Mesenchymal Stromal Cells Over-expression Mutant IL-2 Enhance Treg Function in CIA Mice

Mesenchymal Stromal Cells Over-expression Mutant IL-2 Enhance Treg Function in CIA Mice | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 1 June 2024 V1 Latest version Share on Mesenchymal Stromal Cells Over-expression Mutant IL-2 Enhance Treg Function in CIA Mice Authors : Zhicheng Tang 0009-0003-1890-7209 , Fan Yang , Jingyi Shen , Haolin Wu , Huiming Hong , Yue Wang , Fanzhang Yin , Xiaojun Tang , and Huayong Zhang [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.171722001.19584616/v1 391 views 261 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objectives : Research indicates that low doses of interleukin-2 (IL-2) can effectively mitigate RA symptoms by promoting Treg cells, while high doses may enhance immune responses. Consequently, this study employed mutated IL-2 to minimize its impact on CD8 + T and NK cell activation while preserving its influence on Treg cells. Methods : We constructed IL-2 mutants by overlap PCR and assessed its impact on the proliferation and functionality of Treg cells by flow cytometry and PCR. Furthermore, the synergistic effects of mutated IL-2 and MSC on collagen-induced arthritis (CIA) in mice were evaluated through the infusion of lentiviral-transfected mesenchymal stromal cell (MSC) for CIA treatment and through pathological section staining to assess inflammatory joint injury, cartilage destruction, and osteoclast infiltration. Results : Mutant IL-2 demonstrated targeted enhancement of both the proportion and proliferative activity of Treg cells with a diminished capacity to stimulate the proliferation of CD8 + T cells and NK cells relative to wild-type IL-2. Moreover, MSC-mutant IL-2 significantly augmented the proportion of Treg cells compared to either MSC or mutant IL-2 in isolation. Treatment with MSC-mutant IL-2 infusion in CIA mice ameliorated arthritis symptoms and reduced inflammatory infiltration and cartilage damage in their joints. Conclusion : Mutant IL-2 enhances Treg function and proliferation while exerting reduced effects on CD8 + and NK cell activation. MSC expressing mutant IL-2 demonstrates therapeutic benefits in CIA by increasing the proportion of Treg cells and reducing the proportion of CD8 + T cells. Mesenchymal Stromal Cells Over-expression Mutant IL-2 Enhance Treg Function in CIA Mice Authors: Zhicheng Tang 1 , Fan Yang 1 , Jingyi Shen 1 , Haolin Wu 2 , Huiming Hong 2 , Yue Wang 2 , Fanzhang Yin 2 , Xiaojun Tang 3* , Huayong Zhang 1* Department(s) and institution(s) : 1 Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University. 2 Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine. 3 Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital Author responsible for correspondence： 1.Huayong Zhang PhD. Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, 321 Zhongshan Road,Nanjing, Jiangsu, 210008, China. Tel: +86-25-6818-2428. E-mail: [email protected] 2.Xiaojun Tang Dr. Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, 321 Zhongshan Road,Nanjing, Jiangsu, 210008, China. E-mail: [email protected] Abstract Objectives : Research indicates that low doses of interleukin-2 (IL-2) can effectively mitigate RA symptoms by promoting Treg cells, while high doses may enhance immune responses. Consequently, this study employed mutated IL-2 to minimize its impact on CD8 + T and NK cell activation while preserving its influence on Treg cells. Methods : We constructed IL-2 mutants by overlap PCR and assessed its impact on the proliferation and functionality of Treg cells by flow cytometry and PCR. Furthermore, the synergistic effects of mutated IL-2 and MSC on collagen-induced arthritis (CIA) in mice were evaluated through the infusion of lentiviral-transfected mesenchymal stromal cell (MSC) for CIA treatment and through pathological section staining to assess inflammatory joint injury, cartilage destruction, and osteoclast infiltration. Results : Mutant IL-2 demonstrated targeted enhancement of both the proportion and proliferative activity of Treg cells with a diminished capacity to stimulate the proliferation of CD8 + T cells and NK cells relative to wild-type IL-2. Moreover, MSC-mutant IL-2 significantly augmented the proportion of Treg cells compared to either MSC or mutant IL-2 in isolation. Treatment with MSC-mutant IL-2 infusion in CIA mice ameliorated arthritis symptoms and reduced inflammatory infiltration and cartilage damage in their joints. Conclusion : Mutant IL-2 enhances Treg function and proliferation while exerting reduced effects on CD8 + and NK cell activation. MSC expressing mutant IL-2 demonstrates therapeutic benefits in CIA by increasing the proportion of Treg cells and reducing the proportion of CD8 + T cells. Keywords: interleukin-2; Mesenchymal Stem Cells; Arthritis, Rheumatoid; T-Lymphocytes, Regulatory; Lentivirus; Arthritis, Experimental. Significance Statement This study introduces a mutant IL-2, IL-2 N103R V106D, with a high affinity for Treg cells. This variant preferentially promotes Treg cells over the wild-type IL-2, thus mitigating aberrant autoimmunity. Additionally, utilizing lentiviral transfection to enable MSC expression of mutant IL-2 leverages the inflammatory targeting and immunomodulatory capabilities of MSCs. This approach synergistically controls inflammation and autoimmunity, notably reducing joint inflammation in rheumatoid arthritis patients. When compared to conventional MSC and low-dose IL-2 treatments, this method demonstrates significant advantages, yielding more satisfactory outcomes in both in vitro and animal studies. It offers fresh perspectives and strategies for rheumatoid arthritis treatment. Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by its deleterious effects on bone and cartilage within joints, leading to swelling, pain, and functional impairment[1]. Clinically, treatment involves the use of DMARDs, NSAIDs, glucocorticoids, and biologics; however, their prolonged use may result in a spectrum of adverse effects[2]. Regulatory T cells (Treg), a specialized subset of CD4 + CD25 + FOXP3 + T cells, play a pivotal role in suppressing autoimmune effector cells, thereby preventing autoimmune diseases and maintaining immune homeostasis[3, 4]. A reduction in the proportion of Treg cells and their dysfunction are critical factors in the development and progression of rheumatoid arthritis[5, 6]. Collagen-induced arthritis (CIA) mice, a widely recognized mouse model for rheumatoid arthritis, served as the experimental subjects in this study[7]. Mesenchymal stromal cells (MSC) possess the ability to suppress memory effector T cells[8], promote Treg cells[9], secrete anti-inflammatory cytokines, and migrate to sites of inflammation renders MSC a promising therapeutic candidate for RA[10, 11]. Numerous preclinical and clinical studies have corroborated the efficacy and safety of MSC in treating RA[12]. Interleukin-2 (IL-2) has been clinically demonstrated to be effective in the treatment of cancer (particularly for metastatic renal cell carcinoma and metastatic melanoma) and autoimmune diseases[13, 14]. Clinical studies have corroborated the efficacy of low-dose IL-2 in treating autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis[15, 16]. IL-2, a pleiotropic cytokine produced post-antigen activation, is pivotal in the immune response, promoting the activation and proliferation of various immune cells[17]. The IL-2 receptors vary among immune cells, with trimeric receptors composed of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132) predominantly found on regulatory T cells and activated effector T cells[18-20], and dimeric receptors comprised of IL-2Rα (CD25) and IL-2Rγ (CD132) are primarily expressed on memory T cells, helper T cells, resting effector T cells, and NK cells[21-24]. Treg cells, due to their expression of trimeric receptors, are more responsive than other immune cells to low-dose IL-2, thereby enabling low-dose IL-2 to act as an immunosuppressant through the promotion of Treg cells[25]. However, the dose-dependency and short half-life of IL-2 therapy necessitate frequent administration and raise concerns about the adverse effects of long-term use, thereby limiting its clinical application[26]. As the ability to receive IL-2 signals is determined by the affinity for distinct IL-2-receptor complexes on different cell subsets, selective targeting of regulatory T cells or cytotoxic lymphocytes can be achieved through the construction of IL-2 mutants or fusion proteins that reduce affinity for different IL-2 receptor subunits, offering a promising therapeutic strategy for cancer and autoimmune disease treatments[27-31]. Sun et al. showed that mutant IL-2, characterized by a diminished affinity for IL-2α, led to decreased Treg cell infiltration, thereby enhancing the efficacy of tumor treatment[32]. Similarly, Khoryati, Liliane, et al. demonstrated that IL-2 mutants, possessing a reduced affinity for IL-2β, increased reliance on IL-2Rα. Despite a weaker activation relative to wild-type IL-2, their heightened selectivity for Treg cells contributed to improved treatment outcomes in NOD mice[27]. Our strategy involves expressing IL-2, which has a high affinity for Treg cells, through MSC, while concurrently leveraging the inflammatory tropism of MSC to induce a selective Treg cell-promoting effect[33], thereby exerting anti-inflammatory effects in the affected joints of patients with rheumatoid arthritis. Additionally, this approach facilitated potent and extended resolution of rheumatoid arthritis, potentially minimizing the need for frequent drug administration. Material and methods Prediction of protein structure and physicochemical properties The tertiary structure of wild-type IL-2 was sourced from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB, http://www.rcsb.org), and the structure of mutant IL-2 N103R V106D was predicted using the online tool AlphaFold (https://cosmic-cryoem.org/tools/alphafold2)[38]. A comparison of these two protein structures was conducted using PyMOL (PyMOL Molecular Graphics System, v2.5.7), and the physicochemical properties of the proteins were predicted using Expasy (https://web.expasy.org/protparam/). Construction of lentiviral vectors Cultured 293T cells were seeded into 10-cm dishes. Upon reaching 80% confluency, target plasmids (pLV-puro-IL-2, pLV-puro-IL-2 N103R V106D, control plasmid pLV-puro) and packaging plasmids (PMD2G, PsPAX2) were introduced into serum-free DMEM medium. EZ-Trans transfection reagent was mixed with diluted plasmid DNA to form EZ-Trans-DNA complexes, which were then added to the cell culture dishes. After 6-8 hours, the medium was changed; after 48 hours, the supernatant was collected, then centrifuged and filtered using a 0.45-µm micro-filter. PEG-8000 was added and mixed by inverting 3-5 times, with a 30-minute interval between each inversion. The mixture was left overnight and then centrifuged at high speed, the supernatant was discarded, and the lentiviral precipitates were resuspended in PBS. The suspension was flash-frozen in liquid nitrogen before storage in an ultra-low-temperature freezer. RNA extraction and real-time quantitative PCR Tissues or cells were lysed using RNA isolater Total RNA Extraction Reagent (Vazyme), and the resulting supernatant was collected. Protein was denatured using chloroform and then centrifuged at 12,000 g. The supernatant, containing total RNA, was removed, and total RNA was precipitated using isopropanol, washed with ethanol, and dissolved in RNase-free water. The RNA was reverse-transcribed into cDNA. cDNA was used as a template for PCR amplification, following standard procedures. Data were recorded from the amplification curve generated by the PCR machine, using GAPDH as an internal control. Finally, the ratio between the target gene and GAPDH was calculated and subjected to statistical analysis. Western blot Protein extraction involved lysing splenocytes using RIPA lysis buffer, with subsequent ice incubation for 30 minutes and heating for 15 minutes. This was followed by protein electrophoresis, transfer, and blocking. Subsequently, the cells were incubated overnight with pSTAT5 antibody (1:1000) and STAT5 antibody (1:1000), and then for one hour with the appropriate species-specific secondary antibody, before visualization. Expression levels of pSTAT5 and STAT5 proteins in each group were quantified by gray value analysis using ImageJ software(v1.8.0.). Mice DBA/1J and C57BL/6 mice, aged 8-10 weeks, were acquired from Nanjing Junke Biotechnology Co. These mice were housed under specific pathogen-free conditions and subjected to a 12-hour light/12-hour dark cycle (lights on at 7:00 AM). All experimental protocols involving these mice were approved by the Ethics Committee of Drum Tower Hospital, Nanjing Medical University. CIA induction and joint symptom evaluation On Day 0, 100 µL of Complete Freund’s Adjuvant and bovine collagen Type II were homogenized at high speed and injected into the tail-root area of each DBA/1J mouse, and on Day 21, Incomplete Freund’s Adjuvant and bovine collagen were similarly homogenized and injected. Arthritis scoring commenced on Day 22 and was independently conducted by a blinded professional unaware of the subgroups. Arthritis scoring was based on the following criteria: 0 - normal; 1 - erythema of the foot only, without significant swelling; 2 - erythema with mild swelling of the foot and ankle; 3 - erythema with significant swelling involving the toes and ankle joints; 4 - severe erythema, deformity, rupture, and bleeding. Cells culture Umbilical cord-derived mesenchymal stromal cells (UC-MSC) were cultured in DMEM/F12 complete medium (comprising 90% DMEM/F12 basal medium (Bio-Channel Biotechnology Co., Ltd.), 10% fetal bovine serum (FBS), and 1% Penicillin-Streptomycin Solution) in a cell incubator at 37°C and 5% CO2, and were passaged using trypsin-EDTA digestion upon reaching 80-90% confluency. Subsequent in vivo and in vitro experiments utilized 5th to 6th-generation UC-MSC. Lentivirus and MSC injections The mice were exposed to an infrared lamp for 5-10 minutes to fully dilate their tail veins, and subsequently secured in a mouse restrainer. Pre-prepared lentivirus or MSC was then slowly injected into the lateral tail vein using an insulin syringe, and any bleeding was halted with an alcohol-soaked cotton ball. Each mouse received a lentivirus dose of 10^8 UI and 5 x 10^5 MSC. MSC infusion was scheduled for arthritic mice on days 28, 35, 42, and 49 post-collagen induction. Lentiviral transfection of MSC Lentivirus (MOI=80) was added to well-growing MSC with a cell confluency of 60-70%, and the medium was replaced 12 hours post-transfection, followed by a further 48-hour incubation, after which GFP fluorescence was observed. Subsequently, the cells were screened with puromycin to obtain stably transfected MSC. Histopathology Following euthanasia, the right hind limb joints of the mice were excised and immersed in 4% paraformaldehyde (PFA), decalcified in 10% EDTA for 30 days, and subsequently embedded in paraffin. The joints were then assessed for inflammatory infiltration, cartilage destruction, and osteoclastogenesis using hematoxylin and eosin (H&E), Safranin O/Fast Green staining, and TRAP staining (Servicebio, Wuhan, China). Histological scoring of H&E-stained joint sections followed established scoring systems for synovial inflammation and cartilage destruction.[39]. Flow cytometry Single-cell suspensions were prepared from cultured cells, mouse spleens, and lymph nodes. Dead cells were excluded using eBioscience Fixable Viability Dye eFluor 506. Mouse cell surface antigens were stained with anti-mouse CD4 (FITC, RM4-5), CD25 (APC, PC61), CD8 (PE, S18018E), and NK1.1 (Alexa Fluor 700, S17016D) monoclonal antibodies at 4°C for 25 minutes. For nuclear cytokine and signaling molecule staining, cells were fixed and permeabilized post-surface staining, followed by staining with Foxp3 (Brilliant Violet 421, MF-14), Ki-67 (PE/Cyanine7, 11F6), and pSTAT5 (Tyr694) (PE, A17016B) at 4°C for 40 minutes. Data acquisition was performed using a BD LSR Fortessa (BD Biosciences), and analysis was conducted with FlowJo software (Tree Star). Enzyme-linked immunosorbent assay As per the manufacturer’s instructions for the IL-2 ELISA kit (BioLegend), MSC supernatant, mouse plasma, and gradient-diluted IL-2 standards were added to pre-coated enzyme-linked plates and incubated for 2 hours. These plates were then incubated with detection antibody and horseradish peroxidase (HRP) for 1 hour. Subsequently, the plates were treated with chromogen solution, incubated for 30 minutes in the dark, followed by the addition of the stop solution, and finally read by an enzyme-linked immunosorbent assay (ELISA) reader. A standard curve was plotted based on the concentration of the standards, and the IL-2 concentration in the samples was determined from their absorbance. Statistical Analysis Student’s t-tests and one-way ANOVAs (Analysis of Variance) were utilized for comparisons between two groups and multiple groups, respectively. Analysis was conducted using GraphPad Prism software (version 9.0, San Diego, CA). P-values less than 0.05 were considered statistically significant, denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ’ns’ indicates not significant. Construction of mutant IL-2 N103R V106D To construct the desired mutants, the wild-type IL-2 plasmid underwent site-directed mutagenesis through overlap extension PCR (Fig 1A) [34]. Asparagine at position 103 and valine at position 106 in wild-type IL-2 were substituted with arginine and alanine, respectively (Fig 1B) . The corresponding lentiviruses were generated using a triple plasmid system[35]. IL-2 secretion was confirmed in both wild-type and mutant IL-2 N103R V106D lentivirus-transfected 293T cells through ELISA assays of cell supernatants (Fig 1C) . Structural and physicochemical analyses confirmed that mutations at these two sites did not induce significant alterations in IL-2 structure or function (Table 1, Fig 1D) . IL-2 N103R V106D can target and activate mouse Treg cells We assessed the effect of transfection overexpressing mutant IL-2 on Treg cells in B6 mice by injecting lentivirus into the tail vein of the mice in vivo, Transfection of B6 mice with both wild-type and mutant IL-2 resulted in increased serum IL-2 levels in the mice (Fig 2A) , and mutant IL-2 N103R V106D lentiviral transfection in mice significantly increased the levels of splenic Treg cells compared to the wild-type (Fig 2B, C) . To further assess the effect of IL-2 N103R V106D on the proliferation of different immune cells, cell supernatants from wild-type and mutant IL-2 N103R V106D lentivirus-transfected 293T were injected intraperitoneally into B6 mice, Ki-67 + Treg cell levels in IL-2 N103R V106D-treated B6 mice were comparable to those in wild-type IL-2-treated mice, however, the proportion of Ki-67 + NK cells and CD8 + T cells was significantly lower than in the wild-type IL-2 group, a statistically significant difference (Fig 2D, E) . Conversely, the proportion of CD8 + T cells in IL-2 N103R V106D-treated B6 mice was significantly lower compared to the wild-type IL-2 group, while Treg cell levels remained similar in both (Fig 2F) . This indicates that IL-2 N103R V106D selectively targets Treg cell proliferation and activation, exerting minimal effects on other immune cells. IL-2 N103R V106D Targeted activation of IL-2 downstream PSTAT5 signaling pathway The JAK-STAT5 signaling pathway, recognized as a crucial pathway downstream of the IL-2 receptor, effectively responds to IL-2R activation through STAT5 phosphorylation. We assessed the phosphorylation levels of pSTAT5 in various cells using flow cytometry and western blot. Stimulation of T cells with wild-type IL-2 significantly increased STAT5 phosphorylation downstream of the IL-2 receptor[36], whereas mutant IL-2 N103R V106D stimulation led to reduced STAT5 phosphorylation levels (Fig 3A) . This disparity in phosphorylation levels primarily arises from wild-type IL-2 activating a broader spectrum of cells, including Treg and Tconv cells, while mutant IL-2 N103R V106D selectively activates Treg cells without inducing STAT5 phosphorylation in Tconv cells (Fig 3B) . Co-culturing wild-type IL-2 with spleen cells enhanced the proliferation and proportion of Treg, CD8 + T cells, and NK cells. Conversely, mutant IL-2 N103R V106D solely enhances the proliferation and proportion of Treg cells, and this proliferation is suppressed by STAT5 inhibitors (Figures 3C to H) . Owing to its decreased affinity for the IL-2 receptor subunit β, IL-2 N103R V106D targets the activation of the IL-2 trimeric receptor on Treg cells, thereby activating downstream STAT5 phosphorylation and promoting Treg cell proliferation. MSC overexpressing IL-2 N103R V106D can target and promote Treg cells proliferation and function We transfected MSC with constructed wild-type IL-2 and mutant IL-2 N103R V106D lentiviruses. Green fluorescence protein (GFP) validated the transfection efficiency of MSC (Fig 4A) . Following transfection, a significant increase in IL-2 levels in the MSC supernatant confirmed successful lentiviral transfection and stable expression of both wild-type and mutant IL-2 (Fig 4B) . Transfected MSCs retained their inherent osteogenic, chondrogenic, and lipogenic capabilities [37]. Upon co-culturing with splenocytes, the MSC transfected with mutant IL-2 N103R V106D lentivirus significantly increased Treg cell content (Fig 4C, D) . At both high and low concentrations, MSC-IL-2 N103R V106D supernatant elevated Treg cell content when co-cultured with splenocytes, in contrast to MSC-IL-2 wild-type, which increased the proportion of CD8 + T cells (Fig 4E) . MSC-IL-2 N103R V106D supernatant did not enhance the proportion or proliferation of CD8 + T cells and NK cells, unlike MSC-IL-2, and more effectively promoted a higher proportion of Treg cells compared to MSC or IL-2 N103R V106D alone (Figure 4F, G) . Furthermore, MSC-IL-2 N103R V106D significantly increased the expression of Treg-related functional genes (Fig 4H) . Conversely, wild-type IL-2 lentivirus-transfected MSC significantly elevated the expression of cytotoxic lymphocyte-associated genes, such as perforin and granzyme, a difference that was statistically significant (Fig 4I) . MSC-IL-2 N103R V106D is effective in treating rheumatoid arthritis by elevating Treg cell levels in CIA mice CIA mice were treated via tail vein injection with lentivirus-modified MSC, and joint symptom scores, along with histopathological sections of the ankle joints, were assessed. Lentiviral transfection of MSC overexpressing the mutant IL-2 N103R V106D injection significantly alleviated joint erythema and arthritis scores following injection into CIA mouse models via the tail vein (Figure 5A, B) . Arthropathologic histological sections revealed reduced inflammatory infiltration, cartilage damage, and bone erosion in ankle joints (Figure 5C) , and H&E hematological scores were lower in MSC-IL-2 N103R V106D-treated mice (Figure 5D) . Flow cytometry confirmed significantly elevated Treg cell levels in CIA mice injected with MSC-IL-2 N103R V106D, with a statistically difference (Figure 6A) , and it did not increase effector T cell levels, unlike MSC overexpressing wild-type IL-2 (Figure 6B) . Additionally, functional genes related to effector cells were not elevated in MSC-IL-2 N103R V106D-treated CIA mice (Figure 6C) . This confirms the therapeutic potential of lentiviral transfection of MSC overexpressing mutant IL-2 N103R V106D in treating rheumatoid arthritis, by selectively targeting and enhancing Treg cell proliferation and function without affecting effector T cells and NK cells. Discussion RA, a prevalent immune system disease primarily affecting small and medium-sized joints, has its pathogenesis significantly influenced by the scarcity of Treg cells, a critical immunoregulatory cell type [5, 6]. While Treg cell transplantation has shown efficacy in treating RA [40], this approach may encounter challenges such as low specificity and potential for non-specific rejection. IL-2, a critical immunomodulatory factor, has garnered sustained attention for its role in treating autoimmune diseases and cancer. Thirty years ago, IL-2 became the first immunotherapy drug approved by the US Food and Drug Administration for treating metastatic renal cell carcinoma and metastatic melanoma[41, 42]. Subsequently, the role of IL-2 in regulating Treg cells has been further explored, leading to its use in treating various autoimmune diseases[15, 16, 43]. Given IL-2 being able to activate various immune cells, ensuring its specific targeting in disease treatment is crucial. Additionally, overcoming the short half-life of IL-2 (<15 minutes) to prolong its action in the body and reduce drug administration frequency remains a key research focus. Low-dose IL-2 has demonstrated satisfactory efficacy and safety in clinical trials for the treatment of autoimmune diseases[43]. However, due to the short half-life of low-dose IL-2, prolonged and frequent administration of IL-2 does not result in a stable long-term increase in Treg cells and potentially causes off-target effects[31, 44]. Enhancing the targeting and half-life of IL-2 through engineering technology to achieve better efficacy is a topical area of current research, The four main types of IL-2 engineered proteins include IL-2 muteins(IgG–(IL-2N88D)2), PEGylated IL-2(NKTR-358), IL-2-anti-IL-2 immune complexes(IL-2–JES6) and IL-2-CD25 fusion proteins[27, 31, 45-47]. These engineered proteins preferentially stimulate the IL-2 trimeric receptor expressed on Treg cells by relatively decreasing the capacity of IL-2 to interact with CD122 (IL-2Rβ) and delaying IL-2 receptor-mediated clearing of IL-2 in vivo compared to wild-type IL-2, thereby prolonging half-life. These engineered IL-2 have achieved relatively satisfactory results in clinical trials or animal experiments. In this study, A mutant IL-2 N103R V106D, characterized by a reduced affinity for IL-2Rβ and enhanced dependency on the IL-2α receptor, was explored. This mutation facilitates targeted activation of Treg cells. Additionally, previous research indicates that MSC contribute to an increased proportion of Treg cells in patients with autoimmune diseases[48]. While both MSC infusion and low-dose IL-2 injection are effective individually for treating autoimmune diseases, previous studies suggest that their combination does not yield the desired therapeutic effect, potentially due to off-target activation of effector T cells[49]. Enhanced Treg cell targeting and more stable, sustained IL-2 expression in MSC were achieved by transfecting them with the mutant IL-2 N103R V106D lentivirus, offering a promising therapeutic approach for autoimmune diseases. By selectively targeting and elevating Treg cells, without activating or increasing CD8 + T cells and NK cells, MSC-IL-2 N103R V106D demonstrates an immunosuppressive effect and has proven effective in treating rheumatoid arthritis. The mutant IL-2 N103R V106D demonstrated improved targeting of the trimeric IL-2 receptor and Treg cells compared to wild-type IL-2, resulting in a reduced capacity to stimulate CD8 + T cells and NK cells. Transfecting MSC with this mutant enabled more stable expression and fewer treatment cycles. However, this mutant IL-2 variant increases the selectivity for CD25 (IL-2Rα) but reduces overall activity, necessitating higher treatment doses for efficacy. Additionally, activated effector T cells and CD56 bright NK cells may also express the trimeric IL-2 receptor in vivo, leading to off-target effects[31]. Lentiviral transfection of MSC ensured the efficient expression of IL-2 N103R V106D, Simultaneous inflammatory tropism, and stable IL-2 N103R V106D secretion in MSC reducing frequent drug administration and increasing the targeted delivery and therapeutic effect of IL-2 N103R V106D. Various engineered IL-2 mutants have been utilized in clinical research pertaining to oncology and autoimmune diseases. Our subsequent experimental objectives might involve conducting clinical studies on this specific mutant IL-2-modified MSC. MSCs are genetically modified via lentiviral transfection to secrete mutant IL-2 N103R V106D. This process requires further standardization to ensure its safety for use in clinical trials. Moreover, the therapeutic potential of MSC-IL-2 N103R V106D in autoimmune diseases such as systemic lupus erythematosus, Sjogren’s syndrome, and type 1 diabetes mellitus merits comprehensive exploration. Additionally, future investigations could focus on combining this mutant form of IL-2 with other biologics. This approach could integrate multiple mechanisms to modulate the immune response, potentially offering more extensive immunosuppression and enhanced therapeutic outcomes. An limitation of this study is the inability to purify the mutant IL-2 N103R V106D monomer, coupled with the exclusive reliance on lentivirally transfected 293T cell supernatants. This approach might inadvertently include the effects of extraneous substances present in the cell supernatants. Moreover, further exploration is needed to understand the specific pharmacokinetics and the levels and duration of in vivo IL-2 expression. Additionally, this study did not compare the therapeutic effects of mutant IL-2 N103R V106D with those of low-dose IL-2, despite the broader range of therapeutic doses available. Ultimately, Despite acting mainly on Treg cells, The precise mechanism driving the synergistic interaction between MSC and mutant IL-2 N103R V106D in the treatment of RA requires in-depth study. The study revealed that genetically modified MSC expressing mutant IL-2 N103R V106D effectively alleviated arthritis in CIA mice through targeted engagement of Treg cells. It was also shown that in vivo expansion and activation of Tregs are safer and more efficacious than in vitro expansion and subsequent implantation. Furthermore, to sustain this therapeutic benefit over extended periods, the employment of safer adeno-associated virus-mediated gene therapy emerges as a promising avenue for long-term expression of this mutant IL-2 in patients with the disease. To ensure long-term efficacy, adeno-associated virus-mediated gene therapy presents as a viable strategy for the sustained expression of mutant IL-2 in vivo[50], fostering prolonged and effective proliferation and activation of Treg cells in patients. The utilization of relevant animal models for experimentation further propels this therapeutic strategy, potentially paving the way for its application in clinical trials. Conflict of interest statement This manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. The authors declare that there are no financial or other relationships that might lead to a conflict of interest in the present article. All authors have reviewed the final version of the manuscript and approved it for publication. Author contribution Zhicheng Tang, Fan Yang and Jingyi Shen contributed equally to this manuscript as co-first authors. Zhicheng Tang and Jingyi Shen performed experiments, analyzed the data, and wrote the manuscript. Fan Yang revised the manuscript. Haolin Wu and Huiming Hong performed animal experiments. Yue Wang and Fanzhang Yin performed pilot experiments. Xiaojun Tang conceptualized the idea, provided technical support, and revised the manuscript. Huayong Zhang granted project funding and supervised the project. All authors approved the final manuscript. Ethical Statement Animal experiments are carried out under the project license (No. 2020AE01061). Funding The work was supported by the National Natural Science Foundation of China (grant numbers: 81802126, 81671608) Figure 1 : Construction and verification of the IL-2 Mutein N103R V106D. (A)Construction of the IL-2 Mutein N103R V106D . (B) Sequencing Results of Wild-Type IL-2 and IL-2 N103R V106D. (C) IL-2 Concentration in Cell Supernatants Post-Lentiviral Transfection of 293T Cells. (D) Comparative Analysis of the Structure of IL-2 N103R V106D (Predicted by AlphaFold) and Wild-Type IL-2. ****, P < 0. 0001. Table 1 Physical and chemical property prediction of IL-2 wild type and IL-2 N103R V106D Estimated half-life Extinction coefficients Instability index Aliphatic index Grand average of hydropathicity IL-2 Wild Type 4.4h 8605 70.16 81.81 -0.574 IL-2 N103R V106D 4.4h 8605 68.06 79.87 -0.633 Figure 2 : Targeted Activation of Treg Cells by IL-2 N103R V106D. (A) Plasma IL-2 Levels in Lentivirus-transfected C57BL/6 Mice. (B-C) Proportion of Treg Cells In Vivo in C57BL/6 Mice Transfected with Mutant IL-2 N103R V106D. (D-E) Ki-67 + Treg Cells, CD8 + T Cells, and NK Cells in the Spleen of C57BL/6 Mice Treated with Different Doses of Wild-Type and Mutant IL-2 N103R V106D. (F) Proportions of Treg Cells, CD8 + T Cells, and NK Cells in the Spleen of C57BL/6 Mice Treated with Wild-Type and Mutant IL-2 N103R V106D. *, P < 0.05, **, P < 0.01, ***, P < 0.001; ns, not significant. Figure 3 : Targeted Activation of STAT5 Phosphorylation in Treg Cells by IL-2 N103R V106D to Promote Proliferation. (A-B) Expression of Phosphorylated STAT5 (A) and STAT5 (B) in Splenocytes Stimulated with Wild-Type IL-2 and Mutant IL-2 N103R V106D. (C) Phosphorylation Levels of STAT5 in Treg and Non-Treg Cells Following Stimulation with Wild-Type IL-2 and Mutant IL-2 N103R V106D. (D-F) Proportion of Treg Cells (D), CD8 + T Cells (E), and NK Cells (F) in Splenocytes Co-Cultured with Supernatants from Wild-Type IL-2 and Mutant IL-2 N103R V106D-Transfected 293T Cells. (G-I) Ki-67 MFI of Treg Cells (G), CD8 + T Cells (H), and NK Cells (I) in Splenocytes Cultured with Supernatants from Mutant IL-2 N103R V106D-Transfected 293T Cells Treated with a STAT5 Inhibitor.*, P < 0. 05, **, P < 0. 01; ***, P < 0. 001; ****, P < 0. 0001; ns, not significant. Figure 4 : Enhancement of Treg Cell Function and Proportion by MSC-IL-2 N103R V106D. (A) Validation of MSC Transfection Efficiency by GFP Fluorescence. (B) IL-2 Concentration in Supernatants of Lentivirus-Transfected MSC. (C-D) Proportion of Treg Cells in Splenocytes Co-Cultured with Wild-Type IL-2 and Mutant IL-2 N103R V106D-Transfected MSC. (E) Proportion of Treg and CD8 + T Cells in Splenocytes Co-Cultured with Varying Concentrations of Supernatants from Wild-Type IL-2 and Mutant IL-2 N103R V106D-Transfected MSC. (F-G) Proportion (F) and Ki-67 + Levels (G) of Treg, CD8 + T Cells, and NK Cells in Splenocytes Co-Cultured with Supernatants from MSC and 293T Cells. (H-I) Expression Levels of Treg-Associated (H) and Effector T Cell-Associated Genes (I) in Splenocytes Co-Cultured with MSC-IL-2 N103R V106D and MSC-IL-2 WT. *, P < 0. 05, **, P < 0. 01; ***, P < 0. 001; ****, P < 0. 0001; ns, not significant. Figure 5 : Alleviation of Arthritis Severity and Scores by MSC-IL-2 N103R V106D. In this study, the CIA group received PBS treatment, while various MSC groups received four infusions of 5 x 10^5 cells per mouse via the tail vein. (A) Changes in Arthritis Scores Following MSC Infusion Treatment in Each Group. (B) Representative Joint Images from Different Groups. (C) Representative Images of Ankle Joints Stained with Hematoxylin and Eosin (H&E), Safranin O/Fast Green, and TRAP in Different Treatment Groups. (D) Evaluation of Histological Scores of H&E-Stained Sections in Different Groups. *, P < 0. 05. Figure 6 : Elevation of Treg Cell Proportion by MSC-IL-2 N103R V106D in CIA Mice Without Affecting CD8 + T Cells and Related Genes. 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Keywords arthritis collagen-induced arthritis interleukin-2 lentivirus mesenchymal stem cells regulatory rheumatoid t-lymphocytes Authors Affiliations Zhicheng Tang 0009-0003-1890-7209 Nanjing Medical University View all articles by this author Fan Yang Nanjing Medical University View all articles by this author Jingyi Shen Nanjing Medical University View all articles by this author Haolin Wu Nanjing University of Chinese Medicine View all articles by this author Huiming Hong Nanjing University of Chinese Medicine View all articles by this author Yue Wang Nanjing University of Chinese Medicine View all articles by this author Fanzhang Yin Nanjing University of Chinese Medicine View all articles by this author Xiaojun Tang Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital Department of Rheumatology and Immunology View all articles by this author Huayong Zhang [email protected] Nanjing Medical University View all articles by this author Metrics & Citations Metrics Article Usage 391 views 261 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Zhicheng Tang, Fan Yang, Jingyi Shen, et al. Mesenchymal Stromal Cells Over-expression Mutant IL-2 Enhance Treg Function in CIA Mice. Authorea . 01 June 2024. DOI: https://doi.org/10.22541/au.171722001.19584616/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); Cited by Jinsung Ahn, Bowon Kim, Alvin Bacero Bello, James J. Moon, Yoshie Arai, Soo-Hong Lee, Regenerative Functions of Regulatory T Cells and Current Strategies Utilizing Mesenchymal Stem Cells in Immunomodulatory Tissue Regeneration, Tissue Engineering and Regenerative Medicine, 22 , 2, (167-180), (2025). https://doi.org/10.1007/s13770-024-00690-w Crossref Loading... View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. 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