had achieved complete tumor regression following IL7-TBRII treatment. In 60% of the mice, implanted tumors did not establish, indicating that IL7-TBRII treatment elicited a durable long-term memory response (Fig. 2H). Together, these findings underscore the ability of IL7-TBRII to enhance anti-tumor efficacy beyond IL7-Fc by simultaneously modulating IL-7 and TGF-β signaling pathways.

Enhanced anti-tumor efficacy of IL7-TBRII is induced by the increased number of terminally differentiated CD8⁺ T cells in tumors

We hypothesized that the anti-tumor efficacy of IL7-TBRII might be driven by changes in the immune cell composition. To evaluate the immunological changes in tumors after IL7-TBRII treatment, we harvested tumors one day after the final administration (Fig. 3A). IL7-TBRII treatment increased the overall proportion of T cells (CD8⁺ T cells, non-Treg CD4⁺ T cells, and Tregs) in the tumors, accompanied by a numerical increase in CD8⁺ T cells and non-Treg CD4⁺ T cells. In contrast, among myeloid-lineage cells, the proportion of dendritic cells decreased, and both the proportion and number of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were significantly reduced (Fig. 3B and C). The immune cell composition within tumors after treatment with IL7-TBRII and IL7-Fc exhibited similar trends, characterized by an increase in lymphoid cells and a decrease in myeloid cells. Notably, both IL7-TBRII and IL7-Fc led to a substantial increase in the number of CD8⁺ T cells within tumors compared to PBS. This increase may partly be attributed to either enhanced proliferation of CD8⁺ T cells within the tumors or increased infiltration from the periphery. To assess the proliferative capacity of activated CD8⁺ TILs (PD-1⁺CD8⁺ TILs), we analyzed Ki-67 expression. Neither IL7-TBRII nor IL7-Fc showed a significant difference compared with the PBS group, whereas Fc-TBRII increased the proliferation of PD-1⁺CD8⁺ TILs compared with the PBS and IL7-Fc groups, but not with the IL7-TBRII group (Fig. 3D). This result indicates that enhanced T cell infiltration, rather than local proliferation, may have contributed significantly to the increased CD8⁺ T cell abundance in the tumor. CD8⁺ T cells activated in the tumor-draining lymph nodes (TdLNs) exit the lymph nodes, travel through the vasculature, and infiltrate the tumor tissue (17,24,25). These infiltrating CD8⁺ T cells are typically characterized by high CD44 expression. Consistent with this, we observed a significant increase in the number of CD44⁺CD8⁺ T cells in tumors treated with IL7-TBRII or IL7-Fc (Fig. 3E).

Figure 3. IL7-TBRII treatment increased the number of cytotoxic CD8⁺ T cells in tumors. (A) Experimental scheme of multiple treatment in MC38-bearing Jh (C57BL/6) mice. (B-F) Flow cytometric analysis with tumor-infiltrating immune cells (n=5/group). Each dot indicates the frequency or number of immune cell subsets in each mouse. (B) The frequency of immune cell subsets among CD45⁺ cells in tumors. (C) The number of immune cell subsets in tumors. Each dot indicates the number of immune cell subset in each mouse. (D) The frequency of Ki-67-expressing cells in PD-1⁺CD8⁺ T cells in tumors. (E) The number of CD44⁺CD8⁺ T cells in tumors. (F) The number of PD-1⁺CD8⁺ T cells (left) and PD-1⁻CD8⁺ T cells (right) in tumors. (G) PD-1⁺CD8⁺ T cells gated with indicated markers were shown as representative plot of flow cytometric analysis. (H) Each dot indicates the frequency (left) and number (right) of PD-1⁺TCF-1⁻Tim3⁺ (terminally differentiated) CD8⁺ T cells in tumors. (I) Mean fluorescence intensity of GzmB in terminally differentiated CD8⁺ T cells. One-way ANOVA (B-F, H, and I) with Bonferroni posttests between each group. Data are shown as mean ± SD and representative of 2 independent experiments.

TAM, tumor-associated macrophages; DC, dendritic cells; M-MDSC, monocytic myeloid-derived suppressor cell.

^*p<0.05, ^**p<0.01, ^***p<0.001, and ^****p<0.0001.

Within tumors, we further analyzed tumor-infiltrating CD8⁺ T cells to distinguish tumor-reactive CD8⁺ T cells (PD-1⁺) from bystander CD8⁺ T cells (PD-1⁻). Both IL7-Fc and IL7-TBRII treatments increased the number of tumor-reactive CD8⁺ T cells (Fig. 3F). As previously observed, IL7-Fc treatment also significantly increased the number of bystander CD8⁺ T cells (17). Considering that, among tumor-reactive CD8⁺ T cells, terminally differentiated CD8⁺ T cells (PD-1⁺TCF-1⁻Tim-3⁺) are known to express higher levels of the cytotoxic molecule, GzmB, and involved in tumor clearance, we analyzed the proportion or number of terminally differentiated CD8⁺ T cells (26). Fc-TBRII treatment significantly increased the proportion of terminally differentiated CD8⁺ T cells, a phenomenon potentially explained by the inhibitory role of TGF-β signaling in the differentiation of these cells (27). Although IL7-TBRII treatment showed a slight increase in the proportion of terminally differentiated CD8⁺ T cells compared to PBS and IL7-Fc, the difference was not statistically significant. However, the total number of terminally differentiated CD8⁺ T cells was significantly higher in the IL7-TBRII group than in the PBS and Fc-TBRII-treated groups (Fig. 3G and H). Finally, we compared the expression of GzmB of terminally differentiated CD8⁺ T cells, which showed no significant differences (Fig. 3I). These findings suggest that IL7-TBRII enhances the infiltration of CD44⁺CD8⁺ T cells into tumors and promotes their differentiation into terminally differentiated CD8⁺ T cells through inhibition of TGF-β signaling. The increased number of terminally differentiated CD8⁺ T cells in tumors is likely associated with the superior antitumor efficacy of IL7-TBRII.

Anti-metastatic effect of IL7-TBRII

Metastasis is a major contributor to cancer-related mortality (28,29). Therefore, in addition to controlling tumor growth, regulating metastasis is a crucial objective of anti-tumor therapies. Studies have demonstrated that the cytokine TGF-β1 promotes metastasis by inducing epithelial-to-mesenchymal transition in primary tumors and by modulating stromal cells within metastatic niches (30,31,32). Beyond TGF-β1, immune cells within metastatic niches also play an essential role in the metastatic process (33,34,35,36,37). Thus, preventing metastasis requires modulating both TGF-β signaling and the immune microenvironment of metastatic niches. Since IL7-TBRII can regulate both TGF-β1 levels and immune cell composition, we hypothesized that IL7-TBRII possesses anti-metastatic properties. To test this hypothesis, we established a spontaneous lung metastasis model using the triple-negative breast cancer (TNBC) cell line 4T1 and administered IL7-TBRII twice (Fig. 4A). We observed significant inhibition of breast cancer lung metastasis in the IL7-TBRII-treated group compared to the control group (Fig. 4B).

Figure 4. IL7-TBRII inhibits lung metastasis in 4T1 orthotopic breast cancer mouse model. (A) The experimental scheme for evaluating anti-metastatic effect of IL7-TBRII (n=12/group). (B) Each dot indicates the number of lung nodules in each mouse. (C-F) Transcriptomic analysis of metastatic lung tissues. The metastatic lung tissues were analyzed by RNA-sequencing (n=3/group). (C) Principal component analysis of RNA sequencing. (D) Heatmap of differentially expressed genes associated with “KEGG: TGF-beta signaling pathway – mus musculus” based on RNA-seq data. (E) Top 5 significantly enriched biological process GO terms analyzed with upregulated and downregulated genes from IL7-TBRII-treated metastatic lung tissues 4 days post-treatment. (F) Top 5 significantly enriched biological process GO terms analyzed with upregulated and downregulated genes from IL7-TBRII-treated metastatic lung tissues 7 days post-treatment. (G-K) Flow cytometric analysis with immune cells in metastatic lung tissues and bone marrow. (G) The graph indicates the number of CD8⁺ T cells in metastatic lung cancer. (H) The graph indicates the number of effector CD8⁺ T cells in each mouse at indicated time points. (I) CD8⁺ T cells gated with indicated markers were shown as representative plot of flow cytometric analysis (left). Each dot indicates the number of IFN-γ⁺TNF-α⁺CD8⁺ T cells in each mouse at indicated time points (right). (J) Each dot indicates the number of PMN-MDSC (CD45⁺CD11b⁺Ly6G⁺) in each mouse. (K) The graphs indicate the number of GMP cells (Lin-Sca1-cKit⁺CD34⁺CD16/32⁺, dashed line) and CD11b⁺Ly6G⁺ cells (solid line) in each mouse. One-way ANOVA (B, H, and I) and 2-way ANOVA (G, J, and K) with Bonferroni posttests between each group. Data are shown as mean ± SD.

KEGG, Kyoto Encyclopedia of Genes and Genomes.

^*p<0.05, ^**p<0.01, ^***p<0.001, and ^****p<0.0001.

To investigate the changes in the metastatic lung microenvironment after IL7-TBRII treatment, we conducted transcriptomic analysis on isolated metastatic lung tissues (Fig. 4A). Principal component analysis revealed that the IL7-TBRII-treated samples formed a distinct cluster, separated from the PBS-treated samples (Fig. 4C). First, to determine whether IL7-TBRII effectively regulates TGF-β signaling in metastatic lung tissues, we compared the expression of genes associated with the TGF-β signaling pathway. Although the expression of Tgfb1 was upregulated in the IL7-TBRII-treated group on day 4, this increase was diminished by day 7 post-treatment (Fig. 4D). Given our previous findings on the ability of IL7-TBRII to neutralize TGF-β1, we hypothesized that this transient increase in Tgfb1 might be counter-regulated by IL7-TBRII. In addition to this effect, genes known to suppress TGF-β signaling (Fst, Tgif1, Myc, and Smad7) were upregulated, while genes promoting TGF-β signaling (Bmp6, Ltbp1, Bmpr1a, Smad9, Lrrc32, and Bmp5) were downregulated in the IL7-TBRII-treated group on day 4 (Fig. 4D). These results indicate that despite a transient increase in Tgfb1, the overall TGF-β signaling pathway may be impaired in metastatic lung tissues following IL7-TBRII treatment. Next, we performed Gene Ontology (GO) term analysis to compare biological processes between PBS- and IL7-TBRII-treated groups over time. On day 4, IL7-TBRII treatment upregulated genes involved in cell proliferation, such as those regulating the mitotic cell cycle and metaphase/anaphase transitions, while downregulating genes associated with blood vessel development and myeloid leukocyte migration (Fig. 4E). By day 7, IL7-TBRII treatment upregulated genes related to leukocyte activation and lymphocyte activation while downregulating genes associated with neutrophil chemotaxis (Fig. 4F). These findings suggest that IL7-TBRII can modulate immune cell infiltration within metastatic lung tissues.

To further characterize changes in immune cell composition, we performed flow cytometric analysis on metastatic lung tissues (Fig. 4A). We observed increased infiltration of CD8⁺ T cells, including effector CD8⁺ T cells, in metastatic lung tissues (Fig. 4G and H). Additionally, the cytolytic function of CD8⁺ T cells was enhanced, as evidenced by increased expression of IFN-γ and TNF-α (Fig. 4I). Conversely, the infiltration of pro-metastatic immune cells, PMN-MDSCs, was reduced in metastatic lung tissues (Fig. 4J). Based on previous studies demonstrating that IL7-Fc regulates myelopoiesis in the bone marrow, we further analyzed bone marrow cells (12). We found that the number of granulocyte-monocyte progenitors and CD11b⁺Ly6G⁺ cells decreased following IL7-TBRII treatment (Fig. 4K). Collectively, these findings suggest that IL7-TBRII effectively inhibits breast cancer lung metastasis by modulating the TGF-β signaling and the immune cell composition in metastatic lung niches.

Synergetic anti-tumor effect of IL7-TBRII with radiotherapy and ICB

We next investigated whether IL7-TBRII could enhance the effectiveness of other anti-tumor therapies through combination strategies. We selected radiotherapy and anti-CTLA-4 therapy (αCTLA-4 mAb), both of which enhance anti-tumor efficacy by increasing lymphocyte activation within tumors and TdLNs (38,39,40). However, it has been reported that TGF-β1 diminishes the efficacy of these therapies by impairing CD8⁺ T cell priming in TdLNs and establishing a fibrotic niche within tumors (41,42,43). Consequently, TGF-β1 hinders both CD8⁺ T cell activation and the infiltration of these cells into tumors. Given that IL7-TBRII enhances adaptive immune responses and modulates TGF-β signaling in both tumors and TdLNs, we hypothesized that combining IL7-TBRII with radiotherapy or anti-CTLA-4 therapy could produce a synergistic effect. To test this hypothesis, we utilized a TNBC mouse model using EMT6 cells, which are associated with a fibrotic tumor (44,45). For the radiotherapy experiment, we concurrently administered IL7-TBRII and radiotherapy to EMT6-bearing mice, and observed significant anti-tumor efficacy exclusively in the combination therapy groups (Fig. 5A and B). In a separate experiment, we evaluated the combination of IL7-TBRII with anti-CTLA-4 therapy. IL7-TBRII was administered once, while anti-CTLA-4 therapy was administered 4 times at 3-day intervals in EMT6-bearing mice (Fig. 5C). The group receiving combination therapy exhibited the highest anti-tumor efficacy compared to those receiving either treatment alone (Fig. 5D and E). These results demonstrate that IL7-TBRII can serve as a valuable partner in radiotherapy and anti-CTLA-4 immunotherapy.

Figure 5. IL7-TBRII shows a synergetic effect with radiotherapy or anti-CTLA-4 therapy. (A,B) Combination therapy of IL7-TBRII with radiation therapy. EMT6-bearing BALB/c mice were subcutaneously treated with rhIL-7-hyFc-sTBRII and received radiation therapy. (A) Average-, and (B) individual-tumor growth curve (n=10–11/group). (C-E) Combination therapy of IL7-TBRII with αCTLA-4 mAb (n=11–12/group). (C) Experimental scheme of combination therapy. (D) Average-, and (E) individual-tumor growth curve. Two-way ANOVA (A and D) with Bonferroni posttests between each group. Data are shown as mean ± SD and representative of 2 independent experiments (A-E).

^***p<0.001 and ^****p<0.0001.

DISCUSSION

The presence of cytotoxic CD8⁺ T cells within tumors correlates strongly with favorable clinical outcomes in cancer patients (5). Current immunotherapeutic strategies prioritize increasing CD8⁺ T cell populations and enhancing their tumor infiltration. Despite promising preclinical results, cytokine-based therapies have demonstrated limited clinical efficacy due to inhibitory mechanisms mediated by immunosuppressive cytokines and immune checkpoint molecules within the tumor microenvironment, which often render CD8⁺ T cells dysfunctional upon infiltration (46,47). Therefore, additional strategies incorporating bifunctional molecules, which combine cytokines with moieties that counteract the immunosuppressive tumor microenvironment, are necessary to enhance therapeutic efficacy.

We previously developed IL7-Fc, an IL-7-based cytokine therapy, which effectively promoted tumor clearance by expanding intratumoral CD8⁺ T cells (12). However, our analysis of sc-RNAseq datasets from MC38 and CT26 colorectal cancer models demonstrated significant upregulation of TGF-β signaling genes in CD8⁺ T cells following IL-7 treatment (17,18). Given established evidence that TGF-β signaling suppresses IL-7 activity and induces T cell dysfunction, we reasoned that enhanced TGF-β signaling post-IL-7 therapy limits therapeutic efficacy. Although the detailed mechanisms underlying this phenomenon were not elucidated in the present study, to better understand the immunological effects of IL7-Fc, it will be important to elucidate the mechanisms by which IL-7 enhances TGF-β signaling in CD8⁺ T cells.

Repeated IL7-TBRII administration resulted in significantly improved anti-tumor efficacy compared to IL7-Fc alone, leading to tumor regression in approximately half of the treated mice. Mechanistically, IL7-TBRII notably expanded cytotoxic CD8⁺ T cell subsets within tumors, likely recruited and activated via TdLNs. Although the correlation between TdLN-activated CD8⁺ T cells and tumor-infiltrating cytotoxic T cells is established, the TGF-β1-mediated suppression of CD8⁺ T cell activation in TdLNs poses a significant barrier (24,25,26,48). Notably, IL-7Rα expression is enriched in lymph nodes, as reported by the Human Protein Atlas, suggesting preferential accumulation of IL7-TBRII in these critical immune priming sites. Indeed, our in vitro findings confirmed IL7-TBRII effectively drove effector differentiation of CD8⁺ T cells even in the presence of TGF-β1, enabling their effective tumor infiltration and cytotoxic activity. Thus, we suggests that the IL7-TBRII promotes T cell activation in TdLNs and these cells migrate to tumor tissues and play a critical role in tumor clearance.

Beyond primary tumor control, IL7-TBRII displayed notable anti-metastatic activity in a breast cancer lung metastasis model. Transcriptomic analyses indicated decreased TGF-β signaling and immune modulation within metastatic niches. Treatment with IL7-TBRII significantly enhanced lung infiltration by effector CD8⁺ T cells and increased their pro-inflammatory cytokine production while concurrently reducing pro-metastatic PMN-MDSC accumulation through modulation of bone marrow hematopoiesis. Furthermore, IL7-TBRII’s inhibition of TGF-β signaling may influence stromal cells, including fibroblasts and endothelial cells, which are critical for extracellular matrix remodeling and angiogenesis within pre-metastatic niches (31,49,50). Consistently, our transcriptomic data revealed downregulation of angiogenesis-associated genes, underscoring IL7-TBRII’s potential in limiting tumor-associated vascularization.

Recently, bispecific Abs such as bintrafusp alfa (PD-L1×TBRII) and dalutrafusp alfa (CD73×TBRII), incorporating TGF-β traps linked to Abs targeting tumor Ags, have entered clinical trials (51,52). IL7-TBRII distinctively combines TBRII with IL-7 cytokine, targeting immune cells and preferentially localizing to T-cell enriched organs and lymphoid tissues such as TdLNs, where IL-7Rα is abundantly expressed. This unique localization promotes effective priming and expansion of tumor-specific CD8⁺ T cells by locally attenuating TGF-β signaling. Given that therapeutic modalities such as radiotherapy and immune checkpoint inhibitors critically depend on effective T cell priming—often suppressed by TGF-β signaling—IL7-TBRII provides a strong rationale for clinical application in combination with other anti-cancer therapies (38,39,40). Collectively, our findings highlight IL7-TBRII as a promising dual cytokine-modulating agent that can enhance T cell–mediated anti-tumor immunity when used in rational combination regimens.

Abbreviations

AR2G

amine-reactive 2nd generation

BLI

bio-layer interferometry

DW

distilled water

FPKM

fragments per kb per million reads

GMP

granulocyte-monocyte progenitor

GO

Gene Ontology

GSEA

Gene Set Enrichment Analysis

GzmB

granzyme B

i.p.

intraperitoneally

i.v.

intravenously

ICB

immune-checkpoint blockade

KD

dissociation constants

mIL-7

mouse IL-7

ns

not significant

PB

peripheral blood

PCA

principal component analysis

PMN-MDSCs

polymorphonuclear myeloid-derived suppressor cells

rIL-7

recombinant IL-7

rhIL-7

recombinant human IL-7

s.c.

subcutaneously

scRNA-seq

single-cell RNA-sequencing

TdLN

tumor-draining lymph node

Tgfbr2

TGF-β receptor II

TIL

tumor-infiltrating lymphocyte

TNBC

triple-negative breast cancer

SUPPLEMENTARY MATERIALS

Supplementary Table 1

Data of BLI and pharmacokinetics of IL7-TBRII

Supplementary Figure 1

Treatment of IL-7 therapy enhanced the gene signature associated with the TGF-β signaling pathway in tumor-infiltrating CD8⁺ T cells. (A-D) Public scRNA-seq dataset (GSE237266) analysis. (A) UMAP plot of scRNA-seq data colored by treatment. (B) Violin plot showing module score of IL7 signaling pathway (left) and GSEA analysis with the gene set of BIOCARTA: IL7_PATHWAY (right) in tumor-infiltrating CD8⁺ T cells. (C) Violin plot showing module score of TGF-β signaling pathway (left) and GSEA analysis with the gene set of BIOCARTA: TGFB_PATHWAY (right) in tumor-infiltrating CD8⁺ T cells. (D) Violin plots (left) and dot plot (right) with an expression of Tgfbr2 in tumor-infiltrating CD8⁺ T cells. (E-H) Public scRNA-seq dataset (GSE205307) analysis. (E) UMAP plot of scRNA-seq data colored by cell type. (F) Violin plots with an expression of Tgfbr2 in CD4⁺ T cells, CD8⁺ T cells, and Tregs in CT26 tumors. (G) GSEA analysis with the gene set of BIOCARTA: IL7_PATHWAY in tumor-infiltrating CD8⁺ T cells. (H) GSEA analysis with the gene set of BIOCARTA: TGFB_PATHWAY in tumor-infiltrating CD8⁺ T cells.

Supplementary Figure 2

Comparison of the functionality between IL7-TBRII and TBRII-IL7. (A) The schematic diagrams of structure of bifunctional fusion proteins (left) and amino acid sequence of IL7-TBRII (right). (B) Proliferation assay with 2E8 B cell lymphoma in the presence of IL7-TBRII or TBRII-IL7. (C) Inhibition of TGF-β1 signaling was measured using luciferase reporter assay in the presence of IL7-TBRII or TBRII-IL7. (D) Flow cytometric analysis with CD8⁺ T cells in PB (n=4/group). C57BL/6 mice bearing MC38 were subcutaneously treated with IL7-TBRII or TBRII-IL7. The number of CD8⁺ T cells at indicated time points after treatment (left) or on day 7 (right) after treatment (n=4/group). Data are shown as mean ± SD. Each dot (right) indicates the number of CD8⁺ T cells in each mouse. One-way ANOVA with Bonferroni posttests between each group. (E) TGF-β1 levels in the serum of MC38-bearing mice treated with IL7-TBRII, or TBRII-IL7 (n=4/group). Data are shown as mean ± SD.