Catalpol Reactivates IL-7R–STAT5 Signaling and Reverses Radiation-Induced T Helper 1 Cells Deficiency to Limit Melanoma Metastasis to Lung

Abstract

Background:

Radiation-induced quantitative deficiency of T helper 1 (Th1) cells accelerates lung tumor development. Here, we evaluated IL-7R–STAT5 signaling in Th1 recovery after irradiation and examined how catalpol contributes to Th1 reconstitution by targeting this signaling axis in mice.

Methods:

The recovery characteristics of irradiated Th1 cells, compared with other IFN-γ-producing (IFN-γ⁺) cells, were analyzed in local radiotherapy (LRT) mice challenged with lung melanoma and single low-dose total body irradiation (SLTBI) mice without tumor. IL-7R–STAT5 signaling of Th1 from LRT mice, treated with or without catalpol, was evaluated using flow cytometry and RT-qPCR. The role of IL-7–IL-7R–STAT5 signaling in irradiated Th1 cells was further confirmed in irradiated EL4 cells following administration of IL-7 or catalpol.

Results:

Th1 subsets showed delayed recovery in both LRT and SLTBI mice compared with other IFN-γ⁺ cells, and irradiated Th1 cells exhibited decreased activation of IL-7R–STAT5 signaling. Quantitative reduction of Th1 cells increased lung metastasis of B16 melanoma in irradiated mice. Irradiated EL4 cells also displayed decreased IL-7R–STAT5 signaling and reduced IFN-γ production in vitro. Treatment with catalpol could restore IL-7R–STAT5 signaling and promote Th1 recovery following irradiation both in vivo and in vitro. Catalpol-mediated restoration of IL-7R–STAT5 signaling in Th1 provided superior protection against B16 melanoma metastasis to the lung.

Conclusions:

IL-7R–STAT5 signaling is required for Th1 recovery after irradiation. Catalpol effectively promotes Th1 cell reconstitution and decreases B16 melanoma metastasis to the lung by enhancing IL-7R–STAT5 signaling. These findings provide new strategies for improving radiotherapy and immunotherapy outcomes in cancer.

Introduction

Radiotherapy (RT) is a common component of anti-cancer strategies. ¹ It is widely applied across various clinical settings, including the management of lung metastases, ² yet its effects on the lung immune microenvironment remain to be illustrated. ³ Although advances in RT delivery have minimized damage to normal tissues, irradiation of the surrounding normal tissue is common during RT. ⁴ Moreover, the circulatory and migratory properties of lymphocytes make it difficult to avoid RT-induced injuries, which affects the overall treatment efficacy. ⁵ Thus, the rapid recovery of effective anti-tumor lymphocytes after ionizing radiation (IR) is essential to maintain sufficient cells for tumor clearance and to minimize the risk of tumor metastasis. The lungs contain abundant lymphocytes that maintain immune diversity, which is required for pulmonary immune homeostasis. During innate immune responses, interferon-gamma (IFN-γ) facilitates the activation of macrophages, type 1 innate lymphoid cells (ILC1s), and natural killer cells (NK); induces high expression of major histocompatibility complex (MHC) class I and II molecules by antigen-presenting cell (APC); drives dendritic cell (DC) expansion; and orchestrates a comprehensive anti-tumor innate immune response, especially in the early stages of tumor development.^{6 -8} During adaptive immunity, IFN-γ promotes antibody production by B cells, accelerates T helper 1 (Th1) polarization, and facilitates cytotoxic T cell (Tc) activation and memory T cell (Tm) expansion, thereby enhancing specific and durable anti-tumor immunity essential for controlling tumor metastasis.^5,9,10 Furthermore, the anti-tumor effects of IFN-γ-producing (IFN-γ+) cells also rely on other simultaneously secreted cytokines, such as granzymes, interleukin (IL)-17A, and perforin, to exert their overall anti-tumor activity. ¹¹ Accordingly, in addition to IFN-γ production, the number of IFN-γ⁺ cells can better reflect the overall anti-tumor capacity. ¹² Therefore, controlling tumor progression or metastasis after irradiation depends on the total pool of IFN-γ⁺ cells from both local and non-irradiated lymphoid organs or tissues that can replenish the tumor microenvironment (TME). Among IFN-γ⁺ cells, Th1, Tc1, NK1, and natural killer T type 1 (NKT1) cells are the main cellular sources, accounting for 80% to 90% of total IFN-γ. ¹³ However, the response and recovery characteristics of these cell types, particularly in the lungs, have not been well defined. Thus, characterizing the recovery features of these cells after irradiation is important for optimizing the efficacy of immunotherapy and RT.

Cytokines such as IL-7, IL-12, IL-15, and IL-18 are required for the maintenance and enhancement of IFN-γ⁺ cells, depending on the expression of their respective cytokine receptors and the activation of transcriptional signals, such as signal transducer and activator of transcription (STATs). ¹⁴ The phenotypes of these cytokine receptors on Th1, Tc1, NKT1, and NK1 following irradiation must be characterized, which presumably influence their reconstitution from irradiation, particularly in the lung TME.

Rehmannia Radix Praeparata (RRP), a tonic traditional Chinese medicine, is commonly prescribed to modulate immunological dysfunction in patients with cancer and has possible therapeutic effect on ischemic stroke, cisplatin-induced nephrotoxicity, and sensitivity to chemotherapy. RRP contains several bioactive components, including catalpol.^15,16

This study was conducted to analyze the recovery status and cytokine receptor phenotypes of Th1 cells compared with other IFN-γ⁺ cells in the lungs and spleen following irradiation. Based on cellular and animal model experiments, the role of IL-7R–STAT5 signaling in Th1 reconstitution in response to irradiation, tumor development, and treatment with catalpol was also identified. Our results showed that catalpol can promote Th1 proliferation after low-dose irradiation, but the mechanisms of catalpol on Th1 reconstitution from RT remain to be elucidated.

Materials and Methods

Animals

Specific pathogen-free (SPF) female C57BL/6 mice (6-8 weeks old, RRID:IMSR_JAX:000664) were obtained from an accredited laboratory animal center of Shandong First Medical University (Jinan Pengyue Jinan Pengyue Animal Breeding Center) and housed under controlled environmental conditions (22°C ± 2°C, 50%-60% humidity, 12 h light/dark cycle) with free access to food and water. Mice were acclimatized for at least 1 week before experimentation. All procedures were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Shandong Provincial Hospital Affiliated to Shandong First Medical University(No.NSFC:NO.2020-1107). Animals were randomly assigned to experimental groups. All efforts were made to minimize suffering and reduce the number of animals used.

-X-Ray Irradiation

Irradiation with 160 kV, 25 mA X-rays was performed using a Rad Source RS 2000 Pro X-ray Irradiator (RS 2000 Pro, Cat# RS2000, Rad Source Technologies, Buford, GA, USA). For single low-dose total body irradiation (SLTBI), anesthetized mice were irradiated with a single dose of 2.5 Gy (dose rate 1.3 Gy/min, 0.3 mm copper filtration, distance from the X-ray source to the target: 30 cm). SLTBI mice were sacrificed 2, 4, and 8 days after exposure to analyze IFN-γ⁺ cells. For local radiotherapy (LRT), each mouse, with or without tumor burden, was anesthetized, and the breast was exposed to a lead box to receive 2 × 8 Gy irradiation. SLTBI (2.5 Gy) was employed to evaluate systemic immune reconstitution under non–tumor-bearing conditions and to assess recovery kinetics of IFN-γ⁺ lymphocytes. In contrast, LRT (2 × 8 Gy) was used to simulate clinically relevant local radiotherapy for lung tumors and to examine immune alterations within the tumor microenvironment. These protocols were designed to distinguish systemic immune recovery from localized radiation-induced immune modulation. ¹⁷

Induction of Lung B16 Melanoma and Treatment With Catalpol

Female C57BL/6 mice were intravenously injected with B16 melanoma cells (2 × 10⁵, National Collectionof Authenticated Cell Cultures, RRID:SCSP-5233). Some mice were irradiated with LRT on days 7 and 8 after tumor induction. Tumor-bearing mice that underwent LRT were either administered catalpol (5 mg/kg/day, intraperitoneally; Cat# HY-N0820, MCE, China) for 7 days after LRT until the end of the experiment, or left untreated. To study the effects of SLTBI, mice were intravenously injected with B16 melanoma cells 4 days after irradiation, and the lung metastatic tumor clone was examined 10 days after B16 melanoma injection.

Cell Culture and Irradiation

EL4 murine T-lymphoma cells (RRID:SCSP-5221), human embryonic kidney 293T cells (RRID:SCSP-502), and B16-F10 mouse melanoma cells (RRID:SCSP-5233) were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). All cell lines were authenticated by the supplier using Short Tandem Repeat (STR) profiling and were routinely tested to confirm absence of mycoplasma contamination. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cat# 11965-092, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 IU/ml penicillin–streptomycin (Cat# 15140-122, Gibco), 2 mM L-glutamine (Cat# 25030-081, Gibco), and 10% heat-inactivated fetal bovine serum (Cat# 10099-141, Gibco, USA) at 37°C in a humidified incubator containing 5% CO₂.

EL4 cells were treated with catalpol (50 μM; Cat# HY-N0820, MCE, China), a concentration selected based on preliminary dose–response experiments and previous reports demonstrating effective modulation of intracellular signaling without cytotoxicity.^18,19 The 1 Gy dose was selected to model moderate radiation-induced injury that permits partial T cell recovery, whereas 4 Gy was used to represent more severe irradiation-induced damage, ²⁰ enabling evaluation of dose-dependent effects on IL-7R–STAT5 signaling and IFN-γ production.

Flow Cytometry

Lung tissues were digested using a Tumor Dissociation Kit (Cat# 130-095-929, Miltenyi Biotec, Germany) and processed with a gentleMACS™ Dissociator (Cat# 130-093-235, Miltenyi Biotec). After red blood cell lysis, single cells were blocked with anti-CD16/32 FcR block (Cat# 101320, RRID:AB_1574975, BioLegend, USA) and stained with fluorescently labeled antibodies: APC-Cy7-CD45 (Cat# 103116, RRID:AB_312981, BioLegend), PerCP-Cy5.5-CD3 (Cat# 100328, RRID:AB_893325, BioLegend), FITC-CD4 (Cat# 100406, RRID:AB_312691, BioLegend), PE-Cy7-CD8 (Cat# 100722, RRID:AB_312761, BioLegend), PE-Cy7-CD127 (IL-7Rα; Cat# 135012, RRID:AB_2563456, BioLegend), APC-IFN-γ (Cat# 505810, RRID:AB_315404, BioLegend), Alexa Fluor 488–p-STAT5 (Tyr694; Cat# 9359, RRID:AB_10950988, Cell Signaling Technology). Cells were fixed and permeabilized using the BD Fixation/Permeabilization Kit (Cat# 554714, BD Biosciences, USA) according to the manufacturer’s protocol. Data were acquired using a BD FACS Aria™ II (Cat# 643109, BD Biosciences) and analyzed with FlowJo™ software (RRID:SCR_008520). ²¹

RNA Isolation and RT-qPCR

Total RNA was extracted using TRIzol™ reagent (Cat# 15596026, Thermo Fisher Scientific). RNA concentration was measured using NanoDrop ND-1000 (Thermo Fisher Scientific). Reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit (Cat# K1622, Thermo Fisher Scientific). qPCR was performed using iTaq™ Universal SYBR^® Green Supermix (Cat# 1725121, Bio-Rad). GAPDH was used as an endogenous control. Primer sequences are listed in Supplemental Table S1.

CCK-8 Assay

Cell proliferation was assessed using Cell Counting Kit-8 (Cat# CK04, Dojindo Laboratories, Japan). Absorbance was measured at 450/630 nm using a microplate reader.

Dual-Luciferase Activity Assay

The IFN-γ reporter plasmid and STAT5a expression plasmid were transfected into 293 T cells using Lipofectamine™ 3000 (Cat# L3000015, Thermo Fisher Scientific). Luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Cat# E1910, Promega, Madison, WI, USA). The Renilla/firefly luciferase ratio was calculated.

Statistical Analysis

Statistical data were analyzed using GraphPad Prism 9.0 Software. Results are presented as the mean ± standard deviation (SD). Student’s t-test was used to compare the significance between the 2 groups. Changes among more than 2 groups were analyzed by 1-way analysis of variance (ANOVA) and 2-way ANOVA followed by the Student–Newman–Keuls test. Statistical significance was set at *P < .05, **P < .01, and ***P < .001.

Results

Delayed Recovery of Th1 Cells After SLTBI Promotes Lung Metastasis of B16 Melanoma

To evaluate the recovery of IFN-γ⁺ lymphocytes after SLTBI, splenic IFN-γ⁺ cells from irradiated C57BL/6 mice were analyzed on days 0, 2, 4, and 8 post-irradiations (Figure 1A, Supplemental Figure 1A). The absolute number of IFN-γ⁺ cells among total splenocytes began to increase on day 4 and returned to baseline levels by day 8 (Figure 1B). Although the frequency of IFN-γ⁺ cells significantly increased between days 4 and 8 post-SLTBI (P < .01), absolute cell numbers did not fully recover until day 8 (Figure 1C). Among total IFN-γ⁺ cells, IFN-γ⁺ T, NK, and NKT subsets accounted for approximately 90% of the population (Figure 1D). Notably, IFN-γ⁺ T cells, particularly Th1 cells, exhibited the slowest recovery following SLTBI. As shown in Figure 1E to H, Tc1, NK1, and NKT1 cell numbers returned to baseline by day 8 post-SLTBI, whereas Th1 cells exhibited relatively delayed recovery compared with other subsets. This SLTBI model was used to evaluate systemic immune recovery in the absence of localized tumor burden.

Figure 1.

Delayed recovery of Th1 cells after SLTBI increases lung metastasis of B16 melanoma: (A) Treatment scheme of SLTBI mice. (B) % IFN-γ⁺ cells (of total splenocytes). (C) Absolute numbers of IFN-γ+ cells in spleen. (D) Gating strategies for IFN-γ+ cells involving Th1, Tc1, NK1, and NKT1 cells. (E-H) Percentages and absolute numbers of Th1, Tc1, NK1, and NKT1 cells in IFN-γ+ lymphocytes cells in spleen following SLTBI. (I) Lung tumor induction scheme of SLTBI mice. (J) Lung metastasis of B16 melanoma after SLTBI. Statistics of (K) lung weight and (L) tumor foci.

To assess the functional consequence of impaired IFN-γ⁺ cell recovery, irradiated mice were challenged with B16 melanoma cells on day 4 after SLTBI. Ten days later, irradiated mice exhibited significantly increased lung tumor foci compared with controls (Figure 1I-L). These findings suggest that delayed Th1 cell recovery may contribute to compromised anti-tumor immunity and increased susceptibility to B16 melanoma lung metastasis.

Lung LRT Induces the Quantitative Deficiency of IFN-γ⁺ Cells and Increases Lung B16 Tumor Foci

To evaluate the recovery status of IFN-γ⁺ lymphocytes in response to LRT for lung tumors, we established mouse models with and without B16 lung melanoma tumor burden and treated them with LRT (Figure 2A). LRT significantly reduced the tumor clone area (0.23 ± 0.37 vs 1.69 ± 0.78 mm² in controls, P < .001, Figure 2B) but significantly increased the number of tumor foci (234.2 ± 87.3 vs 125.2 ± 71.4 in controls, P < .05, Figure 2C), while lung weights remained unchanged (Figure 2D). The absolute cell counts of total IFN-γ⁺ cells in both the lung and spleen were significantly decreased after LRT (P < .05, Figure 2G; P < .001, Figure 2J, respectively), whereas the frequency of total IFN-γ⁺ lymphocytes was slightly decreased in the spleen (P < .05, Figure 2I) but not in the lungs (P > .05, Figure 2F).

Figure 2.

Lung LRT induces the quantitative deficiency of IFN-γ+ cells and increases lung B16 tumor foci. (A) Treatment scheme and representative images of B16 melanoma lung metastasis after LRT. Statistics of (B) tumor areas, (C) tumor foci, and (D) lung weight. (E-H) Dot plots of IFN-γ+ cells lineages in lung and spleen. (F-I) Proportions and (G-J) absolute numbers of IFN-γ+ cells in lung and spleen (n = 5/group, 2-way analysis of variance).

Th1 Cells Are Highly Sensitive to Lung LRT

In contrast to the SLTBI model shown in Figure 1, this experiment employed a local radiotherapy (LRT) model to assess immune alterations under tumor-bearing conditions. Along with LRT-induced reduction in IFN-γ⁺ cells, the numbers of Th1, Tc1, and NK1 cells were significantly decreased (Figure 3A-C), and the decline in quantity was more pronounced than the decline in their frequencies. Regarding NKT1 cells, a significant decline was observed only in their absolute counts in the spleen, whereas no significant change was detected in the lungs (Figure 3D). Among IFN-γ⁺ cells, the absolute number of Th1 cells was markedly reduced after irradiation in the lungs and spleen after LRT in mice with or without tumor burden (P < .01, Figure 3A). Notably, the Th1 cell counts in the spleen were reduced to approximately 50% of normal levels. These results suggest that Th1 cells are highly sensitive to LRT-induced damage, and that LRT severely impairs Th1 cell proliferation and reconstitution in both local tissues and lymphoid organs.

Figure 3.

Th1 cells are highly sensitive to lung LRT. (A-D) Percentages and absolute number of Th1, Tc1, NK1, and NKT1 cells in IFN-γ⁺ lymphocytes in lung and spleen.

Reduced IL-7R Expression on Th1 Cells Contributes to Impaired Immune Restoration After Irradiation

Then, we evaluated the transcription levels of IL-12, IL-15, IL-18, and IL-7 using total RNA extracted from whole spleen tissue of irradiated mice. Two days post-SLTBI, all of these cytokines were significantly upregulated. During the recovery period (4-8 days post-IR), the expression of IL-7 and IL-12 returned to baseline levels observed in control mice, while the transcript levels of IL-15 and IL-18 changed dynamically and were significantly upregulated 8 days after irradiation (P < .05, Figure 4A). We also assessed the receptor profiles of these cytokines on Th1, Tc1, and NK1 cells in both SLTBI and LRT mice. NK1 and Tc1 cells, which are characterized by rapid recovery from SLTBI, expressed high levels of IL-7R, IL-12R, IL-15R, and IL-18R. In contrast, Th1 cells displayed lower IL-7R expression compared to Tc1 cells during the recovery period post-IR (P < .05, Figure 4B, Supplemental Figure 1B). In LRT mice, IL-7R expression was significantly decreased in Th1, Tc1, and NK1 cells, whereas IL-12R was significantly elevated in Tc1 (P < .05, Figure 4C). We further observed that lung tumor development suppressed IL-7R expression in Th1 cells in B16 model mice compared with controls, and that LRT applied to lungs with tumor burden exacerbated the reduction of IL-7R on Th1 and Tc1 cells in the lung (Figure 4C).

Figure 4.

Reduced IL-7R expression on Th1 cells contributes to impaired immune restoration after irradiation. (A) Transcriptional levels of IL-7, IL-12, IL-15, and IL-18 in whole spleen tissue after SLTBI. Data were normalized to reference gene GAPDH. (B) Statistics of frequencies of IL-7R, IL-12R, IL-15R, and IL-18R in Th1, Tc1, and NK1 cells analyzed using FACS after SLTBI. (C) Statistics of frequencies of IL-7R, IL-12 and IL-15R in Th1, Tc1, and NK1 cells after LRT (n = 5/group, 2-way analysis of variance).

IL-7R–STAT5 Signaling is Required for T Cells Reconstitution and IFN-γ Production After Irradiation In Vitro

To further demonstrate the importance of IL-7R–STAT5 signaling in IFN-γ⁺ T cell expansion after irradiation in vitro, we evaluated EL4 T cells exposed to 1 or 4 Gy irradiation. Both doses markedly reduced cell viability, with a more pronounced reduction observed at 4 Gy. IL-7 treatment partially restored proliferation at 1 Gy but failed to rescue cell growth following 4 Gy irradiation (Figure 5A and B).

Figure 5.

IL-7R–STAT5 signaling is required for T cells reconstitution and IFN-γ production after irradiation in vitro. (A and B) Irradiated EL4 cells (1 Gy or 4 Gy), were cultured with or without IL-7 (10 ng/ml) for 24 or 48 h. Statistics of cell number and cell viability (*P < .05, compared to irradiated EL4 cells, ^#P < .05, compared to control). (C and D) Transcriptional levels of IL-7R, STAT5(A) and STAT5b in irradiated EL4 cells. (E-G) Dot plots and statistics of percentages of IL-7R and p-STAT5 in IFN-γ-secreting EL4 cells treated with IL-7 and 1 Gy or 4 Gy.

RT-qPCR analysis revealed that irradiation significantly downregulated IL-7R, STAT5a, and STAT5b mRNA expression, with a greater suppression at 4 Gy compared to 1 Gy (Figure 5C and D). Consistently, flow cytometry demonstrated a substantial decrease in IL-7R surface expression and P-STAT5 levels in irradiated EL4 cells, accompanied by a marked reduction in IFN-γ production at 24 h post-irradiation (Figure 5E-G). The decrease in p-STAT5 was particularly evident at 4 Gy, indicating impaired downstream signaling activation. Collectively, these results indicate that radiation-induced suppression of IL-7R–STAT5 signaling directly compromises IFN-γ production and T cell proliferative capacity.

Catalpol Promotes T Cell Proliferation by Enhancing Activation of IL-7R–STAT5 Signal After Irradiation and Prevents Lung Metastasis

Next, we investigated the efficacy of catalpol in T cell reconstitution after irradiation and its mechanisms targeting IL-7R–STAT5. In cell culture system, catalpol administration promoted the proliferation of irradiated EL4 cells (P < .05, Figure 6A) and enhanced IL-7R expression and STAT5 activation compared with the control group exposed to 1 Gy irradiation alone (P < .01, Figure 6B). Moreover, catalpol promoted direct binding of STAT5a to the IFN-γ promoter in 293 T cells with elevated firefly (P < .05, Figure 6C). The IFN-γ production in EL4 cells was also upregulated following catalpol treatment (P < .05, Figure 6D).

Figure 6.

Catalpol promotes T cell proliferation after irradiation and prevents lung metastasis. (A) Irradiated EL4 cells (1 Gy) were cultured with catalpol (50 μM), a non-cytotoxic concentration determined by preliminary dose–response experiments for 24 or 48 hours. Statistics of cell number and cell viability (*P < .05, compared to 1 Gy, ^#P < .05, compared to control). (B) Transcriptional levels of IL-7R, STAT5a, and STAT5b in catalpol and 1 Gy treatments. (C) Luciferase assay demonstrated that STAT5a positively regulated IFN-γ after treatment with catalpol. (D) Dot plots and percentages of IFN-γ+ cells after treatment with catalpol and 1 Gy. (E and F) Treatment scheme and images of the lung metastasis of B16 melanoma. Lung weight and tumor foci statistics. (G-I) Proportions and absolute numbers of Th1 cells, IL-7R, and p-STAT5 in lung Th1 cells in indicated groups (n = 5/group).

We further used FACS to quantify the abundance and activity of Th1 cells in the B16 melanoma lung metastasis model treated with a combination of LRT and catalpol (Figure 6E). The results showed that lung LRT significantly decreased the absolute number of Th1 cells in the lungs and increased the number of lung B16 melanoma foci. Combination treatment with catalpol and LRT in tumor-bearing mice displayed fewer B16 tumor foci and higher Th1 cell counts compared with mice treated with LRT alone (P < .05, Figure 6F and G), and IL-7R and p-STAT5 expression in Th1 cells was also improved by catalpol (P < .01, Figure 6H and I). The signature of high IL-7R–STAT5 levels in Th1 cells following catalpol treatment was associated with improved Th1 proliferation in the lung TME after LRT.

Discussion

The lungs are vulnerable to various pathogens, viruses, and tumor invasion, and the rapid recovery of IFN-γ⁺ lymphocytes after radiation is crucial for maintaining pulmonary immune homeostasis. ²² We found that the IFN-γ⁺ T lymphocytes are hypersensitive to irradiation. During the recovery phase (4-8 days post-IR), the number of IFN-γ⁺ T cells remained lower than that of the normal controls. Previous studies have shown that the radiation-induced reductions in APC signaling impair T cell recovery.^6,9,23 Consistent with these findings we observed that cytokine signals like IL-7, IL-12, IL-15, and IL-18 play a key role in IFN-γ⁺ T cell expansion, depending on the expression levels of cytokine receptors.^14,24 Furthermore, low IL-7R expression in Th1 cells limits IL-7 availability for Th1 proliferation and impairs recovery capacity after radiation. As a result, post-radiation anti-tumor immunity lacks adequate Th1 cells, reducing its specificity and durability and thereby increasing the risk of malignant cells metastatic spread. The observation of more lung metastatic B16 melanoma clones after SLTBI or LRT (Figures 1L and 2C) provides compelling evidence for this finding. Moreover, delayed recovery of Th1 cells disrupts the diversity and balance of T-helper subsets, and competition among T cell subsets further impairs early Th1development.^11,25 It has been reported that the increased regulatory T cell (Treg) and Th17 subsets after radiation also limits Th1 recovery and disturbs overall T cell homeostasis.^5,11,13,26 The relatively elevated levels of Treg and Th17 subsets post-IR provides a suitable environment for tumor development. ²⁷ These findings are consistent with our results, underscoring the critical role of Th1 in maintaining anti-tumor immunity after radiation. These results suggest the importance of Th1 in lung-specific anti-tumor immunity.

Th1 cell growth and functional activation are tightly regulated by coordinated cytokine signaling and transcriptional programming. While IL-12–STAT4 signaling drives initial Th1 polarization and IFN-γ transcription, IL-7–IL-7R–STAT5 signaling is essential for sustaining Th1 survival, metabolic fitness, and homeostatic proliferation, particularly under lymphopenic conditions such as irradiation. ²⁸ STAT5 activation promotes anti-apoptotic gene expression and supports clonal expansion, thereby maintaining the peripheral Th1 pool. ²⁹ Radiation-induced suppression of IL-7R expression, as observed in our study, may therefore limit STAT5 phosphorylation and impair downstream transcriptional programs required for Th1 persistence. In this context, insufficient IL-7R–STAT5 signaling likely compromises both quantitative recovery and functional competence of Th1 cells, weakening IFN-γ–mediated immune surveillance in the lung tumor microenvironment. Restoration of this pathway by catalpol may enhance Th1 proliferative capacity and reinforce anti-tumor immunity following radiotherapy.

T-cell subsets require multiple cytokines to support their polarization, activation, and proliferation, and cytokines are key factors that determine their expansion through the activation of STATs. ³⁰ Among these, IL-7–IL-7R–STAT5 signaling is essential for long-term T cell maintenance. ³¹ Although IL-2 is a classical T cell growth factor that promotes activation-dependent proliferation, IL-7 plays a more prominent role in homeostatic T cell survival and recovery under lymphopenic conditions such as irradiation. IL-2 signaling is closely associated with antigen-driven expansion and T cell receptor activation, whereas IL-7R–STAT5 signaling is critical for maintaining peripheral T cell pools. ³² Therefore, IL-7R was prioritized in this study to investigate mechanisms underlying post-irradiation Th1 reconstitution. Nonetheless, potential interactions between IL-2 and IL-7 signaling pathways warrant further investigation. A significant reduction of IL-7R in Th1 cells after SLTBI, and in both Th1 and Tc1 cells after lung LRT, markedly impairs their reconstitution after radiation. In addition, the progression of tumors in lung tissue can further inhibit IL-7R expression in Th1 cells. This finding was further confirmed by in vitro experiments showing that inhibition of IL-7R–STAT5 signaling was associated with reduced T cell proliferation (Figure 5). In this study, EL4 murine T cells were selected for in vitro mechanistic investigations to maintain species consistency with the C57BL/6 irradiation and B16 melanoma models. EL4 cells retain functional IL-7R–STAT5 signaling and IFN-γ production capacity, allowing targeted evaluation of radiation-induced impairment and catalpol-mediated restoration of this pathway. Because IL-7R signaling predominantly activates STAT5 to regulate T cell survival and homeostatic proliferation, STAT5 was prioritized as the principal downstream mediator. ³³ Although other STAT family members such as STAT1 and STAT4 are involved in IL-12–driven Th1 polarization, their roles were beyond the specific focus of IL-7R–mediated reconstitution examined in this study and warrant further investigation. Accumulating evidence has demonstrated that the reduced IL-7R–STAT5 signaling impairs the anti-tumor function of T cells in the lung TME and is associated with increased sensitivity to chemotherapy and reduced survival in patients with lung cancer.^3,34 Along with the development of cancer immunotherapy, IL-7R has emerged as the focus of T cell immunotherapy for melanoma. ³⁵

Fortunately, catalpol, the active component of the traditional Chinese medicine RRP, could enhance the expression and activation of the IL-7R–STAT5 signaling in Th1 cells, improve T cell reconstitution after radiation, upregulate the strength of Tc1 and NK1 responses, and improve the overall anti-tumor immunity. The reduction in lung metastatic foci and the increase in Th1 counts in catalpol-treated, irradiated mice further confirm the importance of Th1 reconstitution after radiation in antagonizing lung metastasis. The strategies whereby catalpol enhances Th1 regeneration would improve the efficacy of radiotherapy in patients with primary lung cancer by enhancing immune-reactivation. ³⁶

In this study, we focused on the effect of catalpol, while the effects of other ingredients in RRP were not studied. The results of this study demonstrated that the rapid recovery and improvement of IFN-γ⁺ T and NK cells is an important immunopharmacological basis for the efficacy of RRP. Nonetheless, other pharmacodynamic substances and pharmaceutical targets warrant further investigation. In addition, although LRT-induced immune alterations were evaluated in both tumor-bearing and non–tumor-bearing mice, direct statistical comparison between Ctrl + LRT and B16 + LRT groups was not performed to specifically dissect the independent contribution of tumor burden. Future studies are warranted to determine whether tumor-derived factors synergize with radiation to further impair Th1 reconstitution. Moreover, IL-7Rα expression by T cells is regulated by multiple mechanisms such as IL-2, IL-4, IL-15, and T cell receptor activation. ²⁴ . The mechanisms underlying the downregulation of IL-7R by Th1 and Tc1 cells following RT require detailed investigation.

Conclusions

In summary, insufficient reconstitution of Th1 cells with impaired IL-7R–STAT5 signaling following irradiation is a major limitation of anti-tumor responses in the lungs. Catalpol accelerated Th1 recovery following irradiation by enhancing IL-7R–STAT5 signaling in both the lung and spleen, thereby orchestrating a more effective anti-tumor immunity against lung tumors after lung LRT. These findings provide new strategies for improving RT and immunotherapy in patients with primary lung cancer or lung metastases from other malignancies.

Supplemental Material

sj-docx-1-ict-10.1177_15347354261453338 – Supplemental material for Catalpol Reactivates IL-7R–STAT5 Signaling and Reverses Radiation-Induced T Helper 1 Cells Deficiency to Limit Melanoma Metastasis to Lung

Supplemental material, sj-docx-1-ict-10.1177_15347354261453338 for Catalpol Reactivates IL-7R–STAT5 Signaling and Reverses Radiation-Induced T Helper 1 Cells Deficiency to Limit Melanoma Metastasis to Lung by Shan Zhang, Yue Zhang, Mingyan Ma, Junjie Huang, Rongrui Tang and Wenlian Hao in Integrative Cancer Therapies