Anti-TNFR2 Antibody and HMGN1 Combined with TIL Cell Therapy Inhibits Colorectal Cancer Progression by Enhancing Immune Response

Anti-TNFR2 Antibody and HMGN1 Combined with TIL Cell Therapy Inhibits Colorectal Cancer Progression by Enhancing Immune Response | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-TNFR2 Antibody and HMGN1 Combined with TIL Cell Therapy Inhibits Colorectal Cancer Progression by Enhancing Immune Response Hang Lv, Yujie Nie, Huan Gui, Haohua Yuan, Qianyu Jing, Shuanghui Chen, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6366085/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Immunotherapy utilizing tumor-infiltrating lymphocytes (TILs) has demonstrated exceptional effectiveness in the treatment of diverse solid tumors. However, existing procedures typically involve lymphodepleting chemotherapy using cyclophosphamide and high-dose IL-2 to support the proliferation and activity of reintroduced TILs, despite the common occurrence of systemic toxicity. Methods A CT26 colorectal cancer mouse model was established in this research. Tumor tissues were removed, and TILs were isolated and cultured in vitro. The TIL identity was validated via flow cytometry. Mice received treatment with an anti-TNFR2 antibody, HMGN1, and TILs to assess the effectiveness of this new immune-combination therapy against tumors. Flow cytometry was employed to analyze CD8 + T cells, CD4 + T cells, and Treg cells, with TIL function evaluated using CCK8 assays. Results Administration of anti-TNFR2 antibody and HMGN1 not only stimulated TIL proliferation but also suppressed Treg cells within tumor tissues, thereby markedly enhancing TIL-mediated anti-tumor activity in mice. Mice receiving this combination therapy achieved complete tumor eradication and significantly prolonged survival. This immune-combination therapy also demonstrated substantial tumor suppression in the 4T1 breast cancer mouse model. Conclusion The combined treatment of the anti-TNFR2 antibody and HMGN1 therapy synergistically alleviates immunosuppression by decreasing tumor-infiltrating regulatory T cells (Treg cells). This decrease in Treg cells results in the successful eradication of tumors in vivo by promoting the function and expansion of TIL. TNFR2 HMGN1 Tumor-infiltrating lymphocyte Colon cancer Adoptive cell therapy Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Tumor-infiltrating lymphocyte (TIL) therapy is an emerging therapeutic modality that entails the procurement of lymphocytes from a patient's tumor tissue, their expansion in vitro, and subsequent reinfusion into the patient to combat neoplastic disease [ 1 ] . This therapeutic approach is distinguished by several salient advantages, such as its capacity for multi-target engagement, tumor-specific recognition, robust infiltration capabilities, and a minimal incidence of side effects [ 2 ] . Clinical trials have underscored the impressive efficacy of TIL therapy in the management of metastatic melanoma [ 3 ] and advanced cervical carcinoma [ 4 ] . Traditional TIL therapy protocols require lymphodepleting chemotherapy using cyclophosphamide and simultaneous high-dose interleukin-2 (IL-2) administration to enhance the proliferation and function of the reintroduced TILs [ 5 ] . Despite these supportive interventions, the systemic toxicities linked to lymphodepleting chemotherapy and high-dose IL-2 are a significant clinical concern, as evidenced by a study showing that 80% of patients experienced grade 3 adverse events, while 60% experienced grade 4 events [ 6 ] . In patients with advanced cancer, the immunosuppressive milieu in vivo further exacerbates the depletion of TILs, potentially undermining their tumor-killing efficacy [ 7 ] . The TME is an intricate environment containing various immunosuppressive elements, including molecules, cytokines, and cells that hinder the anti-tumor function of TILs. [ 8 ] . Therefore, altering the immunosuppressive TME is a key research area to improve TIL therapy effectiveness. Current approaches to TME modulation primarily involve two strategies: the activation of antigen-presenting cells via Toll-like receptors (TLRs) to augment or restore T-cell anti-tumor responses [ 9 ] , and the targeting of immunosuppressive cells to mitigate immune suppression and bolster T-cell responses to tumors [ 10 ] . Regulatory T cells (Tregs) are notably abundant within the TME of various tumors, where they exert potent immunosuppressive effects, facilitating tumor immune evasion [ 11 ][ 12 ] and correlating with adverse prognostic outcomes [ 13 ][ 14 ][ 15 ] . Tregs accomplish this by releasing immunosuppressive cytokines, inhibiting the production of inflammatory molecules [ 16 ] , This process involves the downregulation of Major Histocompatibility Complex class II (MHC II) molecules and co-stimulatory proteins on dendritic cells (DCs). By reducing the levels of these molecules, tumors can evade the immune system's effector T cells (Teff), thereby impeding an effective immune response against the tumor [ 17 ] . Targeting Tregs within the TME is thus a strategic approach to improving the immunosuppressive microenvironment [ 18 ] . The high expression of TNFR2 on Tregs is implicated in their activation, proliferation, and enhancement of immunosuppressive functions [ 19 ][ 20 ] , and targeting TNFR2 can effectively suppress Treg activity while concomitantly promoting Teff proliferation [ 21 ] . HMGN1, a high-mobility group nucleosome-binding protein, functions as an agonist by inducing dendritic cell maturation through TLR4 activation [ 22 ] , recruiting antigen-presenting cells, and activating NF-κB and mitogen-activated protein kinases [ 23 ] . Elevated cytoplasmic HMGN1 levels are associated with increased tumor-infiltrating lymphocytes and enhanced antitumor immune responses [ 24 ] , suggesting a role for HMGN1 in facilitating TIL infiltration and augmenting immune responses. Our prior research has demonstrated that the combination of anti-TNFR2 with the TLR9 agonist CpG oligodeoxynucleotide (CpG ODN) significantly inhibits the progression of CT26 colon cancer in a murine model [ 25 ] . Moreover, the synergistic immunotherapy comprising HMGN1, R848, and anti-TNFR2 was found to suppress colon cancer through the activation of DCs and the inhibition of Tregs, with mice that were cured exhibiting immune memory against the same tumor [ 26 ] . The current research assesses the effectiveness of tumors and immune reactions induced by the blend of HMGN1, anti-TNFR2 antibodies, and TIL treatment in mice with CT26 tumor. Additionally, we explore the influence of HMGN1 on TIL growth and immune characteristics in a laboratory setting. The results indicate that the combined therapy boosts the anti-tumor response by elevating the presence of CD8 + T cells while decreasing CD4 + T cell levels. Moreover, HMGN1 stimulates TIL growth and aids in the production of CD8 + T cells. 2 Methods and materials 2.1 Mice and cell lines Eight-week-old female Balb/c mice were taken from Guangdong Zhiyuan Biomedical Technology Co., Ltd (Animal quality qualified certificate: SCXK (GD) 2021-0057). They were housed in the SPF animal house of Huarui Pattern Biotechnology (animal permit used number: SYXK(GD)2022 − 0304) and acclimatized for seven days before experimentation. The study project was approved by the IACUC-Shenzhen Huarui Model Biotechnology Limited Company, license APS-241111-025-01. Mouse cell CT26/MC38/4T1 obtained from Wuhan Pricella company was cultured with RPMI 1640 plus FBS (10%), penicillin/gentamycin (100 units/mL) and streptomycin sulfide (100 µg/mL), glutamine (2 mM)). All above culture cells in this experiment grew at 37°C/CO2 saturated conditions. 2.2 Generation of tumor-infiltrating lymphocytes The tumors taken from mice were cut into small pieces (approximately 1 mm3) and subjected to enzymatic digestion in 40 ml PBS with 40 mg collagenase, 100 units hyaluronidase, and 4 mg DNAse for 3–4 hours. The tumor digests were filtered using a cell filtration sieve to yield a single-cell suspension. TIL was separated by density gradient centrifugation using a solution of 75% + 100% Ficoll and further isolated cells with Ficoll density-gradient were suspended in culture medium containing RPMI1640 supplemented with 10% inactivated fetal calf serum, 0.03% natural glutamine, 0.1 mM non-essential amino acids, 5×10 − 5 M 2mercaptoethanol, penicillin-streptomycin (100 ug/ml each), and IL-2 (1000 UI/ml). This complete growth medium was used for cultures requiring proliferation and maintenance of the viabilities of mouse TIL. 2.3 Rapid Expansion Strategies for Tumor-Infiltrating Lymphocytes (REP) The TIL obtained from the above isolation was transferred into T25 culture flasks containing 200-fold the number of TIL in Balb/c mouse spleen cells, which were cultured in complete medium with 30 ng/mL of anti-CD3 antibody, and on the 14th day, the TIL was collected and enriched by centrifugation for use in treatment. 2.4 Chemicals Analytical grade reagents were utilized in the study. Interleukin-2 (IL-2) was obtained from CCK-8. Myeloid growth factor 1 (MGN1) was sourced from Biotechne in Minnesota, USA. The anti-mouse tumor necrosis factor receptor 2 (TNFR2) antibody, also known as CD120b or TR75-54.7, was supplied by MCE in New Jersey, USA. High mobility group nucleosome-binding domain 1 (HMGN1) was procured from Biotechne (R&D Systems, USA). Enzyme-linked immunosorbent assay (ELISA) kits for interferon-gamma (INF-γ) were purchased from Elabscience Biotechnology in Wuhan, China. Fetal bovine serum (FBS) was acquired from Pricella in Wuhan, China. The Penicillin-streptomycin stock solution was bought from New Cell and Molecular Biotech in Suzhou, China. Trypsin-EDTA (0.25%), trypsin, and phenol red (0.25%) were obtained from Servicebio in Wuhan, China. RPMI-1640 medium was sourced from GIBCO BRL in Grand Island, NY, USA. The Zombie NIR™ Fixable Viability Kit and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4 antibody (GK1.5) were purchased from Biolegend in California, USA. A variety of antibodies, including anti-mouse CD16/CD32 (2.4G2), Brilliant Violet 510 (BV510) anti-mouse CD45 (30-F11), Brilliant Violet 605 (BV605) anti-mouse CD3 (17A2), Peridinin-chlorophyll-protein complex (PerCP)-Cy5.5-labeled anti-mouse CD8 (53 − 6.7), Brilliant Violet 421 (BV421) anti-mouse CD49b (DX5), phycoerythrin (PE)-conjugated Foxp3 (MF23), and allophycocyanin (APC)-labeled anti-mouse CD25 (PC61), were all obtained from BD Biosciences in Franklin Lakes, NJ, USA. 2.5 Cell viability assay Tumor-infiltrating lymphocytes (TILs) were isolated at a concentration of 5,000 cells per well and seeded into 96-well plates. After a 24-hour incubation period, the TILs were treated with either anti-TNFR2 or HMGN1 for an additional 24 hours. Following this treatment, the culture medium was substituted with FBS-depleted RPMI-1640 supplemented with 10% CCK-8 solution, and the cells were then incubated at 37°C. Subsequently, optical density measurements were obtained at 450 nm wavelength after a further 1-hour incubation period. 2.6 Establishment and treatment of cancer mouse model CT26 colon cancer cells or 4T1 cells were subcutaneously injected into the right flank of mice at a concentration of 2×10 5 cells in 100 µL per mouse. The term "survival" in this context referred to the duration until the tumor volume reached 2000 mm 3 , at which point euthanasia was required. Throughout the experiment, the weight of the mice and the dimensions of the tumors were measured every three days. Tumor volume was determined using the formula (length × width 2 )/2. Treatment commenced approximately 10 days post-inoculation, when tumors reached 100 mm 3 . Anti-TNFR2 (200 µg/100 µL per mouse) was administered intraperitoneally on days 10, 12, and 14. On day 14, intratumoral injections of TIL (2 × 10 7 cells/100 µL per mouse) and HMGN1 (1 µg/100 µL per mouse) were performed, with additional HMGN1 injections on days 16 and 18. Tumors, draining lymph nodes, and spleens were excised for analysis the day following the final treatment. 2.7 Flow cytometry analysis The distribution of CD8 + T cells, CD4 + T cells, and Treg cells was determined through a multi-step process. Initially, cells were stained with a combination of Zombie NIR™ Fixable Viability Kit, BV510-CD45, BV605-CD3, FITC-CD4, APC-CD25, and PerCPcy5.5-CD8 antibodies to enable their identification and classification. Subsequently, for cytokine analysis, cells were resuspended in 200 µL of medium and then stimulated with PMA (phorbol 12-myristate 13-acetate) and ionomycin, followed by treatment with Brefeldin A to halt the process. The stimulation was terminated after 2.5 hours using a solution of PBS containing 0.2% fetal bovine serum. To assess intracellular cytokine levels, cells underwent surface staining, fixation, and permeabilization using BD fixation and permeabilization solution (BD Cytofix/Cytoperm™, 554714, USA) according to the manufacturer's guidelines. Intracellular staining for PE-IFN-γ and BV421-Foxp3 was then performed to detect the expression of these specific cytokines. Subsequently, flow cytometry was utilized for cell analysis, and the acquired data were analyzed using FlowJo_v10.6.2 software, enabling a comprehensive evaluation of the cellular composition and cytokine profiles within the studied cell populations. 2.8 Statistical analysis Statistical analyses were conducted using a two-tailed Student's t-test, one-way ANOVA, two-way ANOVA, and log-rank test to assess the differences between groups. GraphPad Prism 9 software was employed for all statistical analyses. Statistical significance was defined as p-values less than 0.05. 3 Results 3.1 Promotion of TIL differentiation and proliferation in vitro using IL-2 Following the procedure illustrated in Fig. 1 A, a subcutaneous mouse model of colorectal cancer was established using CT26 cells. When the tumor volume reached 400–500 mm³, the tumor tissue was surgically removed under sterile conditions and cut into 1-mm³ fragments. These fragments were enzymatically digested using a combination of type I and type IV collagenase, DNase I, and collagenase to obtain single-cell suspensions. Subsequently, these suspensions were mixed with a complete medium containing IL-2 and incubated in 24-well plates. As illustrated in Fig. 1 B, cells cultured without IL-2 displayed distinct characteristics of colorectal cancer cells by day 5, indicating that TIL in the single-cell suspensions were unable to differentiate or proliferate without IL-2 stimulation. Conversely, in the presence of IL-2, tumor cells were not microscopically observable. To further validate the identity of our cultured cells as TILs capable of killing tumors, flow cytometry analyses were performed on cells cultured up to day 7, as depicted in Figs. 1 C, D, E, and F. Cells cultured in a medium supplemented with interleukin-2 (IL-2) demonstrated a notable increase in the percentage of CD8 + T cells and showed heightened levels of interferon (IFN) in comparison to the control group. Moreover, there was a noticeable rise in the population of natural killer (NK) cells, accompanied by a significant decrease in the proportion of CD4 + CD25 + T cells. These findings provide strong evidence that the cells cultured in vitro represent TILs with potent antitumor activity. 3.2 Anti-TNFR2 and HMGN1 have a role in promoting TIL proliferation The anti-TNFR2 antibody effectively inhibits the proliferation of Treg cells, thereby mitigating immunosuppression. HMGN1, acting as a TLR4 agonist, stimulates the production of Type I IFNs in dendritic cells by activating IRF3 and IRF7, which enhances the anti-tumor response. Additionally, relevant literature has reported that HMGN1 can promote the proliferation of TIL. To investigate the effects of these two immunotherapeutic agents on TILs, we evaluated their ability to stimulate TIL proliferation in vitro using the CCK8 assay at various concentrations. Cell activity was notably increased by varying concentrations of the anti-TNFR2 antibody and HMGN1, with the most prominent pro-proliferative impact observed at a concentration of 30 µg/ml for the anti-TNFR2 antibody, as depicted in Figs. 2 A and 2 B. This finding supports the conclusion that both the anti-TNFR2 antibody and HMGN1 have a pro-proliferative influence on Tumor-Infiltrating Lymphocytes (TILs) in an in vitro setting. Flow cytometry analysis was conducted to investigate the impact of anti-TNFR2 antibody and HMGN1 on the immunophenotype of tumor-infiltrating lymphocytes TILs. It was treated with anti-TNFR2 antibody (30 µg/100 µl) and HMGN1 (0.2 µg/100 µl) for 24 hours. The ratios of CD8 + T cells and CD4 + T cells relative to CD3 + T cells were assessed, along with the ratio of CD4 + CD25 + T cells relative to CD3 + T cells. The results revealed a significant increase in the proportion of CD8 + T cells and a decrease in the proportion of CD4 + CD25 + T cells in TILs treated with anti-TNFR2 antibody and HMGN1. This shift in the immunophenotype of TILs was associated with enhanced tumor-killing capabilities, suggesting a promising avenue for potential therapeutic interventions. 3.3 HMGN1 combined with anti-TNFR2 enhances the effect of TIL against colorectal cancer tumors in mice in vivo After confirming the successful isolation and expansion of TIL and observing that anti-TNFR2 and HMGN1 promote TIL proliferation, we proceeded to evaluate the therapeutic potential of a novel immune-combination therapy for colorectal cancer in a mouse model. In mice with subcutaneous colorectal cancer (CRC) tumors, we administered three intraperitoneal injections of anti-TNFR2 antibody, followed by intratumoral injection of TIL cells, and then three intratumoral injections of HMGN1 (Fig. 3 A). The results indicated that tumors in the treatment group were completely eradicated compared to the control group, and the dual therapy also showed significant tumor suppression (Fig. 3 B, C, D). Additionally, the survival duration of the mice in the treatment group was significantly extended (Fig. 3 E). No significant weight loss was observed in any group following immunotherapy (Fig. 3 F), and the spleen-to-body-weight ratio was lower in the triple therapy group compared to the PBS group (Fig. 3 G). This suggests that the triple therapy may significantly enhance the systemic immune response and increase immune activity against splenic tumors, whereas the PBS group exhibited splenomegaly or inflammation. The liver-to-body weight and kidney-to-body-weight ratios did not show significant changes in any group, indicating that our therapy did not cause significant organ damage or hepatic and renal toxicities, thus demonstrating superior efficacy compared to other therapeutic approaches. 3.4 TIL combined with HMGN1 and anti-TNFR2 effectively induces anti-tumor immune response in mice in vivo Flow cytometry was employed to assess immune cell distribution in the spleen, draining lymph nodes (DLN), and tumor tissue cells to investigate the effects of a novel immunocombination therapy on systemic immunity and the tumor immune microenvironment. This powerful technique allows for the precise analysis of various immune cell subsets, providing valuable insights into how the novel therapy influences the immune system's response both systemically and locally within the tumor microenvironment. The flow cytometry acquisition strategy is depicted in Fig. 4 A, outlining the specific steps and protocols used to isolate and analyze the immune cells. In comparison to control groups, the treatment group demonstrated significant alterations in the frequency and activation status of key immune cell populations, indicating the potential efficacy of the novel immunocombination therapy in modulating antitumor immunity. These findings highlight the importance of flow cytometry in elucidating the complex interplay between therapeutic interventions and immune cell dynamics in the context of cancer immunology. 3.5 Novel immune combination therapy reduces Tregs ratio and increases Cytotoxic T lymphocyte ratio Flow cytometry was employed to analyze the immune cell phenotypes in the spleens, DLN, and tumors of mice to investigate the effects of the novel immune-combination therapy on the TME. The analysis revealed no significant changes in the proportions of CD4 + CD25 + Foxp3 + Treg and CD8 + INF + CTLs in the spleens of treated mice compared to the control group that received PBS (Figs. 5 A, D).Interestingly, a substantial decrease in Treg cell proportions (Figs. 5 B, C) and a noticeable increase in CTL proportions were observed in the DLNs and tumor tissues (Figs. 5 E, F) of the treated mice. These results indicate that the novel immune-combination therapy effectively reduces the immunosuppressive environment within the TME while concurrently boosting the anti-tumor response. This dual mechanism leads to an enhanced cytotoxic impact on tumor cells, suggesting a promising strategy for cancer immunotherapy. 3.6 Novel immune combination therapy inhibits growth of 4T1 breast cancer in mice To authenticate the effectiveness of the innovative immune-combination treatment, we employed a mouse model of 4T1 breast cancer, utilizing the identical protocol for TIL extraction and delivery as in the CT26 colorectal cancer model. The outcomes indicated a notable suppression of tumor growth and an extension in the lifespan of the mice (Fig. 6 A, B, C). 4 Discussion The objective of this study was to develop a novel TIL immunotherapy regimen that demonstrated remarkable therapeutic efficacy in a murine CT26 colorectal cancer model. Post-treatment, complete tumor eradication was achieved in the mice, with no discernible toxic side effects observed. The therapy involved the use of anti-TNFR2 antibodies to suppress immunosuppressive effects within the tumor immune microenvironment, coupled with HMGN1 to sustain the proliferation and activity of TILs, thereby enhancing their antitumor capabilities in vivo. Current TIL therapeutic protocols often necessitate lymphodepletion chemotherapy (LD) prior to TIL infusion and the administration of high-dose IL-2 to support TIL survival in vivo [ 27 ] . These requirements limit their clinical application due to the associated toxicities of LD and high-dose IL-2 [ 28 ] . A prior research study reported that 80% of patients receiving TIL therapy along with lymph depletion chemotherapy and IL-2 encountered grade 3 adverse events, while 60% experienced grade 4 adverse events [ 5 ] . In contrast, our investigation revealed that mice subjected to our novel therapeutic regimen did not exhibit significant weight loss post-treatment. Furthermore, the ratios of liver-to-body weight and kidney-to-body weight remained stable, indicating no significant organ damage or toxic side effects. These findings suggest that our innovative therapeutic approach for mice does not induce significant toxic side effects. CD8 + T lymphocytes in TILs are vital for immune response against tumors [ 29 ] . Our research findings revealed that the most abundant cell types within TIL were cytotoxic CD8 + INF-γ + T cells and CD3 − CD49 + NK cells, which play crucial roles in tumor eradication. Additionally, a subset of immunosuppressive CD3 + CD4 + CD25 + Treg cells was also identified within the TIL population. The abundance of Tregs that can inhibit anti-tumor immunity in the tumor microenvironment (TME) of many cancers has allowed cancer cells to escape from immune attacks, the role of TME in tumorigenesis, development, immune escape and therapeutic resistance is gradually being emphasized. It not only provides a microenvironment for tumor cell growth and survival, but also influences the function of immune cells through multiple mechanisms and promotes immune escape from tumors, which is associated with poor cancer prognosis [ 12 ][ 13 ] . They secrete immunosuppressive cytokines, inhibit the production and release of inflammatory factors [ 30 ] , and reduce the expression of MHC II molecules and costimulatory molecules (CD80, CD86) on dendritic cell [ 31 ] , inhibit effector T cells (Teff), and thereby promote immune evasion by tumors [ 32 ] . Blocking the immunosuppressive function of Tregs in the TME is considered an effective strategy for improving an immunosuppressive tumor environment [ 33 ] . Studies have demonstrated that TNFR2 is highly expressed on Treg cells and is a key mediator involved in Treg activation, proliferation, and enhanced immunosuppressive function [ 19 ][ 20 ] . Targeting TNFR2 can effectively suppress Treg activity while promoting Teff proliferation [ 34 ] . Dendritic cells are recognized as the most potent professional APCs [ 35 ] , capable of acquiring tumor antigen characteristics and presenting them to lymphocytes to activate naive T cells to kill tumor cells and initiate antitumor immune responses [ 36 ] . However, DCs in the tumor microenvironment are mostly immature [ 37 ] , with poor antigen-presenting capabilities, failing to effectively activate T cells to exert antitumor effects [ 38 ] . HMGN1 acts as an agonist that promotes DC maturation via toll-like receptor 4 (TLR4) [ 39 ] , recruiting antigen-presenting cells (APCs) at the injection site and activating NF-κB and multiple mitogen-activated protein kinases [ 40 ] . Research has reported that high levels of HMGN1 expression in the cytoplasm are significantly associated with increased tumor-infiltrating lymphocyte (TIL) infiltration within tumors, indicating that HMGN1 aids in enhancing TIL infiltration and antitumor immunity [ 41 ] . Our findings showed that in vitro, anti-TNFR2 antibodies and HMGN1 could enhance TIL cell proliferation, elevate CD8 + T cell levels, and reduce Treg cell levels. In vivo, treated mice exhibited a substantial increase in CD8 + T cell levels in the spleen, DLN, and tumor tissues, along with a marked rise in INF-γ + CTLs and a notable decrease in Treg cell levels. To further validate the efficacy of the novel immune combined therapy for various tumors, we administered the same treatment schedule to 4T1 murine breast cancer models. Similar tumor growth inhibition was observed as seen in colorectal cancer models, accompanied by prolonged animal survival. While complete eradication of the xenografted transplanted carcinoma, as demonstrated in colitis-induced CRC, was not achieved, a noticeable inhibitory effect was evident. We postulate that this discrepancy may be attributed to factors beyond the intrinsic properties of the target solid tumors, such as the "cold/tumorigenic" nature characterized by sparse cellularity and limited intra-tumoral immune activation due to deficient effector-immune cell infiltration. Our preliminary findings suggest that the overall functionality of TILs in mammary gland-derived carcinoma patients is notably weaker compared to those in CRC patients. Consequently, we plan to conduct a comprehensive analysis of the immunophenotypic alterations in the immune cell responses of diverse cancers originating from different host tissues. 5 Conclusion In summary, our study demonstrates that the combination of anti-TNFR2 antibodies and HMGN1 significantly improves the antitumor capacity in mice by reducing Treg cells in the TME to alleviate immunosuppression and by maintaining the activity and proliferation of TIL cells in vivo. Declarations Funding The authors declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from Shenzhen Key Laboratory for Cancer Metastasis and Personalized Therapy (ZDSYS20210623091811035), National Natural Science Foundation of China (82060308), “Three Famous Projects" of Shenzhen Health Commission（SZZYSM202411001），Guizhou Provincial Science and Technology Projects (GCC[2022]037-1), and grant from Ministry of Human Resources and Social Security of the People's Republic of China，grant from High-level Talent Team in Guizhou Province. Ethics approval and consent to participate The animal study was approved by the Experimental Animal Care and Use Committee (IACUC) of Shenzhen Huarui Model Biotechnology Co., Ltd (IACUC No.: APS-241111-025-01). The study was conducted in accordance with the local legislation and institutional requirements. CRediT authorship contribution statement YJ.Nie conceived the project and designed the experiments. H Lv., M.J., etc. conducted the experiments and analyzed data, under the supervision of YJ.N. H.Lv, Q.W. etc. maintained the mice and cell lines, performed animal experiments, Flow cytometry, data analysis and visualization. H.L. and YJ.N. prepared and interpreted all the figures， wrote the manuscript. YJ.N. revised and edited the manuscript. All the authors reviewed and approved the final manuscript. Data availability Data will be made available on request. Acknowledgments The authors want to thank the Central Laboratory of Guizhou Provincial People’s Hospital and the Core Laboratory of the University of Hong Kong-Shenzhen Hospital for its help in the research. Declaration of Competing Interest The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper. References Paijens, S. T., Vledder, A., de Bruyn, M., & Nijman, H. W. (2021). Tumor-infiltrating lymphocytes in the immunotherapy era. 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C., Ullrich, E., Saulnier, P., Yang, H., Amigorena, S., Ryffel, B., Barrat, F. J., Saftig, P., Levi, F., Lidereau, R., Nogues, C., Mira, J. P., Chompret, A., … Zitvogel, L. (2007). Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nature medicine , 13 (9), 1050–1059. https://doi.org/10.1038/nm1622 Yang, D., Postnikov, Y. V., Li, Y., Tewary, P., de la Rosa, G., Wei, F., Klinman, D., Gioannini, T., Weiss, J. P., Furusawa, T., Bustin, M., & Oppenheim, J. J. (2012). High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses. The Journal of experimental medicine , 209 (1), 157–171. https://doi.org/10.1084/jem.20101354 Lee, H. J., Kim, J. Y., Song, I. H., Park, I. A., Yu, J. H., Ahn, J. H., & Gong, G. (2015). High mobility group B1 and N1 (HMGB1 and HMGN1) are associated with tumor-infiltrating lymphocytes in HER2-positive breast cancers. Virchows Archiv : an international journal of pathology , 467 (6), 701–709. https://doi.org/10.1007/s00428-015-1861-1 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6366085","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449231769,"identity":"3454ac66-dad4-463a-a1bf-647ea792ab84","order_by":0,"name":"Hang Lv","email":"","orcid":"","institution":"Guizhou University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Lv","suffix":""},{"id":449231770,"identity":"b833a3f0-b9c3-4609-9295-7c14e002567d","order_by":1,"name":"Yujie Nie","email":"","orcid":"","institution":"Guizhou provincial people's 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College","correspondingAuthor":true,"prefix":"","firstName":"Yingjie","middleName":"","lastName":"Nie","suffix":""}],"badges":[],"createdAt":"2025-04-03 05:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6366085/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6366085/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81636955,"identity":"a0b81312-d328-49fa-881b-2f9fe7539ffe","added_by":"auto","created_at":"2025-04-29 12:42:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3522795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromotion of TIL differentiation and proliferation in vitro using IL-2.\u003c/strong\u003e (A) Extraction and in vitro cultivation of TIL procedure. (B) Microscopic images show cells cultured in vitro from single-cell suspensions prepared as depicted in Figure (A), in complete medium with or without IL-2, observed on day 1, day 3, and day 5(Image magnification is 200x) (C) Dot plots and corresponding CD8\u003csup\u003e+\u003c/sup\u003e T cell percentages relative to CD3\u003csup\u003e+\u003c/sup\u003e T cells in the TIL population. Data shown as mean ± SD, n = 3. (D) Dot plots and IFN-γ\u003csup\u003e+ \u003c/sup\u003eT cell percentages relative to CD8\u003csup\u003e+\u003c/sup\u003e T cells in the TIL population. Data shown as mean ± SD, n = 3. (E) Dot plots and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e T cell percentages relative to CD3\u003csup\u003e+\u003c/sup\u003e T cells in the TIL population. Data shown as mean ± SD, n = 3. (F) Dot plot and CD49\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e- \u003c/sup\u003enatural killer (NK) cell percentage in the TIL population. Data shown as mean ± SD, n = 3. Statistical significance assessed using one-way ANOVA, with *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001 denoting significant variations.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/b566b8c2a1b9e750a2ec8088.png"},{"id":81636953,"identity":"1975eb76-67ab-49b3-8d29-37bd4d6a2fad","added_by":"auto","created_at":"2025-04-29 12:42:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1198400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of anti-TNFR2 antibodies and HMGN1 on TIL in vitro.\u003c/strong\u003e (A) and (B) TILs were incubated with varied concentrations of anti-TNFR2 antibody (10, 20, 30, 40, 50 μg) and HMGN1 (0.1, 0.2, 0.3, 0.4, 0.5 μg) for 24 hours. Cell viability was assessed using the CCK8 assay. (C) and (D) TILs were exposed to anti-TNFR2 antibody (30 μg/100 μl) and HMGN1 (0.2 μg/100 μl) for 24 hours. Subsequently, flow cytometry analyses were conducted. (C) Dot plots and the proportions of CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells and CD4\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells relative to CD3\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells are presented. Data means SD, n = 6. (D) Dot plots and the proportions of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells relative to CD3\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T cells are shown. Data means SD, n = 6. Statistical significance was assessed using one-way ANOVA, with *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001 denoting significance.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/4a45ac694ce9ae5b3335ef06.png"},{"id":81636957,"identity":"b7bb35f1-e9bf-4ce8-a8b4-235db17e3adc","added_by":"auto","created_at":"2025-04-29 12:42:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3025308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntitumor effects on CT26 colorectal cancer in mice.\u003c/strong\u003e (A) Depiction of subcutaneous tumor model creation and treatment schedule in 8-week-old Balb/c mice. (B) Tumor growth curves for individual mice, n=10. (C) Tumor growth curves for mice in distinct groups, n=10. Statistical analysis utilized two-factor ANOVA followed by Tukey's post hoc test. (D) Images displaying mice from each group at 4 days post-final treatment (day 12), with CT26 tumor regions marked by solid circles. (E) Survival curves for mice in each group, n=10. Log-rank test applied for comparisons. (F) Body weight changes in mice are monitored at 2-day intervals for each group, n=10. Two-way ANOVA is employed for data analysis. (G) Spleen weight to body weight ratios measured for mice in each group, with accompanying spleen images. Results shown as mean ± SD, analyzed using one-way ANOVA, n=5. (H) Liver weight to body weight ratios determined for mice in each group, with corresponding liver images. Results expressed as mean ± SD, analyzed using one-way ANOVA, n=5. (I) Ratios of kidney weight to body weight measured for each group of mice, with kidney images displayed. Results are expressed as mean ± SD and were analyzed using one-way ANOVA, n=5. ns indicates no significance, while *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001 denote statistically significant differences.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/727db3d0bf73d7503705ed2e.png"},{"id":81637151,"identity":"fa4cdfcf-dc70-4216-a4b4-35741d3d1af6","added_by":"auto","created_at":"2025-04-29 12:50:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1968779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel immunocombination therapy enhances anti-tumor responses in mice in vivo.\u003c/strong\u003e (A) Flow cytometry strategy to detect gating of CD4\u003csup\u003e+\u003c/sup\u003e T, CD8\u003csup\u003e+\u003c/sup\u003e T and Tregs in spleen, DLN, and tumor tissues (B) Representative dot plots of CD8\u003csup\u003e+\u003c/sup\u003e T cells and the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells to CD3\u003csup\u003e+ \u003c/sup\u003eT cells in mouse DLN. (C) Representative dot plots of CD8\u003csup\u003e+\u003c/sup\u003e T cells in mouse spleen and percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells to CD3\u003csup\u003e+\u003c/sup\u003e T cells. (D) Representative dot plots of CD8\u003csup\u003e+\u003c/sup\u003e T cells in mouse tumor tissues and the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells to CD3\u003csup\u003e+\u003c/sup\u003e T cells. (E) Representative dot plots of CD4\u003csup\u003e+\u003c/sup\u003e T cells in mouse DLN and percentage of CD4\u003csup\u003e+ \u003c/sup\u003eT cells to CD3\u003csup\u003e+\u003c/sup\u003e T cells. (F) Representative dot plots of CD4\u003csup\u003e+\u003c/sup\u003e T cells in mouse tumor tissues and the percentage of CD4\u003csup\u003e+\u003c/sup\u003e T cells to CD3\u003csup\u003e+\u003c/sup\u003e T cells. (G) Representative dot plots of CD4\u003csup\u003e+ \u003c/sup\u003eT cells in mouse spleen and percentage of CD4\u003csup\u003e+\u003c/sup\u003e T cells to CD3\u003csup\u003e+\u003c/sup\u003e T cells. All the above data results are expressed as mean ± SD and were analyzed using one-way ANOVA. ns indicates no significance, while *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001 denote statistically significant differences. n=3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/a57fdb0f83eb4a98cced4530.png"},{"id":81637150,"identity":"1471536b-b5c1-42a0-a095-28be865b1208","added_by":"auto","created_at":"2025-04-29 12:50:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2245803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel immunecombination therapy reduces Tregs ratio and increases Cytotoxic T lymphocyte ratio.\u003c/strong\u003e (A) Dot plots showing CTL cells in the spleen of mice and the ratio of IFN\u003csup\u003e+\u003c/sup\u003e T cells to CD8\u003csup\u003e+\u003c/sup\u003e T cells. (B) Dot plots illustrating CTL cells in the DLN of mice and the proportion of IFN\u003csup\u003e+\u003c/sup\u003e T cells to CD8\u003csup\u003e+\u003c/sup\u003e T cells. (C) Dot plots displaying CTL cells in mouse tumor tissues and the ratio of IFN\u003csup\u003e+\u003c/sup\u003e T cells to CD8\u003csup\u003e+\u003c/sup\u003e T cells. (D) Dot plots presenting Treg cells in the spleen of mice and the ratio of CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e T cells to CD4\u003csup\u003e+\u003c/sup\u003e T cells. (E) Dot plots demonstrate Treg cells in the DLN of mice and the proportion of CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e T cells to CD4\u003csup\u003e+\u003c/sup\u003e T cells. (F) Dot plots depicting Treg cells in mouse tumor tissues and the ratio of CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e T cells to CD4\u003csup\u003e+\u003c/sup\u003e T cells. All data are presented as mean ± SD and were assessed using one-way ANOVA. The notation \"ns\" indicates no statistical significance, while *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ***p \u0026lt; 0.0001 indicate statistically significant distinctions. n=3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/591b3b2817cc6796b25ebddb.png"},{"id":81637152,"identity":"d0d593bc-5383-4945-9f79-cc22174fdc41","added_by":"auto","created_at":"2025-04-29 12:50:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":523388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel immune combination therapy inhibits growth of 4T1 breast cancer in mice.\u003c/strong\u003e(A) Tumor growth curves in mice with 4T1 breast cancer, n=5 mice per group. (B) Tumor growth curves in mice across various groups, n=5 mice per group. Data shown as mean ± SEM, analyzed using two-factor ANOVA and Tukey's multiple comparison test. (C) Survival curves of mice in each group, n=5 mice per group. Log-rank test used for comparisons. Statistically significant differences indicated by *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/78d7052ae09a54f10c8f6acc.png"},{"id":84353128,"identity":"090be559-bf8a-4a19-8fc1-ba7b2331674d","added_by":"auto","created_at":"2025-06-11 01:46:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12654536,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6366085/v1/d80ebe93-c072-48ce-9f92-abed45e110d7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-TNFR2 Antibody and HMGN1 Combined with TIL Cell Therapy Inhibits Colorectal Cancer Progression by Enhancing Immune Response","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eTumor-infiltrating lymphocyte (TIL) therapy is an emerging therapeutic modality that entails the procurement of lymphocytes from a patient's tumor tissue, their expansion in vitro, and subsequent reinfusion into the patient to combat neoplastic disease\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. This therapeutic approach is distinguished by several salient advantages, such as its capacity for multi-target engagement, tumor-specific recognition, robust infiltration capabilities, and a minimal incidence of side effects\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Clinical trials have underscored the impressive efficacy of TIL therapy in the management of metastatic melanoma\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e and advanced cervical carcinoma\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Traditional TIL therapy protocols require lymphodepleting chemotherapy using cyclophosphamide and simultaneous high-dose interleukin-2 (IL-2) administration to enhance the proliferation and function of the reintroduced TILs\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Despite these supportive interventions, the systemic toxicities linked to lymphodepleting chemotherapy and high-dose IL-2 are a significant clinical concern, as evidenced by a study showing that 80% of patients experienced grade 3 adverse events, while 60% experienced grade 4 events \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In patients with advanced cancer, the immunosuppressive milieu in vivo further exacerbates the depletion of TILs, potentially undermining their tumor-killing efficacy\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe TME is an intricate environment containing various immunosuppressive elements, including molecules, cytokines, and cells that hinder the anti-tumor function of TILs. \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Therefore, altering the immunosuppressive TME is a key research area to improve TIL therapy effectiveness. Current approaches to TME modulation primarily involve two strategies: the activation of antigen-presenting cells via Toll-like receptors (TLRs) to augment or restore T-cell anti-tumor responses\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and the targeting of immunosuppressive cells to mitigate immune suppression and bolster T-cell responses to tumors \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegulatory T cells (Tregs) are notably abundant within the TME of various tumors, where they exert potent immunosuppressive effects, facilitating tumor immune evasion\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e and correlating with adverse prognostic outcomes\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Tregs accomplish this by releasing immunosuppressive cytokines, inhibiting the production of inflammatory molecules\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, This process involves the downregulation of Major Histocompatibility Complex class II (MHC II) molecules and co-stimulatory proteins on dendritic cells (DCs). By reducing the levels of these molecules, tumors can evade the immune system's effector T cells (Teff), thereby impeding an effective immune response against the tumor\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Targeting Tregs within the TME is thus a strategic approach to improving the immunosuppressive microenvironment\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The high expression of TNFR2 on Tregs is implicated in their activation, proliferation, and enhancement of immunosuppressive functions\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, and targeting TNFR2 can effectively suppress Treg activity while concomitantly promoting Teff proliferation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. HMGN1, a high-mobility group nucleosome-binding protein, functions as an agonist by inducing dendritic cell maturation through TLR4 activation\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, recruiting antigen-presenting cells, and activating NF-κB and mitogen-activated protein kinases \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Elevated cytoplasmic HMGN1 levels are associated with increased tumor-infiltrating lymphocytes and enhanced antitumor immune responses \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, suggesting a role for HMGN1 in facilitating TIL infiltration and augmenting immune responses.\u003c/p\u003e \u003cp\u003eOur prior research has demonstrated that the combination of anti-TNFR2 with the TLR9 agonist CpG oligodeoxynucleotide (CpG ODN) significantly inhibits the progression of CT26 colon cancer in a murine model\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Moreover, the synergistic immunotherapy comprising HMGN1, R848, and anti-TNFR2 was found to suppress colon cancer through the activation of DCs and the inhibition of Tregs, with mice that were cured exhibiting immune memory against the same tumor\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The current research assesses the effectiveness of tumors and immune reactions induced by the blend of HMGN1, anti-TNFR2 antibodies, and TIL treatment in mice with CT26 tumor. Additionally, we explore the influence of HMGN1 on TIL growth and immune characteristics in a laboratory setting. The results indicate that the combined therapy boosts the anti-tumor response by elevating the presence of CD8\u003csup\u003e+\u003c/sup\u003e T cells while decreasing CD4\u003csup\u003e+\u003c/sup\u003e T cell levels. Moreover, HMGN1 stimulates TIL growth and aids in the production of CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e"},{"header":"2 Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Mice and cell lines\u003c/h2\u003e \u003cp\u003eEight-week-old female Balb/c mice were taken from Guangdong Zhiyuan Biomedical Technology Co., Ltd (Animal quality qualified certificate: SCXK (GD) 2021-0057). They were housed in the SPF animal house of Huarui Pattern Biotechnology (animal permit used number: SYXK(GD)2022\u0026thinsp;\u0026minus;\u0026thinsp;0304) and acclimatized for seven days before experimentation. The study project was approved by the IACUC-Shenzhen Huarui Model Biotechnology Limited Company, license APS-241111-025-01. Mouse cell CT26/MC38/4T1 obtained from Wuhan Pricella company was cultured with RPMI 1640 plus FBS (10%), penicillin/gentamycin (100 units/mL) and streptomycin sulfide (100 \u0026micro;g/mL), glutamine (2 mM)). All above culture cells in this experiment grew at 37\u0026deg;C/CO2 saturated conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Generation of tumor-infiltrating lymphocytes\u003c/h2\u003e \u003cp\u003eThe tumors taken from mice were cut into small pieces (approximately 1 mm3) and subjected to enzymatic digestion in 40 ml PBS with 40 mg collagenase, 100 units hyaluronidase, and 4 mg DNAse for 3\u0026ndash;4 hours. The tumor digests were filtered using a cell filtration sieve to yield a single-cell suspension.\u003c/p\u003e \u003cp\u003eTIL was separated by density gradient centrifugation using a solution of 75% \u003csup\u003e+\u003c/sup\u003e 100% Ficoll and further isolated cells with Ficoll density-gradient were suspended in culture medium containing RPMI1640 supplemented with 10% inactivated fetal calf serum, 0.03% natural glutamine, 0.1 mM non-essential amino acids, 5\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;5 M 2mercaptoethanol, penicillin-streptomycin (100 ug/ml each), and IL-2 (1000 UI/ml). This complete growth medium was used for cultures requiring proliferation and maintenance of the viabilities of mouse TIL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Rapid Expansion Strategies for Tumor-Infiltrating Lymphocytes (REP)\u003c/h2\u003e \u003cp\u003eThe TIL obtained from the above isolation was transferred into T25 culture flasks containing 200-fold the number of TIL in Balb/c mouse spleen cells, which were cultured in complete medium with 30 ng/mL of anti-CD3 antibody, and on the 14th day, the TIL was collected and enriched by centrifugation for use in treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Chemicals\u003c/h2\u003e \u003cp\u003eAnalytical grade reagents were utilized in the study. Interleukin-2 (IL-2) was obtained from CCK-8. Myeloid growth factor 1 (MGN1) was sourced from Biotechne in Minnesota, USA. The anti-mouse tumor necrosis factor receptor 2 (TNFR2) antibody, also known as CD120b or TR75-54.7, was supplied by MCE in New Jersey, USA. High mobility group nucleosome-binding domain 1 (HMGN1) was procured from Biotechne (R\u0026amp;D Systems, USA). Enzyme-linked immunosorbent assay (ELISA) kits for interferon-gamma (INF-γ) were purchased from Elabscience Biotechnology in Wuhan, China. Fetal bovine serum (FBS) was acquired from Pricella in Wuhan, China. The Penicillin-streptomycin stock solution was bought from New Cell and Molecular Biotech in Suzhou, China. Trypsin-EDTA (0.25%), trypsin, and phenol red (0.25%) were obtained from Servicebio in Wuhan, China. RPMI-1640 medium was sourced from GIBCO BRL in Grand Island, NY, USA. The Zombie NIR\u0026trade; Fixable Viability Kit and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4 antibody (GK1.5) were purchased from Biolegend in California, USA. A variety of antibodies, including anti-mouse CD16/CD32 (2.4G2), Brilliant Violet 510 (BV510) anti-mouse CD45 (30-F11), Brilliant Violet 605 (BV605) anti-mouse CD3 (17A2), Peridinin-chlorophyll-protein complex (PerCP)-Cy5.5-labeled anti-mouse CD8 (53\u0026thinsp;\u0026minus;\u0026thinsp;6.7), Brilliant Violet 421 (BV421) anti-mouse CD49b (DX5), phycoerythrin (PE)-conjugated Foxp3 (MF23), and allophycocyanin (APC)-labeled anti-mouse CD25 (PC61), were all obtained from BD Biosciences in Franklin Lakes, NJ, USA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell viability assay\u003c/h2\u003e \u003cp\u003eTumor-infiltrating lymphocytes (TILs) were isolated at a concentration of 5,000 cells per well and seeded into 96-well plates. After a 24-hour incubation period, the TILs were treated with either anti-TNFR2 or HMGN1 for an additional 24 hours. Following this treatment, the culture medium was substituted with FBS-depleted RPMI-1640 supplemented with 10% CCK-8 solution, and the cells were then incubated at 37\u0026deg;C. Subsequently, optical density measurements were obtained at 450 nm wavelength after a further 1-hour incubation period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Establishment and treatment of cancer mouse model\u003c/h2\u003e \u003cp\u003eCT26 colon cancer cells or 4T1 cells were subcutaneously injected into the right flank of mice at a concentration of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells in 100 \u0026micro;L per mouse. The term \"survival\" in this context referred to the duration until the tumor volume reached 2000 mm\u003csup\u003e3\u003c/sup\u003e, at which point euthanasia was required. Throughout the experiment, the weight of the mice and the dimensions of the tumors were measured every three days. Tumor volume was determined using the formula (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2. Treatment commenced approximately 10 days post-inoculation, when tumors reached 100 mm\u003csup\u003e3\u003c/sup\u003e. Anti-TNFR2 (200 \u0026micro;g/100 \u0026micro;L per mouse) was administered intraperitoneally on days 10, 12, and 14. On day 14, intratumoral injections of TIL (2 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/100 \u0026micro;L per mouse) and HMGN1 (1 \u0026micro;g/100 \u0026micro;L per mouse) were performed, with additional HMGN1 injections on days 16 and 18. Tumors, draining lymph nodes, and spleens were excised for analysis the day following the final treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Flow cytometry analysis\u003c/h2\u003e \u003cp\u003eThe distribution of CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, and Treg cells was determined through a multi-step process. Initially, cells were stained with a combination of Zombie NIR\u0026trade; Fixable Viability Kit, BV510-CD45, BV605-CD3, FITC-CD4, APC-CD25, and PerCPcy5.5-CD8 antibodies to enable their identification and classification. Subsequently, for cytokine analysis, cells were resuspended in 200 \u0026micro;L of medium and then stimulated with PMA (phorbol 12-myristate 13-acetate) and ionomycin, followed by treatment with Brefeldin A to halt the process. The stimulation was terminated after 2.5 hours using a solution of PBS containing 0.2% fetal bovine serum.\u003c/p\u003e \u003cp\u003eTo assess intracellular cytokine levels, cells underwent surface staining, fixation, and permeabilization using BD fixation and permeabilization solution (BD Cytofix/Cytoperm\u0026trade;, 554714, USA) according to the manufacturer's guidelines. Intracellular staining for PE-IFN-γ and BV421-Foxp3 was then performed to detect the expression of these specific cytokines. Subsequently, flow cytometry was utilized for cell analysis, and the acquired data were analyzed using FlowJo_v10.6.2 software, enabling a comprehensive evaluation of the cellular composition and cytokine profiles within the studied cell populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using a two-tailed Student's t-test, one-way ANOVA, two-way ANOVA, and log-rank test to assess the differences between groups. GraphPad Prism 9 software was employed for all statistical analyses. Statistical significance was defined as p-values less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Promotion of TIL differentiation and proliferation in vitro using IL-2\u003c/h2\u003e \u003cp\u003eFollowing the procedure illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, a subcutaneous mouse model of colorectal cancer was established using CT26 cells. When the tumor volume reached 400\u0026ndash;500 mm\u0026sup3;, the tumor tissue was surgically removed under sterile conditions and cut into 1-mm\u0026sup3; fragments. These fragments were enzymatically digested using a combination of type I and type IV collagenase, DNase I, and collagenase to obtain single-cell suspensions. Subsequently, these suspensions were mixed with a complete medium containing IL-2 and incubated in 24-well plates. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, cells cultured without IL-2 displayed distinct characteristics of colorectal cancer cells by day 5, indicating that TIL in the single-cell suspensions were unable to differentiate or proliferate without IL-2 stimulation. Conversely, in the presence of IL-2, tumor cells were not microscopically observable. To further validate the identity of our cultured cells as TILs capable of killing tumors, flow cytometry analyses were performed on cells cultured up to day 7, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, E, and F. Cells cultured in a medium supplemented with interleukin-2 (IL-2) demonstrated a notable increase in the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells and showed heightened levels of interferon (IFN) in comparison to the control group. Moreover, there was a noticeable rise in the population of natural killer (NK) cells, accompanied by a significant decrease in the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e T cells. These findings provide strong evidence that the cells cultured in vitro represent TILs with potent antitumor activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Anti-TNFR2 and HMGN1 have a role in promoting TIL proliferation\u003c/h2\u003e \u003cp\u003eThe anti-TNFR2 antibody effectively inhibits the proliferation of Treg cells, thereby mitigating immunosuppression. HMGN1, acting as a TLR4 agonist, stimulates the production of Type I IFNs in dendritic cells by activating IRF3 and IRF7, which enhances the anti-tumor response. Additionally, relevant literature has reported that HMGN1 can promote the proliferation of TIL. To investigate the effects of these two immunotherapeutic agents on TILs, we evaluated their ability to stimulate TIL proliferation in vitro using the CCK8 assay at various concentrations. Cell activity was notably increased by varying concentrations of the anti-TNFR2 antibody and HMGN1, with the most prominent pro-proliferative impact observed at a concentration of 30 \u0026micro;g/ml for the anti-TNFR2 antibody, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. This finding supports the conclusion that both the anti-TNFR2 antibody and HMGN1 have a pro-proliferative influence on Tumor-Infiltrating Lymphocytes (TILs) in an in vitro setting. Flow cytometry analysis was conducted to investigate the impact of anti-TNFR2 antibody and HMGN1 on the immunophenotype of tumor-infiltrating lymphocytes TILs. It was treated with anti-TNFR2 antibody (30 \u0026micro;g/100 \u0026micro;l) and HMGN1 (0.2 \u0026micro;g/100 \u0026micro;l) for 24 hours. The ratios of CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD4\u003csup\u003e+\u003c/sup\u003e T cells relative to CD3\u003csup\u003e+\u003c/sup\u003e T cells were assessed, along with the ratio of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e T cells relative to CD3\u003csup\u003e+\u003c/sup\u003e T cells. The results revealed a significant increase in the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells and a decrease in the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e T cells in TILs treated with anti-TNFR2 antibody and HMGN1. This shift in the immunophenotype of TILs was associated with enhanced tumor-killing capabilities, suggesting a promising avenue for potential therapeutic interventions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 HMGN1 combined with anti-TNFR2 enhances the effect of TIL against colorectal cancer tumors in mice in vivo\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter confirming the successful isolation and expansion of TIL and observing that anti-TNFR2 and HMGN1 promote TIL proliferation, we proceeded to evaluate the therapeutic potential of a novel immune-combination therapy for colorectal cancer in a mouse model. In mice with subcutaneous colorectal cancer (CRC) tumors, we administered three intraperitoneal injections of anti-TNFR2 antibody, followed by intratumoral injection of TIL cells, and then three intratumoral injections of HMGN1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results indicated that tumors in the treatment group were completely eradicated compared to the control group, and the dual therapy also showed significant tumor suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C, D). Additionally, the survival duration of the mice in the treatment group was significantly extended (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). No significant weight loss was observed in any group following immunotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and the spleen-to-body-weight ratio was lower in the triple therapy group compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). This suggests that the triple therapy may significantly enhance the systemic immune response and increase immune activity against splenic tumors, whereas the PBS group exhibited splenomegaly or inflammation. The liver-to-body weight and kidney-to-body-weight ratios did not show significant changes in any group, indicating that our therapy did not cause significant organ damage or hepatic and renal toxicities, thus demonstrating superior efficacy compared to other therapeutic approaches.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 TIL combined with HMGN1 and anti-TNFR2 effectively induces anti-tumor immune response in mice in vivo\u003c/h2\u003e \u003cp\u003eFlow cytometry was employed to assess immune cell distribution in the spleen, draining lymph nodes (DLN), and tumor tissue cells to investigate the effects of a novel immunocombination therapy on systemic immunity and the tumor immune microenvironment. This powerful technique allows for the precise analysis of various immune cell subsets, providing valuable insights into how the novel therapy influences the immune system's response both systemically and locally within the tumor microenvironment. The flow cytometry acquisition strategy is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, outlining the specific steps and protocols used to isolate and analyze the immune cells. In comparison to control groups, the treatment group demonstrated significant alterations in the frequency and activation status of key immune cell populations, indicating the potential efficacy of the novel immunocombination therapy in modulating antitumor immunity. These findings highlight the importance of flow cytometry in elucidating the complex interplay between therapeutic interventions and immune cell dynamics in the context of cancer immunology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Novel immune combination therapy reduces Tregs ratio and increases Cytotoxic T lymphocyte ratio\u003c/h2\u003e \u003cp\u003eFlow cytometry was employed to analyze the immune cell phenotypes in the spleens, DLN, and tumors of mice to investigate the effects of the novel immune-combination therapy on the TME. The analysis revealed no significant changes in the proportions of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg and CD8\u003csup\u003e+\u003c/sup\u003eINF\u003csup\u003e+\u003c/sup\u003e CTLs in the spleens of treated mice compared to the control group that received PBS (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, D).Interestingly, a substantial decrease in Treg cell proportions (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C) and a noticeable increase in CTL proportions were observed in the DLNs and tumor tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F) of the treated mice. These results indicate that the novel immune-combination therapy effectively reduces the immunosuppressive environment within the TME while concurrently boosting the anti-tumor response. This dual mechanism leads to an enhanced cytotoxic impact on tumor cells, suggesting a promising strategy for cancer immunotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Novel immune combination therapy inhibits growth of 4T1 breast cancer in mice\u003c/h2\u003e \u003cp\u003eTo authenticate the effectiveness of the innovative immune-combination treatment, we employed a mouse model of 4T1 breast cancer, utilizing the identical protocol for TIL extraction and delivery as in the CT26 colorectal cancer model. The outcomes indicated a notable suppression of tumor growth and an extension in the lifespan of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe objective of this study was to develop a novel TIL immunotherapy regimen that demonstrated remarkable therapeutic efficacy in a murine CT26 colorectal cancer model. Post-treatment, complete tumor eradication was achieved in the mice, with no discernible toxic side effects observed. The therapy involved the use of anti-TNFR2 antibodies to suppress immunosuppressive effects within the tumor immune microenvironment, coupled with HMGN1 to sustain the proliferation and activity of TILs, thereby enhancing their antitumor capabilities in vivo.\u003c/p\u003e \u003cp\u003eCurrent TIL therapeutic protocols often necessitate lymphodepletion chemotherapy (LD) prior to TIL infusion and the administration of high-dose IL-2 to support TIL survival in vivo\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. These requirements limit their clinical application due to the associated toxicities of LD and high-dose IL-2\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. A prior research study reported that 80% of patients receiving TIL therapy along with lymph depletion chemotherapy and IL-2 encountered grade 3 adverse events, while 60% experienced grade 4 adverse events\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In contrast, our investigation revealed that mice subjected to our novel therapeutic regimen did not exhibit significant weight loss post-treatment. Furthermore, the ratios of liver-to-body weight and kidney-to-body weight remained stable, indicating no significant organ damage or toxic side effects. These findings suggest that our innovative therapeutic approach for mice does not induce significant toxic side effects.\u003c/p\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003eT lymphocytes in TILs are vital for immune response against tumors\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Our research findings revealed that the most abundant cell types within TIL were cytotoxic CD8\u003csup\u003e+\u003c/sup\u003eINF-γ\u003csup\u003e+\u003c/sup\u003e T cells and CD3\u003csup\u003e\u0026minus;\u003c/sup\u003eCD49\u003csup\u003e+\u003c/sup\u003eNK cells, which play crucial roles in tumor eradication. Additionally, a subset of immunosuppressive CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eTreg cells was also identified within the TIL population.\u003c/p\u003e \u003cp\u003eThe abundance of Tregs that can inhibit anti-tumor immunity in the tumor microenvironment (TME) of many cancers has allowed cancer cells to escape from immune attacks, the role of TME in tumorigenesis, development, immune escape and therapeutic resistance is gradually being emphasized. It not only provides a microenvironment for tumor cell growth and survival, but also influences the function of immune cells through multiple mechanisms and promotes immune escape from tumors, which is associated with poor cancer prognosis \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. They secrete immunosuppressive cytokines, inhibit the production and release of inflammatory factors\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, and reduce the expression of MHC II molecules and costimulatory molecules (CD80, CD86) on dendritic cell\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, inhibit effector T cells (Teff), and thereby promote immune evasion by tumors \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Blocking the immunosuppressive function of Tregs in the TME is considered an effective strategy for improving an immunosuppressive tumor environment\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Studies have demonstrated that TNFR2 is highly expressed on Treg cells and is a key mediator involved in Treg activation, proliferation, and enhanced immunosuppressive function \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Targeting TNFR2 can effectively suppress Treg activity while promoting Teff proliferation \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Dendritic cells are recognized as the most potent professional APCs\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, capable of acquiring tumor antigen characteristics and presenting them to lymphocytes to activate naive T cells to kill tumor cells and initiate antitumor immune responses \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, DCs in the tumor microenvironment are mostly immature \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, with poor antigen-presenting capabilities, failing to effectively activate T cells to exert antitumor effects \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. HMGN1 acts as an agonist that promotes DC maturation via toll-like receptor 4 (TLR4) \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, recruiting antigen-presenting cells (APCs) at the injection site and activating NF-κB and multiple mitogen-activated protein kinases \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Research has reported that high levels of HMGN1 expression in the cytoplasm are significantly associated with increased tumor-infiltrating lymphocyte (TIL) infiltration within tumors, indicating that HMGN1 aids in enhancing TIL infiltration and antitumor immunity \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Our findings showed that in vitro, anti-TNFR2 antibodies and HMGN1 could enhance TIL cell proliferation, elevate CD8\u003csup\u003e+\u003c/sup\u003e T cell levels, and reduce Treg cell levels. In vivo, treated mice exhibited a substantial increase in CD8\u003csup\u003e+\u003c/sup\u003e T cell levels in the spleen, DLN, and tumor tissues, along with a marked rise in INF-γ\u003csup\u003e+\u003c/sup\u003e CTLs and a notable decrease in Treg cell levels.\u003c/p\u003e \u003cp\u003eTo further validate the efficacy of the novel immune combined therapy for various tumors, we administered the same treatment schedule to 4T1 murine breast cancer models. Similar tumor growth inhibition was observed as seen in colorectal cancer models, accompanied by prolonged animal survival. While complete eradication of the xenografted transplanted carcinoma, as demonstrated in colitis-induced CRC, was not achieved, a noticeable inhibitory effect was evident. We postulate that this discrepancy may be attributed to factors beyond the intrinsic properties of the target solid tumors, such as the \"cold/tumorigenic\" nature characterized by sparse cellularity and limited intra-tumoral immune activation due to deficient effector-immune cell infiltration. Our preliminary findings suggest that the overall functionality of TILs in mammary gland-derived carcinoma patients is notably weaker compared to those in CRC patients. Consequently, we plan to conduct a comprehensive analysis of the immunophenotypic alterations in the immune cell responses of diverse cancers originating from different host tissues.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn summary, our study demonstrates that the combination of anti-TNFR2 antibodies and HMGN1 significantly improves the antitumor capacity in mice by reducing Treg cells in the TME to alleviate immunosuppression and by maintaining the activity and proliferation of TIL cells in vivo.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from Shenzhen Key Laboratory for Cancer Metastasis and Personalized Therapy (ZDSYS20210623091811035), National Natural Science Foundation of China (82060308),\u0026nbsp;“Three Famous Projects\" of Shenzhen Health Commission（SZZYSM202411001），Guizhou Provincial Science and Technology Projects (GCC[2022]037-1), and grant from Ministry of Human Resources and Social Security of the People's Republic of China，grant from High-level Talent Team in Guizhou Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by the Experimental Animal Care and Use Committee (IACUC) of Shenzhen Huarui Model Biotechnology Co., Ltd (IACUC No.: APS-241111-025-01). The study was conducted in accordance with the local legislation and institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYJ.Nie conceived the project and designed the experiments. H Lv., M.J., etc. conducted the experiments and analyzed data, under the supervision of YJ.N. H.Lv, Q.W. etc. maintained the mice and cell lines, performed animal experiments, Flow cytometry, data analysis and visualization. H.L. and YJ.N. prepared and interpreted all the figures，\u0026nbsp;wrote the manuscript. YJ.N. revised and edited the manuscript. All the authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors want to thank the Central Laboratory of Guizhou Provincial People’s Hospital and the Core Laboratory of the University of Hong Kong-Shenzhen Hospital for its help in the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePaijens, S. T., Vledder, A., de Bruyn, M., \u0026amp; Nijman, H. W. (2021). Tumor-infiltrating lymphocytes in the immunotherapy era. 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High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses. \u003cem\u003eThe Journal of experimental medicine\u003c/em\u003e, \u003cem\u003e209\u003c/em\u003e(1), 157\u0026ndash;171. https://doi.org/10.1084/jem.20101354\u003c/li\u003e\n\u003cli\u003eLee, H. J., Kim, J. Y., Song, I. H., Park, I. A., Yu, J. H., Ahn, J. H., \u0026amp; Gong, G. (2015). High mobility group B1 and N1 (HMGB1 and HMGN1) are associated with tumor-infiltrating lymphocytes in HER2-positive breast cancers. \u003cem\u003eVirchows Archiv : an international journal of pathology\u003c/em\u003e, \u003cem\u003e467\u003c/em\u003e(6), 701\u0026ndash;709. https://doi.org/10.1007/s00428-015-1861-1\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TNFR2, HMGN1, Tumor-infiltrating lymphocyte, Colon cancer, Adoptive cell therapy, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6366085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6366085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eImmunotherapy utilizing tumor-infiltrating lymphocytes (TILs) has demonstrated exceptional effectiveness in the treatment of diverse solid tumors. However, existing procedures typically involve lymphodepleting chemotherapy using cyclophosphamide and high-dose IL-2 to support the proliferation and activity of reintroduced TILs, despite the common occurrence of systemic toxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA CT26 colorectal cancer mouse model was established in this research. Tumor tissues were removed, and TILs were isolated and cultured in vitro. The TIL identity was validated via flow cytometry. Mice received treatment with an anti-TNFR2 antibody, HMGN1, and TILs to assess the effectiveness of this new immune-combination therapy against tumors. Flow cytometry was employed to analyze CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, and Treg cells, with TIL function evaluated using CCK8 assays.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAdministration of anti-TNFR2 antibody and HMGN1 not only stimulated TIL proliferation but also suppressed Treg cells within tumor tissues, thereby markedly enhancing TIL-mediated anti-tumor activity in mice. Mice receiving this combination therapy achieved complete tumor eradication and significantly prolonged survival. This immune-combination therapy also demonstrated substantial tumor suppression in the 4T1 breast cancer mouse model.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe combined treatment of the anti-TNFR2 antibody and HMGN1 therapy synergistically alleviates immunosuppression by decreasing tumor-infiltrating regulatory T cells (Treg cells). This decrease in Treg cells results in the successful eradication of tumors in vivo by promoting the function and expansion of TIL.\u003c/p\u003e","manuscriptTitle":"Anti-TNFR2 Antibody and HMGN1 Combined with TIL Cell Therapy Inhibits Colorectal Cancer Progression by Enhancing Immune Response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 12:42:05","doi":"10.21203/rs.3.rs-6366085/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8aa27294-d235-4f11-a382-124df703d794","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-11T01:38:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 12:42:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6366085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6366085","identity":"rs-6366085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}