Inhibition of cGAS in dendritic cells suppresses maturation and prolongs allograft survival in mice

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Recent studies show that cyclic GMP-AMP synthase (cGAS) plays a crucial role in DC maturation. This study investigates the effect of suppressing cGAS expression in DCs on graft immune tolerance. Methods Bioinformatics analysis was conducted to explore the potential role of cGAS in transplant immunity. Immature dendritic cells (imDCs) were isolated, purified, and transduced with an adenovirus vector to suppress cGAS expression.EGFP-DCs group, cGAS-shRNA-DCs group, and control group(PBS), were injected via the tail vein prior to skin and islet transplantation for the establishment of a mouse transplantation model.Analyze graft survival and pathological changes, and use flow cytometry to assess spleen T cell subset proportions. After lipopolysaccharide stimulation, evaluate MHC-II and co-stimulatory molecule expression, antigen phagocytosis, and T cell proliferation in the imDCs (control), EGFP-DCs, and cGAS-shRNA-DCs groups. The supernatants from every group were collected, and the changes in cytokine secretion by DCs were detected using ELISA. Results Bioinformatics analysis shows increased cGAS expression in the allograft group. The cGAS-shRNA-DCs group showed reduced MHC-II and co-stimulatory molecule expression, enhanced phagocytic activity, and decreased T cell activation ability. Levels of IFN-γ, IL-1β, TNF-α, and IL-6 were lower, while IL-10 levels were higher. Mice receiving cGAS-shRNA-DCs had prolonged graft survival and improved graft function. Flow cytometry revealed an increased proportion of regulatory T cells and reduced Th1 and Th17 cell populations. Conclusions Inhibiting cGAS expression in DCs reduces their maturation, antigen-presenting capacity, and T-cell activation, ultimately prolonging graft survival. Biological sciences/Immunology/Translational immunology Biological sciences/Genetics/Gene expression Biological sciences/Genetics/Clinical genetics cGAS immune tolerance dendritic cells transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Organ transplantation serves as a critical therapeutic approach for managing end-stage diseases [ 1 ] . Specifically, islet transplantation has demonstrated efficacy in treating type 1 diabetes by regulating blood glucose levels and improving patient prognosis [ 2 ] .Liver transplantation and kidney transplantation are widely utilized in clinical practice and have become established as effective treatment options for liver cirrhosis [ 3 ] and uremia [ 4 ] , respectively.However, postoperative rejection reactions reduce graft survival time and adversely affect patients' quality of life. Moreover, the use of immunosuppressive drugs after transplantation increases the risk of postoperative infections and tumor recurrence [ 5 ] . Therefore, the exploration of novel strategies to induce immune tolerance is of critical importance for improving graft survival outcomes. Dendritic cells (DCs) serve as a critical link between the innate and adaptive immune systems [ 6 ] . A primary function of dendritic cells is to present antigens to T lymphocytes [ 7 ] . Mature dendritic cells (mDCs) present antigens to CD4 + and CD8 + T cells via MHC class I and class II molecules, respectively, thereby initiating robust T cell responses. In contrast, immature dendritic cells (imDCs) exhibit strong endocytic activity but have limited capacity to activate T cells, which is associated with immune tolerance [ 8 ] . Upon encountering appropriate stimuli, imDCs can differentiate into mDCs. Accumulating evidence suggests that the administration of imDCs prior to transplantation can mitigate postoperative immune rejection [ 9 ] . Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), a member of the nucleotidyltransferase family and a pattern recognition receptor (PRR), is capable of detecting cytosolic DNA fragments and synthesizing cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) [ 10 ] . Emerging evidence indicates that cGAS is predominantly expressed in dendritic cells [ 11 ] and macrophages [ 12 ] , where it plays a pivotal role in inflammatory responses [ 13 ] , tumor immunity [ 14 ] , and ischemia-reperfusion injury [ 15 ] . Upon DNA sensing, cGAS activates the stimulator of interferon genes (STING), thereby promoting the nuclear translocation of transcription factors such as nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3), which induce the production of type I interferons (IFNs) and pro-inflammatory cytokines [ 16 ] . These signaling events are closely associated with immune responses in organ transplantation [ 17 ] . In this study, immature dendritic cells (imDCs) were successfully isolated and purified. The cells were then transduced with an adenoviral vector (AdV-cGAS-shRNA-GFP) to establish a model of dendritic cells with suppressed cGAS expression. Mouse models of skin and islet transplantation were subsequently employed to investigate the role of cGAS in regulating dendritic cell (DC) maturation and its impact on graft immune protection. Materials and Method 2.1Bioinformatics Analysis To preliminarily explore the potential role of cGAS in transplant immune regulation, in R v4.4.1, tidyverse was used to process the transcriptome sequencing data (GSE216869). The DESeq2 package was utilized for differential gene expression analysis, with the criteria for identifying differentially expressed genes set as a log2 fold change > 2 and a corrected p-value < 0.05. After determining these genes, the ClusterProfiler package was applied for GO and KEGG enrichment analysis, with significance levels set at p < 0.05 and q = 1. Additionally, the GSEA package was used for gene set enrichment analysis, with the same thresholds of p < 0.05 and q = 1. 2.2 Animals Male C57BL/6 mice (H-2 b ) and male BALB/c mice (H-2 d ), aged 8–10 weeks and weighing 25–30 g, were obtained from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China), under specific pathogen-free (SPF) conditions.All animal experiments adhered to the guidelines set forth by the Animal Protection and Use Committee of Sun Yat-sen University. The experimental protocol was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.In skin transplantation and islet transplantation experiments, C57BL/6 mice served as the transplant recipients, while BALB/c mice acted as the transplant donors. 2.3 Regents Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) were obtained from PeproTech (Rocky Hill, NJ, USA).The AdV-cGAS-shRNA-GFP adenovirus vector was constructed by Beijing Xibei Hongcheng Biotechnology Co., Ltd. and further optimized to enhance the transduction efficiency DCs. The anti-CD11c and LS microbead-conjugated separation columns were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).Nylon wool fiber syringes were purchased from Polysciences, Inc. (Eppelheim, Germany). Rabbit anti-mouse cGAS primary antibody was obtained from Abcam (Cambridge, MA, USA). Mouse anti-mouse GAPDH primary antibody and goat anti-rabbit and anti-mouse secondary antibodies were purchased from Proteintech Group, Inc. (Wuhan). Cell stimulation cocktail plus protein transport inhibitor, Fixable Viability Dye 780, Transcription Factor Buffer Set, rat anti-mouse fluorescent-conjugated CD45-BUV395, CD3-PerCP-Cy5.5, CD4-BV711, CD25-BB515, Foxp3-PE, IFNγ-BV421, IL−17-AF647, FITC-CD11c, BV421-CD80, PE-Cy7-CD86, PE-MHC-II (I-A/I-E) monoclonal antibodies, and corresponding isotype controls were purchased from BD PharmingenTM (San Diego, CA, USA). Rabbit anti-mouse insulin and glucagon monoclonal antibodies were obtained from Abcam (Cambridge, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for detecting the concentrations of IL−10, IL−6, IL−1β, IFN-γ, and TNF-α were purchased from Jiangsu Meimian Industrial Co., Ltd. (China, Jiangsu). Streptozotocin (STZ), lipopolysaccharide (LPS), Histopaque 1077, and tetramethylrhodamine-dextran (TRITC-dextran) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dithizone (DTZ), acridine orange (AO), and propidium iodide (PI) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Liberase TL, blood glucose meter, and blood glucose test strips were purchased from Roche (Basel, Switzerland). 2.4 Isolation and Purification of Bone Marrow-Derived DCs(BMDCs) Sterile surgery was performed to isolate the tibia and femur from 5 to 8 week-old BALB/c mice, with the bone marrow serving as the source of BMDCs (bone marrow-derived dendritic cells).DCs are differentiated from bone marrow precursor cells through the application of GM-CSF and IL-4. Following red blood cell lysis, the remaining cells were resuspended in complete medium consisting of RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 20 ng/mL recombinant murine GM-CSF, 10 ng/mL IL-4, and 1% penicillin-streptomycin solution.Cells were then seeded at a density of 1 × 10 6 cells/mL in 6-well plates and cultured in a humidified incubator at 37°C with 5% CO 2 .On the 6th day of culture, DCs are fully induced. Subsequently,imDCs are purified via immunomagnetic selection using anti-CD11c-conjugated microbeads to isolate CD11c + cells. 2.5 AdV-cGAS-shRNA-GFP Transfection of DCs Recombinant adenovirus vector (rAd-cGAS-shRNA) or negative control virus vector (rAd-EGFP) was added into purified imDCs at a multiplicity of infection (MOI) of 1:100,and cultured in serum-free OptiMEM medium for 48 hours. Subsequently, two types of cells, EGFP-DCs and cGAS-shRNA-DCs,were obtained. 2.6 Western Blotting A total of 20 µg of protein extracts from imDCs,cGAS-shRNA-DCs and DC-GFP groups were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a nitrocellulose membrane pre-treated with methanol. The membrane was incubated overnight at 4°C with primary antibodies against mouse cGAS(1:1000) and GAPDH(1:10000), followed by a 1-hour incubation with the corresponding secondary antibodies(1:5000). Protein bands were visualized using ECL chemiluminescence, and the gray-scale intensity of the bands was quantified using ImageJ software. 2.7 Pancreatic Islet Transplantation 7 days prior to the islet transplantation, 2×10 6 cells of EGFP-DCs, cGAS-shRNA-DCs, and an equivalent volume of PBS were respectively injected via the tail vein into three groups of C57BL/6 mice and randomly divided in to three groups:EGFP-DCs group, cGAS-shRNA-DCs group and contral group. Diabetes was induced in the C57BL/6 mice through intraperitoneal injection of streptozotocin (200 mg/kg). Islet transplantation was subsequently performed when the recipients' blood glucose levels remained at or above 16.7 mmol/L for 3 consecutive days.Islets were isolated from BALB/c mice. Following clamping of the ampulla of Vater, the common bile duct was punctured, and the pancreas was perfused with Liberase TL. The pancreas was subsequently harvested and stored on ice. After digestion in a 37°C water bath, islet purification was performed using density gradient separation (Histopaque 1077). A total of 350 isolated islets were transplanted under the renal capsule of the recipient using a glass capillary probe. Transplantation was deemed successful if the blood glucose level was maintained below 11.1 mmol/L. Islet graft rejection was defined as a blood glucose level exceeding 16.7 mmol/L for at least two consecutive days and construct the survival curve. 2.8 Intraperitoneal Glucose Tolerance Test 7 days after islet transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, and the kidneys containing transplanted islets were harvested at this time point. Specifically, following a 4–6 hours fasting, mice were intraperitoneally administered a glucose solution (2 g/kg), and tail vein blood glucose levels were measured at 15-minute intervals for a total of 120 minutes. The IPGTT was subsequently repeated after the removal of kidneys harboring the transplanted islets to ensure that the observed recipient blood glucose response was attributable to the functional activity of the donor islet allografts. 2.9 Skin Transplantation C57BL/6 mice were divided into three groups, similar to the grouping strategy used in islet transplantation studies.The tails of BALB/c mice were disinfected with alcohol three times. A 1×1 cm square skin flap was then prepared. The skin flap harvested from BALB/c mice was transplanted onto the back of C57BL/6 mice and sutured using 5 − 0 sutures. Postoperatively, the progression of rejection was assessed by daily observation of changes in the donor skin flaps. Complete rejection was defined as the necrotic area of the successfully transplanted skin flap exceeding 80%. 2.10 Flow Cytometry Obtain purified imDCs and divide them into three groups according to the previous transfection method: imDCs (control group), EGFP-DCs (negative control for transfection), and cGAS-shRNA-DCs (experimental group).3 groups were stimulated to mature through the addition of 1 ug/ml LPS.To evaluate the maturity of cells, in the dark at 4 degrees Celsius for 30 minutes,three groups of cells were stained with BD fluorescein-conjugated monoclonal antibodies against CD86, CD80, and MHC-II and analyzed with a flow cytometer (BD LSRFortessa X-20, USA).After 24 hours of LPS stimulation, to evaluate the phagocytic capacity of DCs, 1 µl of TRITC-Dextran (25 µg/mL) was added to each of the 3 groups of cells. Subsequently, the cells were incubated at 37°C or 4°C for 2 hours. Following this, the treated cells were harvested, and the proportion of TRITC-positive cells was quantified by flow cytometry. On the 7th day post-transplantation, lymphocytes were harvested from the recipient's spleen and stimulated vivo with a cell stimulation cocktail in combination with a protein transport inhibitor for 4 to 6 hours. Subsequently, Th1, Th17,regulatory T cells (Treg), and intracellular cytokine staining analyses were performed. Cells were fixed and permeabilized using a Foxp3/transcription factor staining buffer set, followed by intracellular cytokine staining. Flow cytometry data were analyzed using FlowJo software. CD3 + /CD4 + /IFN-γ + cells were identified as Th1 cells, CD3 + /CD4 + /IL-17 + cells as Th17 cells, and CD4 + /CD25 + /Foxp3 + cells as Treg cells. 2.11 Immunohistochemistry and Hematoxylin and Eosin (HE) Analysis On the 7th day after transplantation, the graft tissues were harvested, fixed with 10% polyformaldehyde, paraffin-embedded, and cut into 3–5 µm thick sections. These sections were stained with HE or anti-mouse insulin or glucagon monoclonal antibodies according to the manufacturer's protocol. HE staining was also performed on the skin grafts.The IHC and H&E experimental results were analyzed using the NanoZoomer® S360 and NDP.view 2.9.22 RUO software. 2.12 ELISA Collect the supernatants of the 3 groups of cells after 24h LPS stimulation. Use the ELISA kit method to detect the concentrations of cytokines (IL-10, IL-6,TNF-α,IL-1β, IFN-γ). 2.13 Mixed Leukocyte Reaction (MLR) Analysis Harvest the spleens from 6 to 8 week-old C57BL/6 mice to prepare a spleen cell suspension. Purify T cells by passing the suspension through a nylon wool column according to the manufacturer's instructions, and collect the filtered T cell suspension. Subsequently, perform cell counting.After LPS stimulation,obtain 3 groups of cells and add mitomycin C (25 µg/mL) and incubate the cells at 37°C for 2 hour. Subsequently, wash the cells 3 times with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Adjust the cell concentration to 1×10 6 cells/mL and resuspend in complete medium.Take a 96-well plate and seed DCs and T cells into each well at ratios of 1:10, 1:20, and 1:50. Establish 3 replicate wells for each group and include a blank control group.The 96-well plates were incubated at 37°C with 5% CO 2 for 72 hours. 4 hours prior to the conclusion of the incubation, 8 µl of CCK-8 reagent was added to each well. Following the completion of the incubation period, the absorbance of each well was measured at 450 nm using a multi-functional microplate reader. The stimulation index (SI)= (OD sample − OD blank control )/ (OD negative control − OD blank control ). 2.14 Statistical Analysis Data are expressed as mean ± standard deviation. Student's t-test was employed to assess the differences between two groups. One-way analysis of variance (ANOVA) was utilized for comparisons among multiple groups, followed by Tukey's post hoc test where applicable. Kaplan-Meier survival curves were constructed, and the Log-rank test was applied to evaluate the differences in graft survival rates. P value < 0.05 was considered indicative of statistical significance. All statistical analyses were conducted using GraphPad Prism 9.0 and SPSS 22.0 software. Results 3.1 Bioinformatics analysis demonstrated that cGAS exhibited elevated expression levels in the allograft group. In the postoperative spleen transcriptome of the allograft group (BALB/c→C57BL/6) and the syngeneic control group (isograft group, C57BL/6→C57BL/6) in the allogeneic heart transplantation model, cGAS was identified as a differentially expressed gene through differential expression analysis, with its expression significantly upregulated in the allograft group compared to the syngeneic controls(Fig. 1A). KEGG enrichment analysis revealed that the primary enriched BP included cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF signaling pathway, Th17 cell differentiation,allograft rejection, and graft-versus-host disease(Fig. 1C).GO enrichment analysis revealed that the BP primarily included the positive regulation of leukocyte activation, regulation of T cell activation, positive regulation of immune effector processes, myeloid cell differentiation, immune response-regulating signaling pathways, antigen receptor-mediated signaling pathways, positive regulation of chemokine production, and regulation of the canonical NF-kappaB signaling pathway(Fig. 1D).GSEA analysis indicated that the differentially expressed genes were predominantly involved in BP such as APC-mediated cell cycle proteins, apoptosis, cell cycle regulation, the innate immune system, and the IL-7 signaling pathway(Fig. 1B). 3.2Genetically modified adenovirus vector increases the transduction efficiency of DCs CD11c + cells were magnetically isolated using anti-CD11c-conjugated microbeads, achieving a purity of over 90% for the CD11c + cell(Fig. 2A, B ).Purified DCs were transduced with AdV-cGAS-shRNA-GFP (cGAS-shRNA-DCs) or AdV-GFP (DC-GFP) at a multiplicity of infection (MOI) of 1:100 for 48 hours. The expression of GFP was confirmed by fluorescence microscopy through the detection of GFP fluorescence(Fig. 2C).Furthermore, the expression of GFP was analyzed via flow cytometry, revealing that over 80% of the DCs were effectively transduced by the adenovirus vector(Fig. 2D).Western blot analysis revealed a significant reduction in the level of cGAS protein in cGAS-shRNA-DCs. These results suggest that the adenovirus vector efficiently delivered the cGAS-shRNA gene into DCs(Fig. 2E, F ). 3.3AdV-cGAS-shRNA-GFP suppressed the maturation of dendritic cells. AdV-cGAS-shRNA reduces the expression of MHC-II and co-stimulatory molecules. In this study, purified imDCs, DC-GFP, and cGAS-shRNA-DCs were treated with LPS (1 µg/mL) for 24 hours. The expression levels of CD80, CD86, and MHC-II were subsequently analyzed by flow cytometry. The results demonstrated that, compared to other two groups, the cGAS-shRNA-DCs group exhibited significantly reduced expression levels of CD80, CD86, and MHC-II(Fig. 3). At the same time, the supernatants of the three groups of cells were collected after 24 hours of LPS treatment. The results demonstrated that cGAS-shRNA-DCs cells secreted significantly higher levels of the immunosuppressive cytokine IL-10 compared to other two groups, while exhibiting reduced secretion of the immunostimulatory cytokines IL-6, IL-γ, TNF-α,and IL-1β(Fig. 4A). TRITC-dextran phagocytosis assay demonstrated that LPS stimulation significantly diminished the phagocytic ability of control and DC-GFP groups, which was associated with increased maturity of DCs. In contrast, LPS stimulation had minimal impact on the phagocytic capacity of cGAS-shRNA-DCs.Notably, the cGAS-shRNA-DCs group exhibited a markedly enhanced phagocytic capacity compared to the other two groups, despite displaying lower levels of DCs maturity(Fig. 4B). 3 groups cells treated with LPS were co-cultured with T cells at different ratios for 72 hours to evaluate the changes in T cell maturation and activation ability and were co-cultured with T cells at different ratios (1:10,1:20 and 1:50) for 72 hours to assess the changes in T cell proliferation and activation capacity. (Fig. 5A)The MLR assay demonstrated that cGAS-shRNA-DCs exhibited a significantly lower stimulatory capacity compared to the other two groups, with statistically significant differences observed at 1:10 and 1:20 ratios(Fig. 5B). These researches demonstrates that inhibiting cGAS expression in DCs not only diminishes the expression of co-stimulatory molecules and MHC-II but also impairs the maturation of DCs, thereby reducing their antigen-presenting capability and the subsequent activation of T cells. 3.4 cGAS-shRNA-DCs preserves the function of allografts and prolongs graft survival. In this study, mouse islet transplantation and skin transplantation models were performed to investigate the effects of cGAS-shRNA-DCs on graft survival and function. Before islet transplantation, DTZ staining and AO-PI staining confirmed that the isolated islets exhibited high viability and purity(Fig. 6B).HE staining, insulin staining, and glucagon staining revealed that the number and activity of residual islets in the cGAS-shRNA-DCs group were significantly higher than those in the other two groups, with less lymphocyte infiltration(Fig. 6C).HE staining of skin grafts revealed that the degree of hair follicle destruction, dermal and subcutaneous tissue edema, and lymphocyte infiltration was less severe in the cGAS-shRNA-DCs group compared to the other two groups(Fig. 7B). To investigate the in vivo functional activity of islet grafts, an intraperitoneal glucose tolerance test (IPGTT) with a dose of 2 g/kg was conducted on the 7th day post-transplantation. Glucose tolerance was assessed after a fasting period of 4–6 hours, and blood glucose levels were measured from tail vein blood samples every 15 minutes for a total duration of 120 minutes.After glucose injection, blood glucose levels in all groups increased sharply, peaking at 15 minutes and then gradually declining over time. Notably, the cGAS-shRNA-DCs group exhibited a more rapid response to glucose compared to the other groups(Fig. 6D).And then, the kidneys containing islet allografts were removed, and the intraperitoneal glucose tolerance test (IPGTT) was repeated. The results demonstrated that the blood glucose response in recipient mice was dependent on the presence of islet allografts(Fig. 6E).In terms of graft survival, both skin and islet grafts in the cGAS-shRNA-DCs group exhibited significantly prolonged survival times compared to those in the other two groups(Fig. 6A,7A). 3.5cGAS-shRNA-DCs modulate the proportions of T cell subsets in the spleen in vivo. To investigate the potential mechanism by which cGAS-shRNA-DCs prolong graft survival, we analyzed the proportions of T cell subsets in mouse islet transplantation model. Spleen single-cell suspensions were collected from recipient mice in each group on 7th day post-transplantation. Following staining with fluorescently-labeled monoclonal antibodies, flow cytometry analysis was conducted. The results demonstrated that, compared with other two groups, the proportions of Th1 and Th17 cells were significantly reduced, whereas the proportion of Treg cells was elevated in the cGAS-shRNA-DCs group(Fig. 8). Discussion This study, using skin and islet transplantation models, showed that inhibiting cGAS expression in DCs reduces co-stimulatory molecules CD80, CD86, and MHC-II [ 18 ] levels and significantly prolongs graft survival. Moreover, flow cytometry analysis of T cell subsets in the recipient spleen revealed an increased proportion of Treg cells alongside decreased proportions of Th1 and Th17 cells, suggesting that the immune response was modulated toward suppression following transplantation.This study utilized western-blot and flow cytometry to confirm that the expression of cGAS was effectively inhibited and the transduction efficiency was notably high. Research has demonstrated that upon activation by dsDNA, cGAS catalyzes the production of cGAMP, which subsequently binds to STING [ 19 , 20 ] , thereby activating downstream signaling pathways and promoting the secretion of cytokines such as IFN-I [ 21 ] and IL-6 [ 22 ] . The cGAS-STING pathway is regarded as a critical bridge connecting innate and adaptive immunity through its regulation of IFN-I-mediated maturation and migration of DCs [ 23 , 24 ] . cGAS is an innovative target in transplantation immunity. To gain deeper insights into the role of cGAS in promoting DC maturation, we employed AdV-cGAS-shRNA-GFP to transduct DCs and establish a cell model with suppressed cGAS expression.The results demonstrate that inhibiting cGAS suppresses DCs maturation, as evidenced by reduced expression of MHC-II and co-stimulatory molecules [ 25 ] . DCs are capable of antigen presentation and play a critical role in activating and maturing T cells [ 26 ] . imDCs exhibit impaired antigen-presenting function, and T cell functionality is significantly influenced by the cytokine profile and the expression levels of co-stimulatory molecules on DCs [ 27 ] . Consequently, the immature state of DCs can induce immune tolerance by attenuating T cell activation and modulating cytokine production. Our research further reveals that inhibiting cGAS in vitro diminishes the antigen-presenting capacity of DCs, restrains T cell proliferation, and decreases the secretion of cytokines such as IL-6 and IFN-γ. The protective effect of cGAS on pancreatic islet grafts and skin grafts in mice was investigated through preoperative tail vein injection of cGAS-shRNA-DCs. The survival duration of the grafts as well as the pathological changes in the grafts post-surgery were analyzed. The results demonstrated that cGAS-shRNA-DCs significantly prolonged graft survival time and mitigated postoperative immune rejection responses. Furthermore, our findings revealed that recipients treated with cGAS-shRNA-DCs exhibited superior glucose tolerance compared to other two groups, which corroborates the prolonged survival of islet grafts.Pathological results demonstrated that in both skin transplantation and islet transplantation, the HE staining revealed a significant reduction in inflammatory infiltration following the reinfusion of cGAS-shRNA-DCs. Additionally, immunohistochemistry analysis showed increased expression levels of insulin and glucagon.Subsequently, we examined the alterations in T cell subsets within the spleen. Specifically, Th1 and Th17 cells are known to mediate pro-inflammatory immune responses [ 28 , 29 ] , whereas Treg cells play a critical role in suppressing immune activation [ 30 ] . Our results demonstrated that in the cGAS-shRNA-DCs group, the proportions of Treg cells was significantly increased, while the proportions of Th1 and Th17 cells were markedly reduced. This transformation in T cell subset composition effectively attenuated immune responses, thereby contributing to the induction of immune tolerance.In this study, we demonstrated through islet transplantation and skin transplantation that cGAS influences adaptive immune responses by regulating the maturation of DCs. Consequently, it plays a role in modulating immune rejection reactions and may serve as a potential target for inducing immune tolerance. However, the underlying mechanisms remain to be fully elucidated, necessitating further investigation. Conclusions Inhibition of cGAS expression in DCs suppresses their maturation, reduces the expression of co-stimulatory molecules and MHC-II, and diminishes their antigen-presenting capacity and ability to activate T cells. In vivo administration of cGAS-shRNA-transfected DCs significantly prolongs graft survival, increases the proportions of Treg cells, and decreases the proportions of Th1 and Th17 cells. This transformation correlates with reduced production of pro-inflammatory factors and enhanced secretion of anti-inflammatory cytokines. Consequently, targeting cGAS may represent a novel strategy for inducing immune tolerance in transplantation. Declarations Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This study was supported by grants from the Natural Science Foundation of Guangdong Province (2023A1515011805 and 2022A1515011052), the National Natural Science Foundation of China (81873591), the Science and Technology Planning Project of Guangdong Province (2018A050506030), the Science and Technology Program of Guangzhou (201704020073), the Guangdong Provincial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology (2013A061401007, 2017B030314018, and 2020B1212060026), and the Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation) (2015B050501002 and 2020A0505020003). References Durand, F., et al., Age and liver transplantation. J Hepatol, 2019. 70 (4): p. 745–758. Shapiro, A.M., M. 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Ablasser, A., et al., Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature, 2013. 503 (7477): p. 530–4. Robb, R.J., et al., Type I-IFNs control GVHD and GVL responses after transplantation. Blood, 2011. 118 (12): p. 3399–409. Hong, C., et al., cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers. Nature, 2022. 607 (7918): p. 366–373. Si, W., et al., Lactobacillus rhamnosus GG induces cGAS/STING- dependent type I interferon and improves response to immune checkpoint blockade. Gut, 2022. 71 (3): p. 521–533. Lam, E., S. Stein, and E. Falck-Pedersen, Adenovirus detection by the cGAS/STING/TBK1 DNA sensing cascade. J Virol, 2014. 88 (2): p. 974–81. Bayer-Santos, E., et al., The Salmonella Effector SteD Mediates MARCH8-Dependent Ubiquitination of MHC II Molecules and Inhibits T Cell Activation. Cell Host Microbe, 2016. 20 (5): p. 584–595. Bosnjak, B., et al., Imaging dendritic cell functions. Immunol Rev, 2022. 306 (1): p. 137–163. Peyneau, M., et al., Quaternary ammoniums activate human dendritic cells and induce a specific T-cell response in vitro. Allergol Int, 2025. 74 (1): p. 105–114. Torchinsky, M.B., et al., Innate immune recognition of infected apoptotic cells directs T(H)17 cell differentiation. Nature, 2009. 458 (7234): p. 78–82. Xanthou, G., et al., Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets. Nat Med, 2007. 13 (5): p. 570–8. Dikiy, S. and A.Y. Rudensky, Principles of regulatory T cell function. Immunity, 2023. 56 (2): p. 240–255. Additional Declarations There is NO conflict of interest to disclose. there is NO conflict of interest to disclose Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Genes & Immunity → Version 1 posted Editorial decision: revise 01 Oct, 2025 Review # 2 received at journal 23 Sep, 2025 Reviewer # 2 agreed at journal 09 Sep, 2025 Review # 1 received at journal 20 Aug, 2025 Reviewer # 1 agreed at journal 18 Aug, 2025 Reviewers invited by journal 17 Aug, 2025 Submission checks completed at journal 13 Aug, 2025 Editor assigned by journal 05 Aug, 2025 First submitted to journal 05 Aug, 2025 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. 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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-7302546","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":501530512,"identity":"9a9ab5fd-85ec-411b-aa25-e197aa33a484","order_by":0,"name":"Yi 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University","correspondingAuthor":false,"prefix":"","firstName":"Hanyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":501530514,"identity":"850494e5-7854-49ee-b0c4-9b1b8cd54933","order_by":2,"name":"Long Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Zhang","suffix":""},{"id":501530515,"identity":"8a1398c0-b54d-4140-a067-6358bf89dc98","order_by":3,"name":"Hanyu Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hanyu","middleName":"","lastName":"Wang","suffix":""},{"id":501530516,"identity":"737aa5c0-90d1-482f-818d-a7fc2153ea44","order_by":4,"name":"Xuzhi Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuzhi","middleName":"","lastName":"Zhang","suffix":""},{"id":501530517,"identity":"8dd75909-4fe8-480c-a86d-3fa9966bbf2f","order_by":5,"name":"Shuai Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Wang","suffix":""},{"id":501530518,"identity":"217a7f4d-5489-45d2-b779-bdc335ec6e83","order_by":6,"name":"Yifang Gao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yifang","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2025-08-05 16:00:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7302546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7302546/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41435-026-00381-7","type":"published","date":"2026-03-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89821838,"identity":"74ec5517-10af-4679-9609-7dd2c997b265","added_by":"auto","created_at":"2025-08-25 11:39:09","extension":"png","order_by":1,"title":"Figure 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5","display":"","copyAsset":false,"role":"figure","size":256568,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7302546/v1/a03e856dead21ba87b8747c4.png"},{"id":89821853,"identity":"38ec4c14-5ccc-469d-8498-6fbc37a118d5","added_by":"auto","created_at":"2025-08-25 11:39:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7080230,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7302546/v1/223b6ffbb2b5f533db61bfbb.png"},{"id":89822002,"identity":"705da40c-fa2e-44c5-9fe3-079fb661b8ae","added_by":"auto","created_at":"2025-08-25 11:47:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3447974,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7302546/v1/70fd0079d2220bcff738a849.png"},{"id":89822000,"identity":"a2acce11-c269-4113-a763-00b1fefe3bd3","added_by":"auto","created_at":"2025-08-25 11:47:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1552222,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7302546/v1/47e74a461dded1b37af70b37.png"},{"id":103892045,"identity":"fdad2792-d95a-48ed-82c5-639764855758","added_by":"auto","created_at":"2026-03-04 08:14:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31192241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7302546/v1/b623899b-aebe-48d3-9011-81ffb675f266.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.\nthere is NO conflict of interest to disclose","formattedTitle":"Inhibition of cGAS in dendritic cells suppresses maturation and prolongs allograft survival in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOrgan transplantation serves as a critical therapeutic approach for managing end-stage diseases\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Specifically, islet transplantation has demonstrated efficacy in treating type 1 diabetes by regulating blood glucose levels and improving patient prognosis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.Liver transplantation and kidney transplantation are widely utilized in clinical practice and have become established as effective treatment options for liver cirrhosis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e and uremia\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, respectively.However, postoperative rejection reactions reduce graft survival time and adversely affect patients' quality of life. Moreover, the use of immunosuppressive drugs after transplantation increases the risk of postoperative infections and tumor recurrence\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, the exploration of novel strategies to induce immune tolerance is of critical importance for improving graft survival outcomes.\u003c/p\u003e\u003cp\u003eDendritic cells (DCs) serve as a critical link between the innate and adaptive immune systems\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. A primary function of dendritic cells is to present antigens to T lymphocytes\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Mature dendritic cells (mDCs) present antigens to CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells via MHC class I and class II molecules, respectively, thereby initiating robust T cell responses. In contrast, immature dendritic cells (imDCs) exhibit strong endocytic activity but have limited capacity to activate T cells, which is associated with immune tolerance\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Upon encountering appropriate stimuli, imDCs can differentiate into mDCs. Accumulating evidence suggests that the administration of imDCs prior to transplantation can mitigate postoperative immune rejection\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), a member of the nucleotidyltransferase family and a pattern recognition receptor (PRR), is capable of detecting cytosolic DNA fragments and synthesizing cyclic guanosine monophosphate-adenosine monophosphate (cGAMP)\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence indicates that cGAS is predominantly expressed in dendritic cells\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e and macrophages\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, where it plays a pivotal role in inflammatory responses\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, tumor immunity\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, and ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Upon DNA sensing, cGAS activates the stimulator of interferon genes (STING), thereby promoting the nuclear translocation of transcription factors such as nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3), which induce the production of type I interferons (IFNs) and pro-inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. These signaling events are closely associated with immune responses in organ transplantation\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, immature dendritic cells (imDCs) were successfully isolated and purified. The cells were then transduced with an adenoviral vector (AdV-cGAS-shRNA-GFP) to establish a model of dendritic cells with suppressed cGAS expression. Mouse models of skin and islet transplantation were subsequently employed to investigate the role of cGAS in regulating dendritic cell (DC) maturation and its impact on graft immune protection.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1Bioinformatics Analysis\u003c/h2\u003e\u003cp\u003eTo preliminarily explore the potential role of cGAS in transplant immune regulation, in R v4.4.1, tidyverse was used to process the transcriptome sequencing data (GSE216869). The DESeq2 package was utilized for differential gene expression analysis, with the criteria for identifying differentially expressed genes set as a log2 fold change\u0026thinsp;\u0026gt;\u0026thinsp;2 and a corrected p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. After determining these genes, the ClusterProfiler package was applied for GO and KEGG enrichment analysis, with significance levels set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and q\u0026thinsp;=\u0026thinsp;1. Additionally, the GSEA package was used for gene set enrichment analysis, with the same thresholds of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and q\u0026thinsp;=\u0026thinsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animals\u003c/h2\u003e\u003cp\u003eMale C57BL/6 mice (H-2\u003csup\u003eb\u003c/sup\u003e) and male BALB/c mice (H-2\u003csup\u003ed\u003c/sup\u003e), aged 8\u0026ndash;10 weeks and weighing 25\u0026ndash;30 g, were obtained from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China), under specific pathogen-free (SPF) conditions.All animal experiments adhered to the guidelines set forth by the Animal Protection and Use Committee of Sun Yat-sen University. The experimental protocol was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.In skin transplantation and islet transplantation experiments, C57BL/6 mice served as the transplant recipients, while BALB/c mice acted as the transplant donors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Regents\u003c/h2\u003e\u003cp\u003eRecombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) were obtained from PeproTech (Rocky Hill, NJ, USA).The AdV-cGAS-shRNA-GFP adenovirus vector was constructed by Beijing Xibei Hongcheng Biotechnology Co., Ltd. and further optimized to enhance the transduction efficiency DCs. The anti-CD11c and LS microbead-conjugated separation columns were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).Nylon wool fiber syringes were purchased from Polysciences, Inc. (Eppelheim, Germany). Rabbit anti-mouse cGAS primary antibody was obtained from Abcam (Cambridge, MA, USA). Mouse anti-mouse GAPDH primary antibody and goat anti-rabbit and anti-mouse secondary antibodies were purchased from Proteintech Group, Inc. (Wuhan). Cell stimulation cocktail plus protein transport inhibitor, Fixable Viability Dye 780, Transcription Factor Buffer Set, rat anti-mouse fluorescent-conjugated CD45-BUV395, CD3-PerCP-Cy5.5, CD4-BV711, CD25-BB515, Foxp3-PE, IFNγ-BV421, IL\u0026minus;17-AF647, FITC-CD11c, BV421-CD80, PE-Cy7-CD86, PE-MHC-II (I-A/I-E) monoclonal antibodies, and corresponding isotype controls were purchased from BD PharmingenTM (San Diego, CA, USA). Rabbit anti-mouse insulin and glucagon monoclonal antibodies were obtained from Abcam (Cambridge, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for detecting the concentrations of IL\u0026minus;10, IL\u0026minus;6, IL\u0026minus;1β, IFN-γ, and TNF-α were purchased from Jiangsu Meimian Industrial Co., Ltd. (China, Jiangsu). Streptozotocin (STZ), lipopolysaccharide (LPS), Histopaque 1077, and tetramethylrhodamine-dextran (TRITC-dextran) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dithizone (DTZ), acridine orange (AO), and propidium iodide (PI) were obtained from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China). Liberase TL, blood glucose meter, and blood glucose test strips were purchased from Roche (Basel, Switzerland).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Isolation and Purification of Bone Marrow-Derived DCs(BMDCs)\u003c/h2\u003e\u003cp\u003eSterile surgery was performed to isolate the tibia and femur from 5 to 8 week-old BALB/c mice, with the bone marrow serving as the source of BMDCs (bone marrow-derived dendritic cells).DCs are differentiated from bone marrow precursor cells through the application of GM-CSF and IL-4. Following red blood cell lysis, the remaining cells were resuspended in complete medium consisting of RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 20 ng/mL recombinant murine GM-CSF, 10 ng/mL IL-4, and 1% penicillin-streptomycin solution.Cells were then seeded at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL in 6-well plates and cultured in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.On the 6th day of culture, DCs are fully induced. Subsequently,imDCs are purified via immunomagnetic selection using anti-CD11c-conjugated microbeads to isolate CD11c\u003csup\u003e+\u003c/sup\u003e cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 AdV-cGAS-shRNA-GFP Transfection of DCs\u003c/h2\u003e\u003cp\u003eRecombinant adenovirus vector (rAd-cGAS-shRNA) or negative control virus vector (rAd-EGFP) was added into purified imDCs at a multiplicity of infection (MOI) of 1:100,and cultured in serum-free OptiMEM medium for 48 hours. Subsequently, two types of cells, EGFP-DCs and cGAS-shRNA-DCs,were obtained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Western Blotting\u003c/h2\u003e\u003cp\u003eA total of 20 \u0026micro;g of protein extracts from imDCs,cGAS-shRNA-DCs and DC-GFP groups were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a nitrocellulose membrane pre-treated with methanol. The membrane was incubated overnight at 4\u0026deg;C with primary antibodies against mouse cGAS(1:1000) and GAPDH(1:10000), followed by a 1-hour incubation with the corresponding secondary antibodies(1:5000). Protein bands were visualized using ECL chemiluminescence, and the gray-scale intensity of the bands was quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Pancreatic Islet Transplantation\u003c/h2\u003e\u003cp\u003e7 days prior to the islet transplantation, 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells of EGFP-DCs, cGAS-shRNA-DCs, and an equivalent volume of PBS were respectively injected via the tail vein into three groups of C57BL/6 mice and randomly divided in to three groups:EGFP-DCs group, cGAS-shRNA-DCs group and contral group. Diabetes was induced in the C57BL/6 mice through intraperitoneal injection of streptozotocin (200 mg/kg). Islet transplantation was subsequently performed when the recipients' blood glucose levels remained at or above 16.7 mmol/L for 3 consecutive days.Islets were isolated from BALB/c mice. Following clamping of the ampulla of Vater, the common bile duct was punctured, and the pancreas was perfused with Liberase TL. The pancreas was subsequently harvested and stored on ice. After digestion in a 37\u0026deg;C water bath, islet purification was performed using density gradient separation (Histopaque 1077). A total of 350 isolated islets were transplanted under the renal capsule of the recipient using a glass capillary probe. Transplantation was deemed successful if the blood glucose level was maintained below 11.1 mmol/L. Islet graft rejection was defined as a blood glucose level exceeding 16.7 mmol/L for at least two consecutive days and construct the survival curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Intraperitoneal Glucose Tolerance Test\u003c/h2\u003e\u003cp\u003e7 days after islet transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, and the kidneys containing transplanted islets were harvested at this time point. Specifically, following a 4\u0026ndash;6 hours fasting, mice were intraperitoneally administered a glucose solution (2 g/kg), and tail vein blood glucose levels were measured at 15-minute intervals for a total of 120 minutes. The IPGTT was subsequently repeated after the removal of kidneys harboring the transplanted islets to ensure that the observed recipient blood glucose response was attributable to the functional activity of the donor islet allografts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Skin Transplantation\u003c/h2\u003e\u003cp\u003eC57BL/6 mice were divided into three groups, similar to the grouping strategy used in islet transplantation studies.The tails of BALB/c mice were disinfected with alcohol three times. A 1\u0026times;1 cm square skin flap was then prepared. The skin flap harvested from BALB/c mice was transplanted onto the back of C57BL/6 mice and sutured using 5\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures. Postoperatively, the progression of rejection was assessed by daily observation of changes in the donor skin flaps. Complete rejection was defined as the necrotic area of the successfully transplanted skin flap exceeding 80%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Flow Cytometry\u003c/h2\u003e\u003cp\u003eObtain purified imDCs and divide them into three groups according to the previous transfection method: imDCs (control group), EGFP-DCs (negative control for transfection), and cGAS-shRNA-DCs (experimental group).3 groups were stimulated to mature through the addition of 1 ug/ml LPS.To evaluate the maturity of cells, in the dark at 4 degrees Celsius for 30 minutes,three groups of cells were stained with BD fluorescein-conjugated monoclonal antibodies against CD86, CD80, and MHC-II and analyzed with a flow cytometer (BD LSRFortessa X-20, USA).After 24 hours of LPS stimulation, to evaluate the phagocytic capacity of DCs, 1 \u0026micro;l of TRITC-Dextran (25 \u0026micro;g/mL) was added to each of the 3 groups of cells. Subsequently, the cells were incubated at 37\u0026deg;C or 4\u0026deg;C for 2 hours. Following this, the treated cells were harvested, and the proportion of TRITC-positive cells was quantified by flow cytometry.\u003c/p\u003e\u003cp\u003eOn the 7th day post-transplantation, lymphocytes were harvested from the recipient's spleen and stimulated vivo with a cell stimulation cocktail in combination with a protein transport inhibitor for 4 to 6 hours. Subsequently, Th1, Th17,regulatory T cells (Treg), and intracellular cytokine staining analyses were performed. Cells were fixed and permeabilized using a Foxp3/transcription factor staining buffer set, followed by intracellular cytokine staining. Flow cytometry data were analyzed using FlowJo software. CD3\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e/IFN-γ\u003csup\u003e+\u003c/sup\u003e cells were identified as Th1 cells, CD3\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e/IL-17\u003csup\u003e+\u003c/sup\u003e cells as Th17 cells, and CD4\u003csup\u003e+\u003c/sup\u003e/CD25\u003csup\u003e+\u003c/sup\u003e/Foxp3\u003csup\u003e+\u003c/sup\u003e cells as Treg cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Immunohistochemistry and Hematoxylin and Eosin (HE) Analysis\u003c/h2\u003e\u003cp\u003eOn the 7th day after transplantation, the graft tissues were harvested, fixed with 10% polyformaldehyde, paraffin-embedded, and cut into 3\u0026ndash;5 \u0026micro;m thick sections. These sections were stained with HE or anti-mouse insulin or glucagon monoclonal antibodies according to the manufacturer's protocol. HE staining was also performed on the skin grafts.The IHC and H\u0026amp;E experimental results were analyzed using the NanoZoomer\u0026reg; S360 and NDP.view 2.9.22 RUO software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 ELISA\u003c/h2\u003e\u003cp\u003eCollect the supernatants of the 3 groups of cells after 24h LPS stimulation. Use the ELISA kit method to detect the concentrations of cytokines (IL-10, IL-6,TNF-α,IL-1β, IFN-γ).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Mixed Leukocyte Reaction (MLR) Analysis\u003c/h2\u003e\u003cp\u003eHarvest the spleens from 6 to 8 week-old C57BL/6 mice to prepare a spleen cell suspension. Purify T cells by passing the suspension through a nylon wool column according to the manufacturer's instructions, and collect the filtered T cell suspension. Subsequently, perform cell counting.After LPS stimulation,obtain 3 groups of cells and add mitomycin C (25 \u0026micro;g/mL) and incubate the cells at 37\u0026deg;C for 2 hour. Subsequently, wash the cells 3 times with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Adjust the cell concentration to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL and resuspend in complete medium.Take a 96-well plate and seed DCs and T cells into each well at ratios of 1:10, 1:20, and 1:50. Establish 3 replicate wells for each group and include a blank control group.The 96-well plates were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 72 hours. 4 hours prior to the conclusion of the incubation, 8 \u0026micro;l of CCK-8 reagent was added to each well. Following the completion of the incubation period, the absorbance of each well was measured at 450 nm using a multi-functional microplate reader. The stimulation index (SI)= (OD\u003csub\u003esample\u003c/sub\u003e \u0026minus; OD\u003csub\u003eblank control\u003c/sub\u003e)/ (OD\u003csub\u003enegative control\u003c/sub\u003e \u0026minus; OD\u003csub\u003eblank control\u003c/sub\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Statistical Analysis\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Student's t-test was employed to assess the differences between two groups. One-way analysis of variance (ANOVA) was utilized for comparisons among multiple groups, followed by Tukey's post hoc test where applicable. Kaplan-Meier survival curves were constructed, and the Log-rank test was applied to evaluate the differences in graft survival rates. P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered indicative of statistical significance. All statistical analyses were conducted using GraphPad Prism 9.0 and SPSS 22.0 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Bioinformatics analysis demonstrated that cGAS exhibited elevated expression levels in the allograft group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the postoperative spleen transcriptome of the allograft group (BALB/c\u0026rarr;C57BL/6) and the syngeneic control group (isograft group, C57BL/6\u0026rarr;C57BL/6) in the allogeneic heart transplantation model, cGAS was identified as a differentially expressed gene through differential expression analysis, with its expression significantly upregulated in the allograft group compared to the syngeneic controls(Fig.\u0026nbsp;1A). KEGG enrichment analysis revealed that the primary enriched BP included cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF signaling pathway, Th17 cell differentiation,allograft rejection, and graft-versus-host disease(Fig.\u0026nbsp;1C).GO enrichment analysis revealed that the BP primarily included the positive regulation of leukocyte activation, regulation of T cell activation, positive regulation of immune effector processes, myeloid cell differentiation, immune response-regulating signaling pathways, antigen receptor-mediated signaling pathways, positive regulation of chemokine production, and regulation of the canonical NF-kappaB signaling pathway(Fig.\u0026nbsp;1D).GSEA analysis indicated that the differentially expressed genes were predominantly involved in BP such as APC-mediated cell cycle proteins, apoptosis, cell cycle regulation, the innate immune system, and the IL-7 signaling pathway(Fig.\u0026nbsp;1B).\u003c/p\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2Genetically modified adenovirus vector increases the transduction efficiency of DCs\u003c/h2\u003e\n \u003cp\u003eCD11c\u0026thinsp;\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells were magnetically isolated using anti-CD11c-conjugated microbeads, achieving a purity of over 90% for the CD11c\u0026thinsp;\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cell(Fig.\u0026nbsp;2A,\u003cstrong\u003eB\u003c/strong\u003e).Purified DCs were transduced with AdV-cGAS-shRNA-GFP (cGAS-shRNA-DCs) or AdV-GFP (DC-GFP) at a multiplicity of infection (MOI) of 1:100 for 48 hours. The expression of GFP was confirmed by fluorescence microscopy through the detection of GFP fluorescence(Fig.\u0026nbsp;2C).Furthermore, the expression of GFP was analyzed via flow cytometry, revealing that over 80% of the DCs were effectively transduced by the adenovirus vector(Fig.\u0026nbsp;2D).Western blot analysis revealed a significant reduction in the level of cGAS protein in cGAS-shRNA-DCs. These results suggest that the adenovirus vector efficiently delivered the cGAS-shRNA gene into DCs(Fig.\u0026nbsp;2E,\u003cstrong\u003eF\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3AdV-cGAS-shRNA-GFP suppressed the maturation of dendritic cells.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAdV-cGAS-shRNA reduces the expression of MHC-II and co-stimulatory molecules. In this study, purified imDCs, DC-GFP, and cGAS-shRNA-DCs were treated with LPS (1 \u0026micro;g/mL) for 24 hours. The expression levels of CD80, CD86, and MHC-II were subsequently analyzed by flow cytometry. The results demonstrated that, compared to other two groups, the cGAS-shRNA-DCs group exhibited significantly reduced expression levels of CD80, CD86, and MHC-II(Fig.\u0026nbsp;3).\u003c/p\u003e\n \u003cp\u003eAt the same time, the supernatants of the three groups of cells were collected after 24 hours of LPS treatment. The results demonstrated that cGAS-shRNA-DCs cells secreted significantly higher levels of the immunosuppressive cytokine IL-10 compared to other two groups, while exhibiting reduced secretion of the immunostimulatory cytokines IL-6, IL-\u0026gamma;, TNF-\u0026alpha;,and IL-1\u0026beta;(Fig.\u0026nbsp;4A).\u003c/p\u003e\n \u003cp\u003eTRITC-dextran phagocytosis assay demonstrated that LPS stimulation significantly diminished the phagocytic ability of control and DC-GFP groups, which was associated with increased maturity of DCs. In contrast, LPS stimulation had minimal impact on the phagocytic capacity of cGAS-shRNA-DCs.Notably, the cGAS-shRNA-DCs group exhibited a markedly enhanced phagocytic capacity compared to the other two groups, despite displaying lower levels of DCs maturity(Fig.\u0026nbsp;4B).\u003c/p\u003e\n \u003cp\u003e3 groups cells treated with LPS were co-cultured with T cells at different ratios for 72 hours to evaluate the changes in T cell maturation and activation ability and were co-cultured with T cells at different ratios (1:10,1:20 and 1:50) for 72 hours to assess the changes in T cell proliferation and activation capacity. (Fig.\u0026nbsp;5A)The MLR assay demonstrated that cGAS-shRNA-DCs exhibited a significantly lower stimulatory capacity compared to the other two groups, with statistically significant differences observed at 1:10 and 1:20 ratios(Fig.\u0026nbsp;5B).\u003c/p\u003e\n \u003cp\u003eThese researches demonstrates that inhibiting cGAS expression in DCs not only diminishes the expression of co-stimulatory molecules and MHC-II but also impairs the maturation of DCs, thereby reducing their antigen-presenting capability and the subsequent activation of T cells.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.4 cGAS-shRNA-DCs preserves the function of allografts and prolongs graft survival.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn this study, mouse islet transplantation and skin transplantation models were performed to investigate the effects of cGAS-shRNA-DCs on graft survival and function. Before islet transplantation, DTZ staining and AO-PI staining confirmed that the isolated islets exhibited high viability and purity(Fig.\u0026nbsp;6B).HE staining, insulin staining, and glucagon staining revealed that the number and activity of residual islets in the cGAS-shRNA-DCs group were significantly higher than those in the other two groups, with less lymphocyte infiltration(Fig.\u0026nbsp;6C).HE staining of skin grafts revealed that the degree of hair follicle destruction, dermal and subcutaneous tissue edema, and lymphocyte infiltration was less severe in the cGAS-shRNA-DCs group compared to the other two groups(Fig.\u0026nbsp;7B).\u003c/p\u003e\n \u003cp\u003eTo investigate the in vivo functional activity of islet grafts, an intraperitoneal glucose tolerance test (IPGTT) with a dose of 2 g/kg was conducted on the 7th day post-transplantation. Glucose tolerance was assessed after a fasting period of 4\u0026ndash;6 hours, and blood glucose levels were measured from tail vein blood samples every 15 minutes for a total duration of 120 minutes.After glucose injection, blood glucose levels in all groups increased sharply, peaking at 15 minutes and then gradually declining over time. Notably, the cGAS-shRNA-DCs group exhibited a more rapid response to glucose compared to the other groups(Fig.\u0026nbsp;6D).And then, the kidneys containing islet allografts were removed, and the intraperitoneal glucose tolerance test (IPGTT) was repeated. The results demonstrated that the blood glucose response in recipient mice was dependent on the presence of islet allografts(Fig.\u0026nbsp;6E).In terms of graft survival, both skin and islet grafts in the cGAS-shRNA-DCs group exhibited significantly prolonged survival times compared to those in the other two groups(Fig.\u0026nbsp;6A,7A).\u003c/p\u003e\u003cstrong\u003e3.5cGAS-shRNA-DCs modulate the proportions of T cell subsets in the spleen in vivo.\u003c/strong\u003e\u003cbr\u003e\n \u003cp\u003eTo investigate the potential mechanism by which cGAS-shRNA-DCs prolong graft survival, we analyzed the proportions of T cell subsets in mouse islet transplantation model. Spleen single-cell suspensions were collected from recipient mice in each group on 7th day post-transplantation. Following staining with fluorescently-labeled monoclonal antibodies, flow cytometry analysis was conducted. The results demonstrated that, compared with other two groups, the proportions of Th1 and Th17 cells were significantly reduced, whereas the proportion of Treg cells was elevated in the cGAS-shRNA-DCs group(Fig. 8).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study, using skin and islet transplantation models, showed that inhibiting cGAS expression in DCs reduces co-stimulatory molecules CD80, CD86, and MHC-II\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e levels and significantly prolongs graft survival. Moreover, flow cytometry analysis of T cell subsets in the recipient spleen revealed an increased proportion of Treg cells alongside decreased proportions of Th1 and Th17 cells, suggesting that the immune response was modulated toward suppression following transplantation.This study utilized western-blot and flow cytometry to confirm that the expression of cGAS was effectively inhibited and the transduction efficiency was notably high.\u003c/p\u003e\u003cp\u003eResearch has demonstrated that upon activation by dsDNA, cGAS catalyzes the production of cGAMP, which subsequently binds to STING\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, thereby activating downstream signaling pathways and promoting the secretion of cytokines such as IFN-I\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e and IL-6\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The cGAS-STING pathway is regarded as a critical bridge connecting innate and adaptive immunity through its regulation of IFN-I-mediated maturation and migration of DCs\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ecGAS is an innovative target in transplantation immunity. To gain deeper insights into the role of cGAS in promoting DC maturation, we employed AdV-cGAS-shRNA-GFP to transduct DCs and establish a cell model with suppressed cGAS expression.The results demonstrate that inhibiting cGAS suppresses DCs maturation, as evidenced by reduced expression of MHC-II and co-stimulatory molecules\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. DCs are capable of antigen presentation and play a critical role in activating and maturing T cells\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. imDCs exhibit impaired antigen-presenting function, and T cell functionality is significantly influenced by the cytokine profile and the expression levels of co-stimulatory molecules on DCs\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Consequently, the immature state of DCs can induce immune tolerance by attenuating T cell activation and modulating cytokine production. Our research further reveals that inhibiting cGAS in vitro diminishes the antigen-presenting capacity of DCs, restrains T cell proliferation, and decreases the secretion of cytokines such as IL-6 and IFN-γ.\u003c/p\u003e\u003cp\u003eThe protective effect of cGAS on pancreatic islet grafts and skin grafts in mice was investigated through preoperative tail vein injection of cGAS-shRNA-DCs. The survival duration of the grafts as well as the pathological changes in the grafts post-surgery were analyzed. The results demonstrated that cGAS-shRNA-DCs significantly prolonged graft survival time and mitigated postoperative immune rejection responses. Furthermore, our findings revealed that recipients treated with cGAS-shRNA-DCs exhibited superior glucose tolerance compared to other two groups, which corroborates the prolonged survival of islet grafts.Pathological results demonstrated that in both skin transplantation and islet transplantation, the HE staining revealed a significant reduction in inflammatory infiltration following the reinfusion of cGAS-shRNA-DCs. Additionally, immunohistochemistry analysis showed increased expression levels of insulin and glucagon.Subsequently, we examined the alterations in T cell subsets within the spleen. Specifically, Th1 and Th17 cells are known to mediate pro-inflammatory immune responses\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, whereas Treg cells play a critical role in suppressing immune activation\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Our results demonstrated that in the cGAS-shRNA-DCs group, the proportions of Treg cells was significantly increased, while the proportions of Th1 and Th17 cells were markedly reduced. This transformation in T cell subset composition effectively attenuated immune responses, thereby contributing to the induction of immune tolerance.In this study, we demonstrated through islet transplantation and skin transplantation that cGAS influences adaptive immune responses by regulating the maturation of DCs. Consequently, it plays a role in modulating immune rejection reactions and may serve as a potential target for inducing immune tolerance. However, the underlying mechanisms remain to be fully elucidated, necessitating further investigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eInhibition of cGAS expression in DCs suppresses their maturation, reduces the expression of co-stimulatory molecules and MHC-II, and diminishes their antigen-presenting capacity and ability to activate T cells. In vivo administration of cGAS-shRNA-transfected DCs significantly prolongs graft survival, increases the proportions of Treg cells, and decreases the proportions of Th1 and Th17 cells. This transformation correlates with reduced production of pro-inflammatory factors and enhanced secretion of anti-inflammatory cytokines. Consequently, targeting cGAS may represent a novel strategy for inducing immune tolerance in transplantation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Natural Science Foundation of Guangdong Province (2023A1515011805 and 2022A1515011052), the National Natural Science Foundation of China (81873591), the Science and Technology Planning Project of Guangdong Province (2018A050506030), the Science and Technology Program of Guangzhou (201704020073), the Guangdong Provincial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology (2013A061401007, 2017B030314018, and 2020B1212060026), and the Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation) (2015B050501002 and 2020A0505020003).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDurand, F., et al., \u003cem\u003eAge and liver transplantation.\u003c/em\u003e J Hepatol, 2019. \u003cstrong\u003e70\u003c/strong\u003e(4): p. 745\u0026ndash;758.\u003c/li\u003e\n\u003cli\u003eShapiro, A.M., M. 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Rudensky, \u003cem\u003ePrinciples of regulatory T cell function.\u003c/em\u003e Immunity, 2023. \u003cstrong\u003e56\u003c/strong\u003e(2): p. 240\u0026ndash;255.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cGAS, immune tolerance, dendritic cells, transplantation","lastPublishedDoi":"10.21203/rs.3.rs-7302546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7302546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eDendritic cells (DCs) regulate immune responses. Recent studies show that cyclic GMP-AMP synthase (cGAS) plays a crucial role in DC maturation. This study investigates the effect of suppressing cGAS expression in DCs on graft immune tolerance.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eBioinformatics analysis was conducted to explore the potential role of cGAS in transplant immunity. Immature dendritic cells (imDCs) were isolated, purified, and transduced with an adenovirus vector to suppress cGAS expression.EGFP-DCs group, cGAS-shRNA-DCs group, and control group(PBS), were injected via the tail vein prior to skin and islet transplantation for the establishment of a mouse transplantation model.Analyze graft survival and pathological changes, and use flow cytometry to assess spleen T cell subset proportions. After lipopolysaccharide stimulation, evaluate MHC-II and co-stimulatory molecule expression, antigen phagocytosis, and T cell proliferation in the imDCs (control), EGFP-DCs, and cGAS-shRNA-DCs groups. The supernatants from every group were collected, and the changes in cytokine secretion by DCs were detected using ELISA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBioinformatics analysis shows increased cGAS expression in the allograft group. The cGAS-shRNA-DCs group showed reduced MHC-II and co-stimulatory molecule expression, enhanced phagocytic activity, and decreased T cell activation ability. Levels of IFN-γ, IL-1β, TNF-α, and IL-6 were lower, while IL-10 levels were higher. Mice receiving cGAS-shRNA-DCs had prolonged graft survival and improved graft function. Flow cytometry revealed an increased proportion of regulatory T cells and reduced Th1 and Th17 cell populations.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eInhibiting cGAS expression in DCs reduces their maturation, antigen-presenting capacity, and T-cell activation, ultimately prolonging graft survival.\u003c/p\u003e","manuscriptTitle":"Inhibition of cGAS in dendritic cells suppresses maturation and prolongs allograft survival in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 11:39:05","doi":"10.21203/rs.3.rs-7302546/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-01T14:17:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-23T12:47:11+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-09T14:16:44+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-20T11:28:20+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-18T10:52:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-08-17T16:58:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-13T17:21:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-05T15:57:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes \u0026 Immunity","date":"2025-08-05T15:57:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1024d246-180a-4145-8b95-63168832cabf","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53266434,"name":"Biological sciences/Immunology/Translational immunology"},{"id":53266435,"name":"Biological sciences/Genetics/Gene expression"},{"id":53266436,"name":"Biological sciences/Genetics/Clinical genetics"}],"tags":[],"updatedAt":"2026-03-04T08:13:55+00:00","versionOfRecord":{"articleIdentity":"rs-7302546","link":"https://doi.org/10.1038/s41435-026-00381-7","journal":{"identity":"genes-and-immunity","isVorOnly":false,"title":"Genes \u0026 Immunity"},"publishedOn":"2026-03-03 05:00:00","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-08-25 11:39:05","video":"","vorDoi":"10.1038/s41435-026-00381-7","vorDoiUrl":"https://doi.org/10.1038/s41435-026-00381-7","workflowStages":[]},"version":"v1","identity":"rs-7302546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7302546","identity":"rs-7302546","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}