Donor MHC-specific Thymus Vaccination for Immunocompatible Allotransplantation

Donor MHC-specific Thymus Vaccination for Immunocompatible Allotransplantation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Donor MHC-specific Thymus Vaccination for Immunocompatible Allotransplantation Yang Liu, Hexi Feng, Ke Li, Ruiyi Li, Xiao-Jie Zhang, Ye Tian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4080522/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Organ transplantation is the last-resort option to treat organ failure. However, less than 10% of patients benefit from this only option due to lack of major histocompatibility complex (MHC)-matched donor organs and 25-80% of donated organs could not find MHC-matched recipients. T cell allorecognition is the principal mechanism for allogeneic graft rejection. We herein present a “donor MHC-specific thymus vaccination” (DMTV) strategy to induce T cell tolerance to both autologous and allogeneic donor MHC. Allogeneic MHC molecules were expressed in the recipient thymus through adeno-associated virus infection, which led to stable expression of allogeneic MHC together with the autologous MHC in the engineered thymus. During local T cell education, those T cells recognizing either autologous MHC or allogeneic MHC were equally depleted. We constructed C57BL/6-MHC and BALB/c-MHC dual immunocompatible mice via thymus vaccination of C57BL/6-MHC into the BALB/c thymus and observed long-term tolerance after transplantation of C57BL/6 skin and C57BL/6 mouse embryonic stem cells into the vaccinated BALB/c mice. We also validated our DMTV strategy in a bone marrow, liver, thymus (BLT)-humanized mouse model for immunocompatible allotransplantation of human embryonic stem cells. Our study suggests that DMTV is a potent avenue to introduce a donor compatible immune system in recipients, which overcomes the clinical dilemma over the extreme shortage of MHC-matched donor organs for treating patients with end-stage organ failure. Allotransplantation Immunotolerance MHC Thymic vaccination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Organ transplantation is the last-resort option to treat patients with end-stage organ failure. According to 2021 global report published by the Global Observatory on Donation and Transplantation 1 , less than 10% of patients left with this only option received organ transplantation treatment owing to an extreme shortage of major histocompatibility complex (MHC, in human MHC is generally recognized as human leukocyte antigens or HLA)-matched donor organs. On the other hand, the acceptance rate is just 0.6% in U.S. when the electronically transmitted offers for patients are ahead of the ultimate acceptor, and the nonuse rate among recovered organs for transplant or organs potentially available per donor approaches 25–80% 2–4 . This organ underutilization is attributable to the post-donation challenges to the allocation, storage and transport of recovered organs to waitlisted and MHC-matched patients 5 , 6 . MHC mismatch is the leading cause of transplant rejection 5 , 7 – 10 . There are two major types of MHC molecules. MHC class I molecules (MHC I) are expressed on the surface of all nucleated cells while MHC class II molecules (MHC II) are restricted to professional antigen-presenting cells (APCs) 11 , 12 . Allogeneic MHC molecules expressed on donor cells after organ transplantation are directly recognized by T cell receptors (TCRs) of recipient T cells, which is the primary mechanism for immune rejection 13 – 17 . MHC I and MHC II also present antigen peptides periodically broken down during normal or diseased cellular processes to the immune cells for antigen recognition, which partially contributes to immune rejection 18 , 19 . Successful organ transplantation largely relies on the degree of MHC matching 5 , 10 . However, as MHC is highly polymorphic, fully MHC-matched donors are rarely available 20 , 21 . Therefore, immunosuppressants are regularly used to reduce the intensity of immune responses after allotransplantation, albeit this might lead to increased risk of infections and malignancies 22 – 24 . T cells are central to the process of transplant rejection. The features of conventional T cells discriminating between MHC-mismatched “non-self” cells and MHC-matched “self” cells are acquired during their thymic development 25 – 27 . In the thymus, immature T cells (thymocytes) undergo a positive-negative selection 27 – 30 . During positive selection, T cells will undergo apoptosis by neglect when there is a low affinity between their surface TCRs and MHCs expressed on thymic epithelial cells (TECs) and dendritic cells (DCs) 31 – 35 . Those T cells are also programmed to undergo apoptosis during negative selection when TCRs on immature T cells bind with a high affinity to MHC or presented antigenic peptides in order to prevent autoimmune response 31 , 36 – 38 . Here, we report a d onor M HC-specific t hymus v accination (DMTV) strategy to recapitulate TCR-MHC adaptation during T cell development in the thymus. We hypothesize that ectopic expression of allogeneic MHC (MHC allo ) in TECs and DCs in recipient thymus will drive “non-self” to “self” T cell antigen discrimination via specific depletion of the donor reactive T cells during positive and negative selection ( Fig. 1 ). The recipient receiving DMTV is expected to tolerate allotransplantation of the donor organs or tissues bearing the vaccinated MHC with no immunosuppressant required. RESULTS Ectopic expression of allogeneic MHC in recipient thymus To test this hypothesis in allotransplantation between BALB/c (recipient) and C57BL/6 (donor) mice, two MHC completely mismatched allogeneic strains (Fig. 2 a), AAV2/8-CMV-H2-K b α chain-IRES-H2-D b α chain and AAV2/8-CMV-I-A b α chain-IRES-I-A b β chain bearing both C57BL/6 donor MHC I and MHC II expression cassettes were constructed and viruses were then packaged and concentrated for thymus vaccination (Supplementary information, Fig. S1). 1×10 11 viral genomes (vg) for each virus in 10 µl were intrathymically injected for each lobe of BALB/c mouse (DMTV C57 ), and an empty AAV2/8 virus was similarly injected in the thymus at a concentration of 2×10 11 vg for each lobe for control (Ctrl TV). 7 days after injection, C57BL/6 donor MHC I H2-K b and H2-D b as well as MHC II I-A b were detected in 88.7–93.1% of TECs and 15.5–39.9% DCs in the vaccinated BALB/c thymus as evaluated by fluorescence-activated cell sorting (FACS) (Fig. 2 b-d). The ectopic expression of donor MHC in TECs lasted for more than 90 days, although those expressed in DCs eventually decreased, which is likely due to the rapid turnover rate of DCs in the thymus (Fig. 2 c and 2 d) 39 . These data suggest that the delivery system was efficient and the stable expression pattern of donor MHC in TECs of recipients would therefore ensure a long-term effect for introducing donor MHC tolerance. DMTV leads to alloreactive T cell clonal depletion To evaluate the functional outcome of DMTV, BALB/c mice received Ctrl TV or DMTV C57 for 7 days were treated with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs) as well as 3 Gy total body irradiation (TBI) to eliminate pre-existing T cell repertoire. Newly developed CD4 + T cells and CD8 + T cells completely reconstituted the cell repertoire in peripheral 2 months after depletion (Supplementary information, Fig. S2). The reconstituted peripheral blood mononuclear cells (PBMCs) from Ctrl TV and DMTV C57 BALB/c mice were then used for mixed lymphocyte reaction (MLR) analysis to evaluate their reactiveness to donor cells (Supplementary information, Fig. S3a). Both PBMCs from Ctrl TV and DMTV C57 BALB/c mice did not show any responsiveness to irradiation-inactivated PBMCs from BALB/c mice (Fig. 3 a, 3 b), suggesting no autoimmunity was acquired after DMTV. PBMCs from both groups equally responded to common antigen phytohemagglutinin (PHA) and irradiation-inactivated PBMCs from C3H/He mice, another mouse strain with distinct MHC background (H2 k , Fig. 3 a, 3 b and Supplementary information, Fig. S3b), suggesting normal immune responsiveness remained in both Ctrl and donor-specific MHC vaccinated mice. Remarkably, PBMCs from DMTV C57 BALB/c mice had a much lower responsiveness towards irradiation-inactivated PBMCs from C57BL/6 mice as compared with that of PBMCs from Ctrl TV BALB/c mice (Fig. 3 a, 3 b). These data strongly suggest that DMTV results in efficient and specific T cell tolerance towards cells expressing vaccinated MHC. To confirm specific depletion of donor-reactive T cells, clonally expanded T cells from PBMCs of Ctrl TV BALB/c mice were FACS enriched and subjected to integrative single-cell RNA sequencing and TCR sequencing (scTCR-seq & scRNA-seq) after priming with irradiated C57BL/6 PBMCs (Fig. 3 a, Supplementary information, Fig. S3a and S3c). As compared with the non-primed T cells, a cluster of CD4 + T cells (cluster #1) and CD8 + T cells (cluster #9) were specifically expanded after irradiated C57BL/6 PBMCs priming (Fig. 3 c, 3 d and Supplementary information, Fig. S3d). Meanwhile, proliferating T cells (cluster #10) were also obviously elevated after priming with allogeneic PBMCs (Fig. 3 d and Supplementary information, Fig. S3d). The most dominant TCR profiles in clonally expanded CD4 + T cells and CD8 + T cells were then successfully retrieved and considered as the potential candidates that specifically responded to donor MHC (Fig. 3 e). We then isolated peripheral T cells from Ctrl TV BALB/c mice and DMTV C57 BALB/c mice 3 months after vaccination for integrated scRNA-seq & scTCR-seq. There were no differences on the overall constitutions in peripherally re-populated T cells after thymus vaccination (Fig. 3 d) 36 , suggesting intact intrinsic T cell developmental programs remained after DMTV. Intriguingly, the proportions of TRBV31 -bearing CD4 + T cells in cluster #1 and TRAV13-1 -bearing CD8 + T cells in cluster #9 were significantly reduced in thymus vaccinated mice (Fig. 3 f and 3 g). Together, these results indicate that donor reactive T cells are specifically depleted during T cell development in the thymus of mice receiving DMTV. DMTV leads to blunted immune responses to donor cells in vivo After allotransplantation, T cells are initially activated either directly or indirectly, which subsequently leads to activation of B cells, resulting in both cell-mediated and antibody-mediated immune rejection 40 . Monitoring donor-specific antibodies (DSA) is therefore widely used for detecting the overall levels of immune rejection and a guide for tailored immunosuppressive treatment 41 , 42 . To analyze the immune responses to donor cells after DMTV in vivo , we monitored DSA in Ctrl TV and DMTV C57 BALB/c mice (Fig. 4 a). After tail vein injection of irradiation-inactivated PBMCs from BALB/c mice for 10 days, no DSA was detected in the serum of both Ctrl TV and DMTV C57 BALB/c mice (Fig. 4 b, 4 c). As expected, the injection of irradiation-inactivated PBMCs from C3H/He mice resulted in comparable and prominent induction of DSA in both groups. On the other hand, the injection of irradiation-inactivated C57BL/6 PBMCs led to robust DSA induction in Ctrl TV BALB/c mice and DMTV C57 treatment almost fully abolished DSA induction (Fig. 4 b, 4 c), highlighting the robustness of DMTV in blunting the overall donor-specific immune responses in vivo . DMTV mitigates immune rejection of skin allotransplants To examine whether DMTV could provide an immune competent while donor-specific immunotolerant condition for allotransplantation, we transplanted the skin from C57BL/6 mice to BALB/c mice 2 months after thymus vaccination (Fig. 5 a). In Ctrl TV BALB/c mice, the transplanted skin from C57BL/6 mice showed prominent rejection starting from post transplantation day (PTD) 6 (Fig. 5 b-e). The transplanted skin from C3H/He mice was similarly rejected in DMTV C57 BALB/c mice in PTD 6–15. In contrast, the transplanted skin donated from C57BL/6 mice in DMTV C57 BALB/c mice showed tolerance even 30 days after transplantation, which was almost comparable to that in autologous transplantation (Fig. 5 b-e). These data suggest that DMTV constructs a donor-specific immune tolerance environment and supports long-term survival of allotransplanted organs with no need of immunosuppressive treatment. To confirm that DMTV induces a donor-specific immune tolerance environment, histological analyses were performed to check immune cell infiltration after transplantation. There were massive CD3 + T cells, CD4 + T cells and CD8 + T cells infiltrated in the C57BL/6 mice skin tissue transplanted into Ctrl TV BALB/c mice and the C3H/He mice skin tissue transplanted into DMTV C57 BALB/c mice (Fig. 5 f-i and Supplementary information, Fig. S4). Similar to the autologous transplantation group, very rare T cell infiltration was observed in the C57BL/6 mice skin tissue transplanted into DMTV C57 BALB/c mice even 30 days after transplantation (Fig. 5 f-i and Supplementary information, Fig. S4). DMTV mitigates immune rejection of allogeneic mouse embryonic stem cell transplants Given their pluripotency, mouse embryonic stem cells (mESCs) develop into almost all types of tissues or lineages after transplantation into immune compromised mice. To investigate whether DMTV supports successful allogeneic transplantation of different tissues, we subcutaneously transplanted C57BL/6 mESCs into C57BL/6 mice (autologous transplantation) and Ctrl TV or DMTV C57 BALB/c mice (allogeneic transplantation) (Fig. 6 a). In the autologous transplantation group, mESCs efficiently survived and developed into different lineages, such as ectodermal cells (region of interest 1, ROI 1) and endodermal cells (ROI 2) (Fig. 6 b-e). In Ctrl TV BALB/c mice, allotransplanted C57BL/6 mESCs were completely rejected, which resulted with no transplants for further analyses. However, allotransplanted C57BL/6 mESCs in DMTV C57 BALB/c mice largely survived and normally developed into multi-lineages (Fig. 6 b-e). Histological analyses further confirmed low T cell infiltration in both autologous transplants and allogeneic transplants in donor MHC thymus vaccinated mice (Fig. 6 d, 6 e and Supplementary information, Fig. S5a, S5b). These results suggest that the donor-specific immune tolerance environment introduced by DMTV supports survival and normal development of mESCs. Meanwhile, DMTV induced allotransplantation tolerance is universal and might be suitable for transplantation of various organs or tissues. Humanized DMTV also induces allotransplantation immune tolerance To validate whether the DMTV strategy also functions in a human context, we vaccinated human donor MHC (referred to HLA hereafter) in the reconstructed human thymus of a bone marrow, liver, thymus (BLT)-humanized mouse model 43 , 44 . NOD-Prkdc scid Il2rg em1 /Smoc (M-NSG) mice were exposed to 1 Gy irradiation and transplanted with foetal thymus and liver tissues under the renal capsules. The thymus/liver-transplanted M-NSG mice were then tail vein injected with 5.0×10 5 huCD34 + hemopoietic stem cells (HSCs) isolated from the liver of the same foetal donor (Fig. 6 f). HLA typing revealed that HLA alleles of the foetal tissues used for constructing BLT-humanized mice were completely mismatched from both H1 and H9 human embryonic stem cells (WA01 and WA09 hESCs, authorized from WiCell, Madison) (Fig. 6 g). Expression vectors for H9 HLA types were then constructed (AAV2/8-CMV-HLA-A*02:01-IRES-HLA-A*03:01, AAV2/8-CMV-HLA-B*35:03-IRES-HLA-B*44:02 and AAV2/8-CMV-HLA-Cw*04:01-IRES-HLA-Cw*07:04, Supplementary information, Fig. S1). To construct a humanized DMTV system, 1×10 11 vg in 10 µl for each virus were co-injected into a block of foetal thymus (around 100 mg) and the thymus was further incubated in 1 ml basal medium containing 2×10 11 vg viruses for 2 h before renal subcapsular transplantation together with the liver tissue. FACS analysis revealed that human immune cells (huCD45 + and huCD3 + ) efficiently populated the peripheral blood of the humanized mice 2 months after H9 HLA DMTV (DMTV H 9 ) and BLT humanized mouse model construction (Supplementary information, Fig. S5c, S5d). H1 or H9 hESCs were then subcutaneously injected into Ctrl TV or DMTV H 9 BLT humanized mice. 2 months after hESC transplantation, the sizes of transplants recovered from DMTV H 9 BLT humanized mice allotransplanted with H9 hESCs were obviously larger than those from DMTV H 9 BLT humanized mice allotransplanted with H1 hESCs or Ctrl TV BLT humanized mice transplanted with H9 hESCs (Fig. 6 h, 6 i). Of note, there were prominent infiltration of huCD3 + T cells, huCD4 + T cells and huCD8 + T cells in ectodermal tissues (ROI 1) and endodermal tissues (ROI 2) of H1 hESC transplants from DMTV H 9 BLT humanized mice and H9 transplants from Ctrl TV BLT humanized mice, whereas, T cell infiltration could rarely be detected in tissues from H9 hESC transplants after DMTV H 9 pre-treatment (Fig. 6 j, 6 k and Supplementary information, Fig. S5e, S5f). These data highlight that the DMTV strategy also functions in human context and could therefore serves as a potential strategy to bypass MHC-matching in organ allotransplantation. DISCUSSION Organ transplantation is well-acknowledged as the last-resort option to treat organ failure. However, the organ transplantation rate in patients left with this only option was extremely low owing to a lack of efficient system in finding and locating MHC-matched donors and subsequent difficulties in allocating, storing and transporting of recovered organs, which also resulted in severe underutilization of donated organs. Meanwhile, transplantation of partially MHC-matched organs always leads to allograft rejection, which requires lifelong immunosuppressive treatment, shortening the survival period of transplanted organs and causing unwanted risks of infections and malignancies. One can therefore reasonably foresee that organ transplantation will ultimately become a regular treatment for patients with organ failure if the availability of donor organs is no longer an issue and the dilemma of immune rejection is overcome. In the current study, we have developed a DMTV strategy by ectopic expression of donor-specific MHC molecules in the recipient thymus to deplete donor reactive T cells. Both in vitro and in vivo studies reveal that after DMTV the recipient immune system becomes tolerant to donor organs or tissues although they bear a mismatched MHC profile. DMTV works together with the endogenous selection system in the thymus and educates T cells to tolerate to both self MHC and donor MHC. Our designed DMTV strategy therefore avoids stringent recipient-donor MHC matching and simplifies all other tedious procedures after recovering an organ for transplantation since it fills the gap standing between the recipient and the donor either spatially or temporally. In a non-immune tolerant allotransplantation, T cells are activated directly or indirectly, which leads to activation of other immune cells and causes immune rejection 45 , 46 . Removing donor reactive T cells is therefore the key to ensure a successful allotransplantation. In the current study, DMTV efficiently removes donor reactive T cells during their development in the thymus. This is evidenced by in vitro MLR analysis, in vivo DSA monitoring, and very low infiltration of T cells in the grafted tissues after DMTV even without any immunosuppressive intervention. Integrated scRNA-seq & scTCR-seq further reveals clonal depletion of T cells harboring potential donor reactive TCRs. Notably, DMTV only introduces immune tolerance to donor-specific MHC and animals receiving DMTV treatment show almost complete immune responses to general antigens as well as other allogeneic MHC types. ScRNA-seq & scTCR-seq also showed that in the peripheral T cell repertoire, the composition of T cell populations remained intact. Together, these data indicate that DMTV treatment has minor effects on overall T cell development and maturation. In the future, it will be intriguing to test whether the thymus vaccination strategy is also efficient to induce designed immune tolerance by expression of targeted antigens other than donor MHC in the thymus. In humanized mice receiving DMTV treatment, cognate hESCs tolerate allogeneic transplantation and normally develop into multi-lineages. This raises a point that DMTV is suitable not only for allogeneic organ transplantation but also for cell-based therapy utilizing products derived from human pluripotent stem cells (hPSCs). Although individualized human induced pluripotent stem cells have been proposed for immunotolerant transplantation, it is expensive and needs long-period reprogramming procedures and safety validations. Engineering surface HLA molecule profiles or other immune modulatory molecules in hPSCs has also been proposed to generate universal cells for allogeneic transplantation 47 – 50 . However, these engineered cells might be endowed with the capability of immune surveillance evasion. Therefore, DMTV is another practical option for application of hPSCs for cell therapies with no need of extra genetic engineering in these cells or immunosuppressive intervention. According to the European Molecular Biology Laboratory’s European Bioinformatics Institute (EMBL-EBI), 14,956 types of classical HLA-I proteins have so far been officially recognized in all populations 51 . Considering future allogeneic transplantation between recipients and donors locally, the required expression vectors for variable HLA-I types will be much reduced in a specific area. It is therefore doable for advanced banking of all spectrums of AAVs for each HLA-I protein expression, which will no doubt save waiting time for efficient transplantation. In the future, it will be necessary to test the DMTV strategy for allogeneic organ transplantation in large animals and eventually in clinical reality. MATERIALS AND METHODS Animals Female C57BL/6 (SM-001), BALB/c (SM-003), C3H/He (SM-008) and NOD-Prkdc scid Il2rg em1 /Smoc (M-NSG) mice were purchased from Shanghai Model Organisms Center Inc, China. All mice were housed in groups of 5 individuals per cage and maintained on a 12-h light-dark cycle at 22–25°C under specific-pathogen free (SPF) conditions. All animal experiments were approved by the Laboratory Animal Research Center, Tongji University. All procedures involving animals were carried out in compliance with the Guide for the Care and Use of Laboratory Animals, and ethical approval was granted by the Ethics Committee, Tongji University (approval number: 2020YANYUSHEN093). The investigators were blinded to allocations during experiments and outcome assessment. Thymus vaccination For donor MHC expression in recipient thymus, adeno-associated virus (AAV) packaging system was used. In brief, donor MHC cassettes driven by the CMV promoter were constructed in the AAV2/8 vector (Supplementary information,Fig.S1). After packaging, viruses were concentrated through gradient centrifugation and viral titer was detected by qRT-PCR (for rAAV genome). Mice were anesthetized through intraperitoneal (i.p.) injection of Avertin. Hair on the chest was removed with depilatory cream. Mice were then intubated and connected to a small animal ventilator (RWD, cat. no. R420). After skin disinfection with povidone-iodine, a central skin incision at the level of 2 nd intercostal space was made. To expose the thymus, a horizontal incision at the mouse sternum was introduced and set apart with a retractor. AAVs for Ctrl TV or DMTV (1×10 13 vg/ml, 10 μl for each MHC expression virus, and the same total dosage was applied for Ctrl in each group) were intrathymically injected with a 30-gauge Hamilton syringe. During the thymus vaccination procedure, mice were maintained inflated with a ventilator before thoracic cavity was closed and the opening was closely sutured. Carprofen (5mg/kg, subcutaneous injection) and enrofloxacin (10mg/kg, i.p. injection) were used to provide analgesia or prevent infection for 3 days after surgery. T cell depletion 1.5 mg anti-CD4 (BioXCell, New Hampshire, USA, BP0003-1) and 0.8 mg anti-CD8 (BioXCell, BP0061) monoclonal antibodies (mAbs) were i.p. injected into Ctrl TV mice or DMTV mice twice (7 days and 10 days after thymus vaccination) to deplete pre-existing CD4 + and CD8 + T cells. On day 10 after thymus vaccination, mice were subjected to a 3 Gy total body irradiation (TBI). The populations of CD4 + and CD8 + T cells were then assessed by flow cytometry analysis. Thymus epithelial cell, dendritic cell and peripheral blood mononuclear cell isolation To validate ectopic MHC expression in thymus epithelial cells (TECs) and dendritic cells (DCs), vaccinated thymus was isolated and cut into small pieces with a scissor. Thymus tissues were then digested with 0.5 mg/ml papain (Sangon, Shanghai, China, cat. no. A003124), 2.5 mg/ml collagenase IV (R&D, cat. no. 9001-12-1) and 0.1 mg/ml DNase I (Sigma-Aldrich, Darmstadt, Germany, cat. no. 11284932001) in basal DMEM/F12 medium at 37°C for 30 min. 10% feal bovine serum (FBS) in DMEM/F12 was applied to stop the digestion and cells were passed through a 70-μm cell strainer before flow cytometry analysis. Peripheral blood mononuclear cells (PBMCs) were collected for mixed lymphocyte reaction (MLR) assay, detecting donor specific antibodies (DSA), single-cell sequencing and immune cell composition detection. Peripheral blood cells and spleen homogenates (passed through a 40-μm cell strainer) were collected in tubes prefilled with EDTA. Histopaque®-1083 (Sigma-Aldrich, cat. no. 10831-100ml) and Histopaque®-1077 (Sigma-Aldrich, cat. no. 10771-100ml) were then used to enrich mouse and human PBMCs via density gradient centrifugation, respectively. Flow cytometry Flow cytometry analyses were performed as previously described. Cells were washed with flow cytometry buffer (PBS containing 2% BSA and 2 mM EDTA) and collected by centrifugation at 400g for 5 min. Cells resuspended in flow cytometry buffer were then incubated with anti-mouse CD16/CD32 mAb (BD, New Jersey, USA，553142) for 10 min on ice to block nonspecific FcR binding, followed by incubation of fluorescently labeled antibodies for 30 min on ice. Flow cytometry was performed on a FACSVerse™ flow cytometer (BD). FlowJo Software was used for data analysis. Antibodies used in flow cytometry analyses are as follows, CD45 (eBioscience, California, USA, cat. no. 12-0451-82; isotype: Rat IgG2b kappa), CD45 (eBioscience, cat. no. 17-0451; isotype: Rat IgG2b kappa), CD326 (BD, cat. no. 563478; isotype: Rat IgG2a κ), CD11c (eBioscience, cat. no. 12-0114-81; isotype: Armenian Hamster IgG), H2-Kb (eBioscience, cat. no. 11-5958-82; isotype: Mouse IgG2a kappa), H2-Db (BD, cat. no. 553573; isotype: Mouse IgG2b, κ), IA-b (Biolegend, San Diego, CA, cat. no. 116405; isotype: Mouse IgG2a, κ), H2-Kd/Dd (Biolegend, cat. no. 34-1-2S; isotype: Mouse IgG2a), CD3 (eBioscience, cat. no. 11-0031-63; isotype: Armenian Hamster IgG), CD4 (BD, cat. no. 553051; isotype: Rat IgG2a κ), and CD8 (eBioscience, cat. no. 12-0081-82; isotype: Rat IgG2a, κ), huCD45 (BD, cat. no. 555485; isotype: Mouse IgG1, κ), huCD3 (eBioscience, cat. no. 11-0038-42; isotype: Mouse IgG1, κ). MLR assay Isolated PBMCs were collected in X-VIVO™ 15 medium (Lonza, Visp, Switzerland, cat. no. 04-418Q) supplemented with 10% FBS and 200U/ml pen/strep (Gibco, Massachusetts, USA, cat.no. 15140122). 1.0×10 5 PBMCs from either Ctrl TV BALB/c or DMTV C57 BALB/c mice were labeled with CellTrace™ CFSE (Thermo Fisher Scientific, Massachusetts, USA, cat. no. C34554) and incubated with irradiation (25 Gy)-inactivated 1.0×10 5 PBMCs from BALB/c, C57BL/6 or C3H/He mice for 10 days in a well of 96-well flat-bottom culture plate in culture medium (X-VIVO™ 15, 10% FBS, 2mM L-glutamine (Gibco, cat. no. A2916801), 50μM β-mercaptoethanol (Sigma-Aldrich, cat. no. 60-24-2), 20U/ml IL-2 (Biolegend, cat. no. 575402) and 200U/ml pen/strep). Phytohemagglutinin-L (PHA-L) (Invitrogen, Massachusetts, USA, cat. no. 00-4977-93) was also served as a positive control. CFSE intensity in CD3 + T cells was checked on a FACSVerse flow cytometer (BD) and data were processed with FlowJo. Integrated single cell RNA-sequencing and TCR-sequencing 10× Genomics platform was used for integrated single cell RNA-sequencing and TCR-sequencing (scRNA-seq & scTCR-seq) according to the manufacturer’s protocols. Libraries were sequenced by Illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run. Cellranger (v7.1.0) was used to align reads to mm10 genome and generate feature-barcode matrices. Genes expressed in fewer than 3 cells were filtered from expression matrices. Cells with a mitochondrial fraction not in the highest confidence interval were filtered out, which results in removal of cells with a mitochondrial percentage of more than 5%. Doublets were excluded with DoubletFinder (v2.0.3). Artificial doublets were generated from raw RNA count matrices based on the average of gene expression profiles of randomly sampled cell pairs. After merging artificial doublets with real existing scRNA-seq data using Seurat (v4.0.4), euclidean distance matrix was obtained from cell embeddings in principal component (PC) spaces. The proportion of artificial nearest neighbors (pANN) is computed by dividing its number of artificial neighbors by the neighborhood size (pK). Cells with the highest pANN were identified as doublets. The parameters were set as follows, where doublets proportion pN=0.25, PCs=30, pK was determined using mean-variance-normalized bimodality coefficient. Seurat package (v4.0.4) was used for clustering. Raw RNA count matrices were normalized using SCTransform function with mitochondrial fraction as a variable to regress out. Top 2 000 features that are repeatedly variable across datasets for integration were then identified with SelectIntegration function. Anchors were then determined using the FindIntegrationAnchors() function and datasets were integrated together with IntegrateData() function. Dimensionality reduction was performed on the integrated data with principal component analysis (PCA). First 20 principal components were then used further for UMAP visualization and clustering procedure. The resolution of 0.1 was used in FindClusters function after computing the nearest neighbors by FindNeighbors function. Differentially expressed genes (DEGs) between clusters were identified using FindAllMarkers function. Cell type annotation was carried out with the expression of canonical gene markers. Single-cell VDJ receptor sequences were assembled and analysis with Cell Ranger’s vdj pipeline (v7.1.0). T cells with inappropriate combinations of α- and β-chains were removed. The expression levels of specific TCRs were assigned to cell populations defined with scRNA-seq clustering and visualized with UMAP. Monitoring donor-specific antibodies Monitoring donor-specific antibodies (DSA) is used for analyzing the overall immunoreactivity in DMTV-treated mice upon donor cell priming. Irradiated PBMCs from BALB/c, C57BL/6 and C3H/He mice were tail vein injected into the Ctrl TV BALB/c mice and DMTV C57 BALB/c mice. 10 days after inoculation, serum was collected and incubated with the corresponding irradiated PBMCs at 37 °C overnight. FITC-anti-mouse secondary antibody (JacksonImmuno, Pennsylvania, USA, cat. no. 115-095-003) incubation was performed on the next day for 1 h at room temperature followed by flow cytometry analysis. Skin transplantation For skin transplantation, mice were anaesthetized with 4% isoflurane (RWD, Shenzhen, China, cat. no. R510-22-10) in medical air and maintained under anaesthesia using a nose cone with 1.5% isoflurane. Animals were placed on a heat pad set at 37 °C and hair was trimmed from the back of both recipient and donor. A 9 mm × 9 mm piece of full thickness skin was then cut off from the recipient back, and a 10 mm × 10 mm full thickness skin collected from the back of a donor was laid smoothly to coincide with the edge of the cut skin and was subsequently sutured together. Recipients after transplantation were patched with 3M nexcare (3M, Kleinostheim, Germany, cat. no. CBGBLRUS1509) and 3M athletic wrap (3M, cat. no. CBGBLRUS1507) was used for secondary fixation to make the skin better fit into the recipient graft bed. Carprofen and enrofloxacin were used to provide analgesia or prevent infection. Staining Skin grafts and surrounding tissues or recovered teratomas were collected and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight followed by gradient sucrose (in PBS) treatment. Tissues were then embedded in OCT compound (Sakura, California, USA, cat. no. 4583) and sectioned at 10-μm thickness using LEICA CM3050 S. For immunofluorescence staining, slides were incubated in blocking buffer (10% donkey serum, 0.1% Triton X-100 in PBS) for 1 h and incubated with primary antibodies at 4 °C overnight. After adequate washing with PBS, slides were incubated with fluorescently conjugated secondary antibodies for at room temperature for 1 hr. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, D9542). Slides were then mounted with Fluoromount-G Mounting Medium (Southern Biotech, Alabama, USA, cat. no. 0100-01). Images were captured using confocal microscope (Leica SP8). For hematoxylin and eosin (H&E) staining, tissues were fixed in 4% PFA and subjected to paraffin embedding. Paraffin-embedded tissues were sectioned at 3-μm thickness, and sections were processed for H&E staining. The following primary antibodies were used for staining analyses in the current study, anti-CD3 (Servicebio, Wuhan, China, GB13014), anti-CD4 (Servicebio, GB13064-2), and anti-CD8 (Servicebio, GB114196). Construction of humanized mice Bone marrow, liver, thymus (BLT)-humanized mouse model was constructed according to a previously published protocol 44 . Normal aborted fetuses were obtained from Shanghai First Maternity and Infant Hospital or Jing'an District Hospital of Traditional Chinese Medicine with agreement of the donors and approval of related ethical review and informed consent documents. All the procedures were approved by the Ethics Committee of School of Medicine, Tongji University, and complied with the fundamental guidelines for the proper conduct of Interim Measures for the Administration of Human Genetic Resources and related activities in academic research institutions under the jurisdiction of the Chinese Ministry of Health 52 . HLA typing (HLA-A, -B and -Cw) of human foetus was performed by the Shanghai Tissuebank Biotechnology Co., Ltd. M-NSG mice were exposed to 1Gy irradiation right before renal subcapsular transplantation of foetal liver and thymus tissues at the right side. The thymus/liver transplanted M-NSG mice were then tail vein injected with 5.0×10 5 huCD34 + hemopoietic stem cells (HSCs) enriched with magnetic beads (Meltenyi, Bergisch Gladbach, Germany, 130-056-701) from the liver of the same foetal donor. Teratoma formation Mouse embryonic stem cells (mESCs) or human embryonic stem cells (hESCs) were injected subcutaneously over the scapula in recipient mice as indicated at a dosage of 1.0 × 10 6 cells for mESCs and 2.0 × 10 6 cells for hESCs per injection site. Statistics Statistical analyses were carried out with Prism. Differences between groups were evaluated by Student’s t -test, One-way ANOVA or Log-rank test as indicated in each figure legend. Data are mean ± SEM. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05. Declarations Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Code availability The code used in this study are available from the corresponding author upon reasonable request. ACKNOWLEDGEMENTS We thank Lin Ma at School of Medicine, Tongji University for her kind assistance and advice. Aborted foetuses were obtained from Shanghai First Maternity and Infant Hospital and Jing’an District Hospital of Traditional Chinese Medicine. This work is supported by grants from the National Natural Science Foundation of China (82025020, 82230072 and 32270874), the National Key Research and Development Program of China (2021YFA1100400 and 2021YFC2701400), the Science and Technology Commission of Shanghai Municipality (21140902300 and 22ZR1464000), Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (ZJ2018-ZD-004), Medical Research Project of Jing'an District, Shanghai (2023ZX03)，Shanghai Municipal Health Commission (202240011) and Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai. This work is also sponsored by Shanghai Blue Cross Brain Hospital Co., Ltd., and Shanghai Tongji University Education Development Foundation. AUTHOR CONTRIBUTIONS Y.L. and X.Z. conceived and initiated the project. Y.L., H.F. and X.Z. designed the experiments. Y.L., H.F., K.L., Y.Z. and Y.F. conducted the experiments. R.L. performed the single-cell RNA sequencing and TCR sequencing analysis. X.J.Z. supports the construction of BLT-humanized mice. Y.L., H.F. and K.L. carried out the data analysis. Y.L. and Y.T. designed conceptual figures. Y.L., Y.T. and X.Z. prepared the manuscript with input from all authors. L.L. and X.Z. supervised the project. COMPETING INTERESTS The authors declare no conflict of interests in this study. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at … Correspondence and requests for materials should be addressed to Xiaoqing Zhang. Reprints and permission information is available at http://www.nature.com/reprints References Global Observatory on Donation and Transplantation (GODT). Organ donation and transplantation activities 2021 report. G. O. D. T. https://www.transplant-observatory.org/2021-global-report-5 (2021). Stewart, D., Hasz, R. & Lonze, B. Beyond donation to organ utilization in the USA. Curr. Opin. 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J . https://doi.org/10.15252/embj.2020107277 (2021). Additional Declarations The authors declare no competing interests. Supplementary Files SUPPLEMENTARYINFORMATION.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4080522","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278582949,"identity":"48cf0147-240a-41ce-822b-4fae783dcbee","order_by":0,"name":"Yang Liu","email":"","orcid":"","institution":"Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""},{"id":278582950,"identity":"9606d183-c35f-4236-a967-ed6363f5e8ea","order_by":1,"name":"Hexi 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05:59:28","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4080522/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4080522/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52617386,"identity":"9d271f3f-4127-4c4e-a3b0-c996dd7a9098","added_by":"auto","created_at":"2024-03-13 16:19:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":405763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDonor MHC-specific thymus vaccination-induced immune tolerance for allogeneic transplantation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder physiological conditions, the interactions between the T cell receptors (TCRs) on thymocytes and autologous MHC (MHC\u003csup\u003eauto\u003c/sup\u003e) on both thymic epithelial cells (TECs) and dendritic cells (DCs) drive clonal deletion of self-reactive thymocytes and self-tolerance in mature T cells via positive and negative selection. MHC-mismatched allogeneic organ transplantation results in immune rejection given the presence of donor reactive T cells. In donor MHC-specific thymus vaccination (DMTV), ectopic expression of donor allogeneic MHC (MHC\u003csup\u003eallo\u003c/sup\u003e) in TECs and/or DCs in the recipient thymus will recapitulate the selection process, which leads to specific depletion of donor reactive T cells and promise immune\u0026nbsp;tolerance in an allogeneic transplant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/3a4cb031409cb46eadab8dbb.png"},{"id":52618130,"identity":"5e843d0f-ca44-493f-95d3-64c37bfeb29a","added_by":"auto","created_at":"2024-03-13 16:27:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1169155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficient expression of allogeneic MHC in TECs and DCs for DMTV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e MHC profiles of C57BL/6 mice and BALB/c mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e FACS analysis of TECs and DCs in the thymus of BALB/c mice after DMTV of C57BL/6 mice MHC (DMTV\u003csup\u003eC57\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Representative flow cytometry plots illustrating TECs expressing self-MHC (BALB/c) and donor-MHC (C57BL/6) at day 1 before vaccination and day 7 or day 90 after vaccination (left panel), and percentages of cells expressing of self-MHC and donor-MHC are quantified (right panel). Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Representative flow cytometry plots illustrating DCs expressing self-MHC and donor-MHC at day 1 before vaccination and day 7 or day 90 after vaccination (left panel), and percentages of cells expressing of self-MHC and donor-MHC are quantified (right panel). Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/135479fc69291533b2c57291.png"},{"id":52618128,"identity":"c85c6171-85f8-4457-9457-d549972a6c86","added_by":"auto","created_at":"2024-03-13 16:27:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1340252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClonal depletion of donor-reactive T cells mediated by DMTV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Peripheral blood mononuclear cells (PBMCs) from Ctrl TV BALB/c mice or DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice were labeled with CFSE and primed with irradiation-inactivated PBMCs from BALB/c mice, C57BL/6 mice or C3H/He mice. CD3\u003csup\u003e+\u003c/sup\u003e CFSE\u003csup\u003elow\u003c/sup\u003e T cells were then analyzed by FACS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Quantification of proportions of CD3\u003csup\u003e+\u003c/sup\u003e CFSE\u003csup\u003elow\u003c/sup\u003e clonally expanded T cells following allogeneic MHC stimulation. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; non-significant (n.s.); One-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Clonally expanded T cells in mixed lymphocyte reaction (MLR, \u003cem\u003en\u003c/em\u003e=2, 23 709 cells) and peripheral T cells from Ctrl TV (\u003cem\u003en\u003c/em\u003e=3, 383 485 cells) and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice (\u003cem\u003en\u003c/em\u003e=3, 330 241 cells) were subjected to integrated scRNA-seq \u0026amp; scTCR-seq, and were categorized into 11 clusters as visualized by UMAP plot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Quantification of proportions of each cluster in \u003cstrong\u003ec\u003c/strong\u003e. Clusters #1, #9 and #10 were clonally expanded in MLR after priming with irradiation-inactivated C57BL/6 PBMCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e TRAV-TRBV pairing profiles in clonally expanded Cluster #1 T cells (left panel) and Cluster #9 T cells (right panel) in MLR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e FeaturePlot showing representative \u003cem\u003eTRBV31\u003c/em\u003e and \u003cem\u003eTRAV13-1\u003c/em\u003e TCR gene expression in T cell clusters from Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice. Downsampling to 42 130 cells in total and 3 991 cells in Cluster #1 in Ctrl TV group, and 30,241 cells in total and 4 082 cells in Cluster #1 in DMTV\u003csup\u003eC57\u003c/sup\u003e group (top panel); downsampling to 35 755 cells in total and 1 016 cells in Cluster #9 in Ctrl TV group, and 30,241 cells in total and 1 054 cells in Cluster #9 in DMTV\u003csup\u003eC57\u003c/sup\u003e group (lower panel). Cluster #1 and # 9 of interest were broken line circled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e Quantification of proportions of \u003cem\u003eTRBV31\u003c/em\u003e and \u003cem\u003eTRAV9-1\u003c/em\u003e TCR-bearing T cells in Cluster #1, and \u003cem\u003eTRAV13-1\u003c/em\u003e and \u003cem\u003eTRBV13-1\u003c/em\u003e TCR-bearing T cells in Cluster #9 in Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e groups. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; non-significant (n.s.); Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/5d8c4754044d78f176621ce3.png"},{"id":52617389,"identity":"c93c015d-9cee-44e0-be76-7d9fa2875f2a","added_by":"auto","created_at":"2024-03-13 16:19:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":628287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDonor-specific tolerance mediated by DMTV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic representation of designed processes for detecting donor-specific antibodies (DSA). Irradiated PBMCs from BALB/c, C57BL/6 or C3H/He mice were tail vein injected into Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice. 10 days after inoculation, DSA in the serum was monitored by incubation with PBMCs isolated from BALB/c, C57BL/6, and C3H/He mice, incubation with FITC-conjugated anti-mouse secondary antibody and FACS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Representative flow cytometry plot for DSA detection, where Affinity\u003csup\u003ehigh\u003c/sup\u003e indicates the presence of DSA in the serum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Quantification of proportions of cells with high DSA affinity in \u003cstrong\u003eb\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; non-significant (n.s.); One-way ANOVA.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/7a896078ba936c55b9e5090d.png"},{"id":52617385,"identity":"30dd9e7c-d602-413b-8763-e04813de5b17","added_by":"auto","created_at":"2024-03-13 16:19:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2195261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDonor-specific tolerance of allotransplanted skin mediated by DMTV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic representation of skin transplantation in thymus vaccinated mice. Day 0, thymus vaccination of Ctrl or C57BL/6 MHC; day 3, IFN-g administration; day 7, T cell depletion via anti-T cell antibodies and irradiation; day 60 (post-transplantation day 0, PTD 0), skin transplantation. Recipient, BALB/c mice; donor, BALB/c mice, C57BL/6 mice or C3H/He mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Representative skin tissues 7, 15 or 30 days after transplantation. 30 days after transplantation, no grafts survived in Ctrl TV BALB/c mice transplanted with C57BL/6 skin or in DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice transplanted with C3H/He skin. Scale bar, 1cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e 5-point rating scale for evaluation of the status of the donor skin grafts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Survival scores of the donor skin grafts. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=6 independent experiments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Survival curves of the donor skin grafts. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=6 independent experiments). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; non-significant (n.s.); Log-rank test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e Representative H\u0026amp;E and immunohistochemical (IHC) staining of CD3\u003csup\u003e+\u003c/sup\u003e T cells in adjacent slices from mice 7 days post-transplantation. Region of interest 1 (ROI 1), donor skin graft region; ROI 2, junction region between recipient skin and donor skin. Scale bars of the top two panels, 500 μm; scale bar of the lower panel, 40 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg \u003c/strong\u003eQuantification analyses of\u0026nbsp;CD3\u003csup\u003e+ \u003c/sup\u003epositively\u0026nbsp;stained\u0026nbsp;area 7 days after transplantation in ROI 1 and ROI 2 in \u003cstrong\u003ef\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; One-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e Representative H\u0026amp;E and IHC staining of CD3\u003csup\u003e+\u003c/sup\u003e T cells in adjacent slices from mice 30 days after transplantation. ROI 1, donor skin graft region; ROI 2, junction region between recipient skin and donor skin. Scale bars of the top two panels, 500 μm; scale bar of the lower panel, 40 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei \u003c/strong\u003eQuantification analyses of\u0026nbsp;CD3\u003csup\u003e+ \u003c/sup\u003epositively\u0026nbsp;stained\u0026nbsp;area 30 days after transplantation in ROI 1 and ROI 2 in \u003cstrong\u003eh\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). Non-significant (n.s.); Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/9c2278176b9abbc158a90551.png"},{"id":52617391,"identity":"5a704098-b9ee-453e-828a-04667f7b6602","added_by":"auto","created_at":"2024-03-13 16:19:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2012691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDonor-specific tolerance of allotransplanted multi-lineages mediated by DMTV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic representation of mouse embryonic stem cell (mESC) transplantation. Day 0, thymus vaccination of Ctrl or C57BL/6 MHC; day 3, IFN-g administration; day 7, T cell depletion via anti-T cell antibodies and irradiation; day 60, mESC transplantation. Recipient, BALB/c mice receiving Ctrl TV or DMTV\u003csup\u003eC57\u003c/sup\u003e or C57BL/6 mice; donor, C57BL/6 mESC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Spontaneously developed teratomas generated from C57BL/6 mESC grafts in C57BL/6 mice and DMTV\u003csup\u003eC57\u003c/sup\u003e-treated BALB/c mice. No teratomas were generated in C57BL/6 mESC grafts in Ctrl TV-treated BALB/c mice. Scale bar, 1cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Quantification of the transverse diameters of teratomas in \u003cstrong\u003eb\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=8 and 6 in C57BL/6 mESC grafts from C57BL/6 mice and DMTV\u003csup\u003eC57\u003c/sup\u003e-treated BALB/c mice, respectively). non-significant (n.s.); Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Representative H\u0026amp;E and IHC staining of CD3\u003csup\u003e+\u003c/sup\u003e T cells in teratomas generated from C57BL/6 mESC grafts in C57BL/6 mice and DMTV\u003csup\u003eC57\u003c/sup\u003e-treated BALB/c mice. ROI 1, neural tube-like ectodermal tissues; ROI 2, intestine-like endodermal tissues. Scale bars of the top two panels, 400 μm; scale bar of the lower panel, 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Quantification analyses of\u0026nbsp;CD3\u003csup\u003e+ \u003c/sup\u003epositively\u0026nbsp;stained\u0026nbsp;area in \u003cstrong\u003ed\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef \u003c/strong\u003eSchematic representation of human embryonic stem cells (hESCs) transplantation in a bone marrow, liver, thymus (BLT) humanized mouse model with either Ctrl TV or DMTV\u003csup\u003eH9\u003c/sup\u003e in the reconstructed human thymus. Day 0, the M-NSG mice were transplanted with foetal thymus and liver tissues under the renal capsules and were tail vein injected with hemopoietic stem cells (HSCs) isolated from the same foetal liver. Before transplantation, the foetal thymus tissues were vaccinated with either Ctrl or H9 hESC HLA; day 30, the proportions of reconstituted human immune cells in the peripheral blood of the BLT-humanized mouse were validated via FACS; day 60, the BLT-humanized mouse with either Ctrl TV or DMTV\u003csup\u003eH9\u003c/sup\u003e were subcutaneously transplanted with H1 or H9 hESCs. Recipient, BLT-humanized mouse treated with either Ctrl TV or DMTV\u003csup\u003eH9\u003c/sup\u003e; donor, H1 or H9 hESCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e HLA typing of the foetal tissues, H1 hESCs and H9 hESCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh \u003c/strong\u003eSpontaneously developed teratomas generated from H1 and H9 hESC grafts in BLT-humanized mice treated with either Ctrl TV or DMTV\u003csup\u003eH9\u003c/sup\u003e. Scale bar, 1cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei \u003c/strong\u003eQuantification of the transverse diameters of teratomas in \u003cstrong\u003eh\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 for each group). *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; non-significant (n.s.); One-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej \u003c/strong\u003eRepresentative H\u0026amp;E and IHC staining of huCD3\u003csup\u003e+\u003c/sup\u003e T cells in teratomas generated from teratomas generated from H1 and H9 hESC grafts in BLT-humanized mice treated with either Ctrl TV or DMTV\u003csup\u003eH9\u003c/sup\u003e. ROI 1, neural tube-like ectodermal tissues; ROI 2, intestine-like endodermal tissues. Scale bars of the top two panels, 400 μm; scale bar of the lower panel, 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek\u003c/strong\u003e Quantification analyses of\u0026nbsp;huCD3+ positively\u0026nbsp;stained\u0026nbsp;area in\u003cstrong\u003e j\u003c/strong\u003e. Data are mean ± SEM (\u003cem\u003en\u003c/em\u003e=3 independent experiments). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; One-way ANOVA.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/617cc9958ae0d4aa027351fc.png"},{"id":52620143,"identity":"47e5a9c3-e3ff-4ac9-b935-10b466f4903a","added_by":"auto","created_at":"2024-03-13 16:43:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5486102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/cd81f6ba-7c94-4483-ba95-8f323dcb11bc.pdf"},{"id":52619291,"identity":"34b8f5f2-6b7b-47c4-be9a-cf5ddb461550","added_by":"auto","created_at":"2024-03-13 16:35:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1428091,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYINFORMATION.docx","url":"https://assets-eu.researchsquare.com/files/rs-4080522/v1/d1de1e338b9dd63b848890a2.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eDonor MHC-specific Thymus Vaccination for Immunocompatible Allotransplantation\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOrgan transplantation is the last-resort option to treat patients with end-stage organ failure. According to 2021 global report published by the Global Observatory on Donation and Transplantation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, less than 10% of patients left with this only option received organ transplantation treatment owing to an extreme shortage of major histocompatibility complex (MHC, in human MHC is generally recognized as human leukocyte antigens or HLA)-matched donor organs. On the other hand, the acceptance rate is just 0.6% in U.S. when the electronically transmitted offers for patients are ahead of the ultimate acceptor, and the nonuse rate among recovered organs for transplant or organs potentially available per donor approaches 25\u0026ndash;80%\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. This organ underutilization is attributable to the post-donation challenges to the allocation, storage and transport of recovered organs to waitlisted and MHC-matched patients\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMHC mismatch is the leading cause of transplant rejection\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. There are two major types of MHC molecules. MHC class I molecules (MHC I) are expressed on the surface of all nucleated cells while MHC class II molecules (MHC II) are restricted to professional antigen-presenting cells (APCs)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Allogeneic MHC molecules expressed on donor cells after organ transplantation are directly recognized by T cell receptors (TCRs) of recipient T cells, which is the primary mechanism for immune rejection\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. MHC I and MHC II also present antigen peptides periodically broken down during normal or diseased cellular processes to the immune cells for antigen recognition, which partially contributes to immune rejection\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Successful organ transplantation largely relies on the degree of MHC matching\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, as MHC is highly polymorphic, fully MHC-matched donors are rarely available\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, immunosuppressants are regularly used to reduce the intensity of immune responses after allotransplantation, albeit this might lead to increased risk of infections and malignancies\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eT cells are central to the process of transplant rejection. The features of conventional T cells discriminating between MHC-mismatched \u0026ldquo;non-self\u0026rdquo; cells and MHC-matched \u0026ldquo;self\u0026rdquo; cells are acquired during their thymic development\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In the thymus, immature T cells (thymocytes) undergo a positive-negative selection\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. During positive selection, T cells will undergo apoptosis by neglect when there is a low affinity between their surface TCRs and MHCs expressed on thymic epithelial cells (TECs) and dendritic cells (DCs)\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Those T cells are also programmed to undergo apoptosis during negative selection when TCRs on immature T cells bind with a high affinity to MHC or presented antigenic peptides in order to prevent autoimmune response\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we report a \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ed\u003c/span\u003eonor \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eHC-specific \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003et\u003c/span\u003ehymus \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ev\u003c/span\u003eaccination (DMTV) strategy to recapitulate TCR-MHC adaptation during T cell development in the thymus. We hypothesize that ectopic expression of allogeneic MHC (MHC\u003csup\u003eallo\u003c/sup\u003e) in TECs and DCs in recipient thymus will drive \u0026ldquo;non-self\u0026rdquo; to \u0026ldquo;self\u0026rdquo; T cell antigen discrimination via specific depletion of the donor reactive T cells during positive and negative selection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The recipient receiving DMTV is expected to tolerate allotransplantation of the donor organs or tissues bearing the vaccinated MHC with no immunosuppressant required.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEctopic expression of allogeneic MHC in recipient thymus\u003c/h2\u003e \u003cp\u003eTo test this hypothesis in allotransplantation between BALB/c (recipient) and C57BL/6 (donor) mice, two MHC completely mismatched allogeneic strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), AAV2/8-CMV-H2-K\u003csup\u003eb\u003c/sup\u003e α chain-IRES-H2-D\u003csup\u003eb\u003c/sup\u003e α chain and AAV2/8-CMV-I-A\u003csup\u003eb\u003c/sup\u003e α chain-IRES-I-A\u003csup\u003eb\u003c/sup\u003e β chain bearing both C57BL/6 donor MHC I and MHC II expression cassettes were constructed and viruses were then packaged and concentrated for thymus vaccination (Supplementary information, Fig. S1). 1\u0026times;10\u003csup\u003e11\u003c/sup\u003e viral genomes (vg) for each virus in 10 \u0026micro;l were intrathymically injected for each lobe of BALB/c mouse (DMTV\u003csup\u003eC57\u003c/sup\u003e), and an empty AAV2/8 virus was similarly injected in the thymus at a concentration of 2\u0026times;10\u003csup\u003e11\u003c/sup\u003e vg for each lobe for control (Ctrl TV). 7 days after injection, C57BL/6 donor MHC I H2-K\u003csup\u003eb\u003c/sup\u003e and H2-D\u003csup\u003eb\u003c/sup\u003e as well as MHC II I-A\u003csup\u003eb\u003c/sup\u003e were detected in 88.7\u0026ndash;93.1% of TECs and 15.5\u0026ndash;39.9% DCs in the vaccinated BALB/c thymus as evaluated by fluorescence-activated cell sorting (FACS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). The ectopic expression of donor MHC in TECs lasted for more than 90 days, although those expressed in DCs eventually decreased, which is likely due to the rapid turnover rate of DCs in the thymus (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ed)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These data suggest that the delivery system was efficient and the stable expression pattern of donor MHC in TECs of recipients would therefore ensure a long-term effect for introducing donor MHC tolerance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDMTV leads to alloreactive T cell clonal depletion\u003c/h2\u003e \u003cp\u003eTo evaluate the functional outcome of DMTV, BALB/c mice received Ctrl TV or DMTV\u003csup\u003eC57\u003c/sup\u003e for 7 days were treated with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs) as well as 3 Gy total body irradiation (TBI) to eliminate pre-existing T cell repertoire. Newly developed CD4\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells completely reconstituted the cell repertoire in peripheral 2 months after depletion (Supplementary information, Fig. S2). The reconstituted peripheral blood mononuclear cells (PBMCs) from Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice were then used for mixed lymphocyte reaction (MLR) analysis to evaluate their reactiveness to donor cells (Supplementary information, Fig. S3a). Both PBMCs from Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice did not show any responsiveness to irradiation-inactivated PBMCs from BALB/c mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), suggesting no autoimmunity was acquired after DMTV. PBMCs from both groups equally responded to common antigen phytohemagglutinin (PHA) and irradiation-inactivated PBMCs from C3H/He mice, another mouse strain with distinct MHC background (H2\u003csup\u003ek\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Supplementary information, Fig. S3b), suggesting normal immune responsiveness remained in both Ctrl and donor-specific MHC vaccinated mice. Remarkably, PBMCs from DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice had a much lower responsiveness towards irradiation-inactivated PBMCs from C57BL/6 mice as compared with that of PBMCs from Ctrl TV BALB/c mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These data strongly suggest that DMTV results in efficient and specific T cell tolerance towards cells expressing vaccinated MHC.\u003c/p\u003e \u003cp\u003eTo confirm specific depletion of donor-reactive T cells, clonally expanded T cells from PBMCs of Ctrl TV BALB/c mice were FACS enriched and subjected to integrative single-cell RNA sequencing and TCR sequencing (scTCR-seq \u0026amp; scRNA-seq) after priming with irradiated C57BL/6 PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Supplementary information, Fig. S3a and S3c). As compared with the non-primed T cells, a cluster of CD4\u003csup\u003e+\u003c/sup\u003e T cells (cluster #1) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (cluster #9) were specifically expanded after irradiated C57BL/6 PBMCs priming (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Supplementary information, Fig. S3d). Meanwhile, proliferating T cells (cluster #10) were also obviously elevated after priming with allogeneic PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Supplementary information, Fig. S3d). The most dominant TCR profiles in clonally expanded CD4\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells were then successfully retrieved and considered as the potential candidates that specifically responded to donor MHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). We then isolated peripheral T cells from Ctrl TV BALB/c mice and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice 3 months after vaccination for integrated scRNA-seq \u0026amp; scTCR-seq.\u0026nbsp;There were no differences on the overall constitutions in peripherally re-populated T cells after thymus vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ed)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, suggesting intact intrinsic T cell developmental programs remained after DMTV. Intriguingly, the proportions of \u003cem\u003eTRBV31\u003c/em\u003e-bearing CD4\u003csup\u003e+\u003c/sup\u003e T cells in cluster #1 and \u003cem\u003eTRAV13-1\u003c/em\u003e-bearing CD8\u003csup\u003e+\u003c/sup\u003e T cells in cluster #9 were significantly reduced in thymus vaccinated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Together, these results indicate that donor reactive T cells are specifically depleted during T cell development in the thymus of mice receiving DMTV.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDMTV leads to blunted immune responses to donor cells\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter allotransplantation, T cells are initially activated either directly or indirectly, which subsequently leads to activation of B cells, resulting in both cell-mediated and antibody-mediated immune rejection\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Monitoring donor-specific antibodies (DSA) is therefore widely used for detecting the overall levels of immune rejection and a guide for tailored immunosuppressive treatment\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. To analyze the immune responses to donor cells after DMTV \u003cem\u003ein vivo\u003c/em\u003e, we monitored DSA in Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After tail vein injection of irradiation-inactivated PBMCs from BALB/c mice for 10 days, no DSA was detected in the serum of both Ctrl TV and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). As expected, the injection of irradiation-inactivated PBMCs from C3H/He mice resulted in comparable and prominent induction of DSA in both groups. On the other hand, the injection of irradiation-inactivated C57BL/6 PBMCs led to robust DSA induction in Ctrl TV BALB/c mice and DMTV\u003csup\u003eC57\u003c/sup\u003e treatment almost fully abolished DSA induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), highlighting the robustness of DMTV in blunting the overall donor-specific immune responses \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDMTV mitigates immune rejection of skin allotransplants\u003c/h2\u003e \u003cp\u003eTo examine whether DMTV could provide an immune competent while donor-specific immunotolerant condition for allotransplantation, we transplanted the skin from C57BL/6 mice to BALB/c mice 2 months after thymus vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In Ctrl TV BALB/c mice, the transplanted skin from C57BL/6 mice showed prominent rejection starting from post transplantation day (PTD) 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-e). The transplanted skin from C3H/He mice was similarly rejected in DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice in PTD 6\u0026ndash;15. In contrast, the transplanted skin donated from C57BL/6 mice in DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice showed tolerance even 30 days after transplantation, which was almost comparable to that in autologous transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-e). These data suggest that DMTV constructs a donor-specific immune tolerance environment and supports long-term survival of allotransplanted organs with no need of immunosuppressive treatment.\u003c/p\u003e \u003cp\u003eTo confirm that DMTV induces a donor-specific immune tolerance environment, histological analyses were performed to check immune cell infiltration after transplantation. There were massive CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells infiltrated in the C57BL/6 mice skin tissue transplanted into Ctrl TV BALB/c mice and the C3H/He mice skin tissue transplanted into DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i and Supplementary information, Fig. S4). Similar to the autologous transplantation group, very rare T cell infiltration was observed in the C57BL/6 mice skin tissue transplanted into DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice even 30 days after transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i and Supplementary information, Fig. S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDMTV mitigates immune rejection of allogeneic mouse embryonic stem cell transplants\u003c/h2\u003e \u003cp\u003eGiven their pluripotency, mouse embryonic stem cells (mESCs) develop into almost all types of tissues or lineages after transplantation into immune compromised mice. To investigate whether DMTV supports successful allogeneic transplantation of different tissues, we subcutaneously transplanted C57BL/6 mESCs into C57BL/6 mice (autologous transplantation) and Ctrl TV or DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice (allogeneic transplantation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In the autologous transplantation group, mESCs efficiently survived and developed into different lineages, such as ectodermal cells (region of interest 1, ROI 1) and endodermal cells (ROI 2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-e). In Ctrl TV BALB/c mice, allotransplanted C57BL/6 mESCs were completely rejected, which resulted with no transplants for further analyses. However, allotransplanted C57BL/6 mESCs in DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice largely survived and normally developed into multi-lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-e). Histological analyses further confirmed low T cell infiltration in both autologous transplants and allogeneic transplants in donor MHC thymus vaccinated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and Supplementary information, Fig. S5a, S5b). These results suggest that the donor-specific immune tolerance environment introduced by DMTV supports survival and normal development of mESCs. Meanwhile, DMTV induced allotransplantation tolerance is universal and might be suitable for transplantation of various organs or tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHumanized DMTV also induces allotransplantation immune tolerance\u003c/h2\u003e \u003cp\u003eTo validate whether the DMTV strategy also functions in a human context, we vaccinated human donor MHC (referred to HLA hereafter) in the reconstructed human thymus of a bone marrow, liver, thymus (BLT)-humanized mouse model\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. NOD-Prkdc\u003csup\u003escid\u003c/sup\u003eIl2rg\u003csup\u003eem1\u003c/sup\u003e/Smoc (M-NSG) mice were exposed to 1 Gy irradiation and transplanted with foetal thymus and liver tissues under the renal capsules. The thymus/liver-transplanted M-NSG mice were then tail vein injected with 5.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e huCD34\u003csup\u003e+\u003c/sup\u003e hemopoietic stem cells (HSCs) isolated from the liver of the same foetal donor (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). HLA typing revealed that HLA alleles of the foetal tissues used for constructing BLT-humanized mice were completely mismatched from both H1 and H9 human embryonic stem cells (WA01 and WA09 hESCs, authorized from WiCell, Madison) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Expression vectors for H9 HLA types were then constructed (AAV2/8-CMV-HLA-A*02:01-IRES-HLA-A*03:01, AAV2/8-CMV-HLA-B*35:03-IRES-HLA-B*44:02 and AAV2/8-CMV-HLA-Cw*04:01-IRES-HLA-Cw*07:04, Supplementary information, Fig. S1).\u003c/p\u003e \u003cp\u003eTo construct a humanized DMTV system, 1\u0026times;10\u003csup\u003e11\u003c/sup\u003e vg in 10 \u0026micro;l for each virus were co-injected into a block of foetal thymus (around 100 mg) and the thymus was further incubated in 1 ml basal medium containing 2\u0026times;10\u003csup\u003e11\u003c/sup\u003e vg viruses for 2 h before renal subcapsular transplantation together with the liver tissue. FACS analysis revealed that human immune cells (huCD45\u003csup\u003e+\u003c/sup\u003e and huCD3\u003csup\u003e+\u003c/sup\u003e) efficiently populated the peripheral blood of the humanized mice 2 months after H9 HLA DMTV (DMTV\u003csup\u003eH\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e) and BLT humanized mouse model construction (Supplementary information, Fig. S5c, S5d). H1 or H9 hESCs were then subcutaneously injected into Ctrl TV or DMTV\u003csup\u003eH\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e BLT humanized mice. 2 months after hESC transplantation, the sizes of transplants recovered from DMTV\u003csup\u003eH\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e BLT humanized mice allotransplanted with H9 hESCs were obviously larger than those from DMTV\u003csup\u003eH\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e BLT humanized mice allotransplanted with H1 hESCs or Ctrl TV BLT humanized mice transplanted with H9 hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eh, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). Of note, there were prominent infiltration of huCD3\u003csup\u003e+\u003c/sup\u003e T cells, huCD4\u003csup\u003e+\u003c/sup\u003e T cells and huCD8\u003csup\u003e+\u003c/sup\u003e T cells in ectodermal tissues (ROI 1) and endodermal tissues (ROI 2) of H1 hESC transplants from DMTV\u003csup\u003eH\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e BLT humanized mice and H9 transplants from Ctrl TV BLT humanized mice, whereas, T cell infiltration could rarely be detected in tissues from H9 hESC transplants after DMTV\u003csup\u003eH\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e pre-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ej, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ek and Supplementary information, Fig. S5e, S5f). These data highlight that the DMTV strategy also functions in human context and could therefore serves as a potential strategy to bypass MHC-matching in organ allotransplantation.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOrgan transplantation is well-acknowledged as the last-resort option to treat organ failure. However, the organ transplantation rate in patients left with this only option was extremely low owing to a lack of efficient system in finding and locating MHC-matched donors and subsequent difficulties in allocating, storing and transporting of recovered organs, which also resulted in severe underutilization of donated organs. Meanwhile, transplantation of partially MHC-matched organs always leads to allograft rejection, which requires lifelong immunosuppressive treatment, shortening the survival period of transplanted organs and causing unwanted risks of infections and malignancies. One can therefore reasonably foresee that organ transplantation will ultimately become a regular treatment for patients with organ failure if the availability of donor organs is no longer an issue and the dilemma of immune rejection is overcome. In the current study, we have developed a DMTV strategy by ectopic expression of donor-specific MHC molecules in the recipient thymus to deplete donor reactive T cells. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies reveal that after DMTV the recipient immune system becomes tolerant to donor organs or tissues although they bear a mismatched MHC profile. DMTV works together with the endogenous selection system in the thymus and educates T cells to tolerate to both self MHC and donor MHC. Our designed DMTV strategy therefore avoids stringent recipient-donor MHC matching and simplifies all other tedious procedures after recovering an organ for transplantation since it fills the gap standing between the recipient and the donor either spatially or temporally.\u003c/p\u003e \u003cp\u003eIn a non-immune tolerant allotransplantation, T cells are activated directly or indirectly, which leads to activation of other immune cells and causes immune rejection\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Removing donor reactive T cells is therefore the key to ensure a successful allotransplantation. In the current study, DMTV efficiently removes donor reactive T cells during their development in the thymus. This is evidenced by \u003cem\u003ein vitro\u003c/em\u003e MLR analysis, \u003cem\u003ein vivo\u003c/em\u003e DSA monitoring, and very low infiltration of T cells in the grafted tissues after DMTV even without any immunosuppressive intervention. Integrated scRNA-seq \u0026amp; scTCR-seq further reveals clonal depletion of T cells harboring potential donor reactive TCRs. Notably, DMTV only introduces immune tolerance to donor-specific MHC and animals receiving DMTV treatment show almost complete immune responses to general antigens as well as other allogeneic MHC types. ScRNA-seq \u0026amp; scTCR-seq also showed that in the peripheral T cell repertoire, the composition of T cell populations remained intact. Together, these data indicate that DMTV treatment has minor effects on overall T cell development and maturation. In the future, it will be intriguing to test whether the thymus vaccination strategy is also efficient to induce designed immune tolerance by expression of targeted antigens other than donor MHC in the thymus.\u003c/p\u003e \u003cp\u003eIn humanized mice receiving DMTV treatment, cognate hESCs tolerate allogeneic transplantation and normally develop into multi-lineages. This raises a point that DMTV is suitable not only for allogeneic organ transplantation but also for cell-based therapy utilizing products derived from human pluripotent stem cells (hPSCs). Although individualized human induced pluripotent stem cells have been proposed for immunotolerant transplantation, it is expensive and needs long-period reprogramming procedures and safety validations. Engineering surface HLA molecule profiles or other immune modulatory molecules in hPSCs has also been proposed to generate universal cells for allogeneic transplantation\u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, these engineered cells might be endowed with the capability of immune surveillance evasion. Therefore, DMTV is another practical option for application of hPSCs for cell therapies with no need of extra genetic engineering in these cells or immunosuppressive intervention.\u003c/p\u003e \u003cp\u003eAccording to the European Molecular Biology Laboratory\u0026rsquo;s European Bioinformatics Institute (EMBL-EBI), 14,956 types of classical HLA-I proteins have so far been officially recognized in all populations\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Considering future allogeneic transplantation between recipients and donors locally, the required expression vectors for variable HLA-I types will be much reduced in a specific area. It is therefore doable for advanced banking of all spectrums of AAVs for each HLA-I protein expression, which will no doubt save waiting time for efficient transplantation. In the future, it will be necessary to test the DMTV strategy for allogeneic organ transplantation in large animals and eventually in clinical reality.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale C57BL/6 (SM-001), BALB/c (SM-003), C3H/He (SM-008) and\u0026nbsp;NOD-Prkdc\u003csup\u003escid\u003c/sup\u003eIl2rg\u003csup\u003eem1\u003c/sup\u003e/Smoc (M-NSG)\u0026nbsp;mice were purchased from Shanghai Model Organisms Center Inc, China. All mice were housed in groups of 5 individuals per cage and maintained on a 12-h light-dark cycle at 22–25°C under specific-pathogen free (SPF) conditions. All animal experiments were approved by the Laboratory Animal Research Center, Tongji University. All procedures involving animals were carried out in compliance with the Guide for the Care and Use of Laboratory Animals, and ethical approval was granted by the Ethics Committee, Tongji University\u0026nbsp;(approval\u0026nbsp;number:\u0026nbsp;2020YANYUSHEN093).\u0026nbsp;The investigators were blinded to allocations during experiments and outcome assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThymus vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor donor MHC expression in recipient thymus, adeno-associated virus (AAV) packaging system\u0026nbsp;was used. In brief, donor MHC cassettes driven by the CMV promoter were constructed in the AAV2/8 vector (Supplementary information,Fig.S1). After packaging, viruses were concentrated through gradient centrifugation and viral titer was detected by qRT-PCR\u0026nbsp;(for rAAV genome).\u003c/p\u003e\n\u003cp\u003eMice were anesthetized through intraperitoneal (i.p.) injection of Avertin.\u0026nbsp;Hair on the chest was removed with depilatory cream. Mice were then intubated and connected to a small animal ventilator (RWD, cat. no. R420). After skin disinfection\u0026nbsp;with povidone-iodine, a central skin incision at the level of 2\u003csup\u003end\u003c/sup\u003e intercostal space was made. To expose the thymus, a horizontal incision at the mouse sternum was introduced and set apart with a retractor. AAVs for Ctrl TV or DMTV (1×10\u003csup\u003e13\u003c/sup\u003e vg/ml, 10 μl for each MHC expression virus, and the same total dosage was applied for Ctrl in each group) were intrathymically injected with a 30-gauge Hamilton syringe. During the thymus vaccination procedure, mice were maintained inflated with a ventilator before thoracic cavity was closed and the opening was closely sutured. Carprofen (5mg/kg, subcutaneous injection) and enrofloxacin (10mg/kg, i.p. injection) were used to provide analgesia or prevent infection for 3 days after surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT cell depletion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1.5 mg anti-CD4 (BioXCell, New Hampshire, USA, BP0003-1) and 0.8 mg anti-CD8 (BioXCell, BP0061) monoclonal antibodies (mAbs) were i.p. injected into Ctrl TV mice or DMTV mice twice (7 days and 10 days after thymus vaccination) to deplete pre-existing CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells. On day 10 after thymus vaccination, mice were subjected to a\u0026nbsp;3 Gy total body irradiation (TBI). The\u0026nbsp;populations of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells were then assessed by flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThymus epithelial cell, dendritic cell and peripheral blood mononuclear cell isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate ectopic MHC expression in\u0026nbsp;thymus epithelial cells (TECs) and dendritic cells (DCs), vaccinated thymus was isolated and cut into small pieces with a scissor. Thymus tissues were then digested with 0.5 mg/ml papain (Sangon, Shanghai, China, cat. no. A003124), 2.5 mg/ml collagenase IV (R\u0026amp;D, cat. no. 9001-12-1) and 0.1 mg/ml DNase I (Sigma-Aldrich, Darmstadt,\u0026nbsp;Germany, cat. no. 11284932001) in basal DMEM/F12 medium at 37°C for 30 min. 10% feal bovine serum (FBS) in DMEM/F12 was applied to stop the digestion and cells were passed through a 70-μm cell strainer before flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were collected for mixed lymphocyte reaction (MLR) assay, detecting donor specific antibodies (DSA), single-cell sequencing and immune cell composition detection. Peripheral blood cells and spleen homogenates (passed through a 40-μm cell strainer) were collected in tubes prefilled with EDTA. Histopaque®-1083 (Sigma-Aldrich, cat. no. 10831-100ml) and Histopaque®-1077 (Sigma-Aldrich, cat. no. 10771-100ml) were then used to enrich mouse and human PBMCs via density gradient centrifugation, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry analyses were performed as previously described. Cells were washed with flow cytometry buffer (PBS containing 2% BSA and 2 mM EDTA) and collected by centrifugation at 400g for 5 min. Cells resuspended in flow cytometry buffer were then incubated with anti-mouse\u0026nbsp;CD16/CD32\u0026nbsp;mAb (BD, New Jersey, USA，553142) for 10 min on ice to block nonspecific FcR binding, followed by incubation of fluorescently labeled antibodies for 30 min on ice. Flow cytometry was performed on a FACSVerse™ flow cytometer (BD). FlowJo Software was used for data analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntibodies used in flow cytometry analyses are as follows, CD45 (eBioscience, California, USA, cat.\u0026nbsp;no. 12-0451-82; isotype: Rat IgG2b kappa), CD45 (eBioscience, cat.\u0026nbsp;no. 17-0451; isotype: Rat IgG2b kappa), CD326 (BD, cat.\u0026nbsp;no. 563478; isotype: Rat IgG2a κ), CD11c (eBioscience, cat.\u0026nbsp;no. 12-0114-81; isotype: Armenian Hamster IgG), H2-Kb (eBioscience, cat.\u0026nbsp;no. 11-5958-82; isotype: Mouse IgG2a kappa), H2-Db (BD, cat.\u0026nbsp;no. 553573; isotype: Mouse IgG2b, κ), IA-b (Biolegend, San Diego, CA,\u0026nbsp;cat.\u0026nbsp;no. 116405; isotype: Mouse IgG2a, κ), H2-Kd/Dd (Biolegend, cat.\u0026nbsp;no. 34-1-2S; isotype: Mouse IgG2a), CD3 (eBioscience, cat.\u0026nbsp;no. 11-0031-63; isotype: Armenian Hamster IgG), CD4 (BD, cat.\u0026nbsp;no. 553051; isotype: Rat IgG2a κ), and CD8 (eBioscience, cat.\u0026nbsp;no. 12-0081-82; isotype: Rat IgG2a, κ), huCD45 (BD, cat.\u0026nbsp;no. 555485; isotype: Mouse IgG1, κ), huCD3 (eBioscience, cat.\u0026nbsp;no. 11-0038-42; isotype: Mouse IgG1, κ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolated PBMCs were collected in X-VIVO™ 15 medium (Lonza, Visp, Switzerland, cat. no. 04-418Q) supplemented with 10% FBS and 200U/ml pen/strep (Gibco, Massachusetts, USA, cat.no. 15140122).\u0026nbsp;1.0×10\u003csup\u003e5\u003c/sup\u003e PBMCs from either Ctrl TV BALB/c or DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice were labeled with CellTrace™ CFSE (Thermo Fisher Scientific, Massachusetts, USA, cat. no. C34554) and incubated with irradiation (25 Gy)-inactivated\u0026nbsp;1.0×10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ePBMCs from BALB/c, C57BL/6 or C3H/He mice for 10 days in a well of 96-well flat-bottom culture plate in culture\u0026nbsp;medium (X-VIVO™ 15, 10% FBS, 2mM L-glutamine (Gibco, cat. no. A2916801), 50μM β-mercaptoethanol (Sigma-Aldrich, cat. no. 60-24-2), 20U/ml IL-2 (Biolegend, cat. no. 575402) and 200U/ml pen/strep). Phytohemagglutinin-L (PHA-L) (Invitrogen, Massachusetts, USA, cat.\u0026nbsp;no.\u0026nbsp;00-4977-93) was also served as a positive control. CFSE intensity in CD3\u003csup\u003e+\u003c/sup\u003e T cells was checked on a FACSVerse flow cytometer (BD) and data were processed with FlowJo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntegrated single cell RNA-sequencing and TCR-sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10× Genomics platform was used for integrated single cell RNA-sequencing and TCR-sequencing (scRNA-seq \u0026amp; scTCR-seq) according to the manufacturer’s protocols. Libraries were sequenced by Illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.\u003c/p\u003e\n\u003cp\u003eCellranger (v7.1.0) was used to align reads to mm10 genome and generate feature-barcode matrices. Genes expressed in fewer than 3 cells were filtered from expression matrices. Cells with a mitochondrial fraction not in the highest confidence interval were filtered out, which results in removal of cells with a mitochondrial percentage of more than 5%.\u003c/p\u003e\n\u003cp\u003eDoublets were excluded with DoubletFinder (v2.0.3). Artificial doublets were generated from raw RNA count matrices based on the average of gene expression profiles of randomly sampled cell pairs. After merging artificial doublets with real existing scRNA-seq data using Seurat (v4.0.4), euclidean distance matrix was obtained from cell embeddings in principal component (PC) spaces. The proportion of artificial nearest neighbors (pANN) is computed by dividing its number of artificial neighbors by the neighborhood size (pK). Cells with the highest pANN were identified as doublets. The parameters were set as follows, where doublets proportion pN=0.25, PCs=30, pK was determined using mean-variance-normalized bimodality coefficient.\u003c/p\u003e\n\u003cp\u003eSeurat package (v4.0.4) was used for clustering. Raw RNA count matrices were normalized using SCTransform function with mitochondrial fraction as a variable to regress out. Top 2 000 features that are repeatedly variable across datasets for integration were then identified with SelectIntegration function. Anchors were then determined using the FindIntegrationAnchors() function and datasets were integrated together with IntegrateData() function. Dimensionality reduction was performed on the integrated data with principal component analysis (PCA). First 20 principal components were then used further for UMAP visualization and clustering procedure. The resolution of 0.1 was used in FindClusters function after computing the nearest neighbors by FindNeighbors function. Differentially expressed genes (DEGs) between clusters were identified using FindAllMarkers function. Cell type annotation was carried out with the expression of canonical gene markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSingle-cell VDJ receptor sequences were assembled and analysis with Cell Ranger’s vdj pipeline (v7.1.0). T cells with inappropriate combinations of α- and β-chains were removed. The expression levels of specific TCRs were assigned to cell populations defined with scRNA-seq clustering and visualized with UMAP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonitoring donor-specific antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMonitoring donor-specific antibodies (DSA) is used for analyzing the overall immunoreactivity in DMTV-treated mice upon donor cell priming. Irradiated PBMCs from BALB/c, C57BL/6 and C3H/He mice were tail vein injected into the Ctrl TV BALB/c mice and DMTV\u003csup\u003eC57\u003c/sup\u003e BALB/c mice. 10 days after inoculation, serum was collected and incubated with the corresponding irradiated PBMCs at 37\u0026nbsp;°C\u0026nbsp;overnight. FITC-anti-mouse secondary antibody (JacksonImmuno, Pennsylvania, USA, cat. no. 115-095-003) incubation was performed on the next day for 1 h at room temperature followed by flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSkin transplantation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor skin transplantation, mice were anaesthetized with 4% isoflurane (RWD, Shenzhen, China, cat. no. R510-22-10) in medical air and maintained under anaesthesia using a nose cone with 1.5% isoflurane. Animals were placed on a heat pad set at 37 °C and hair was trimmed from the back of both recipient and donor. A 9\u0026nbsp;mm × 9 mm piece of full thickness skin was then cut off from the recipient back, and a\u0026nbsp;10 mm × 10 mm full thickness skin collected from the\u0026nbsp;back of a donor was laid smoothly to coincide with the edge of the cut skin and was subsequently sutured together. Recipients after transplantation were patched with\u0026nbsp;3M nexcare (3M, Kleinostheim, Germany, cat. no. CBGBLRUS1509) and 3M athletic wrap (3M, cat. no. CBGBLRUS1507) was used for secondary fixation to make the skin better fit into the recipient graft bed.\u0026nbsp;Carprofen and enrofloxacin were used to provide analgesia or prevent infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkin grafts and surrounding tissues or recovered teratomas were collected and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight followed by gradient sucrose (in PBS) treatment. Tissues were then embedded in OCT compound (Sakura, California, USA, cat. no. 4583) and sectioned at 10-μm thickness using LEICA CM3050 S. For immunofluorescence staining, slides were incubated in blocking buffer (10% donkey serum, 0.1% Triton X-100 in PBS) for 1 h and incubated with primary antibodies at 4 °C overnight. After adequate washing with PBS, slides were incubated with fluorescently conjugated secondary antibodies for at room temperature for 1 hr. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, D9542). Slides were then mounted with Fluoromount-G Mounting Medium (Southern Biotech, Alabama, USA, cat. no. 0100-01). Images were captured using confocal microscope (Leica SP8).\u003c/p\u003e\n\u003cp\u003eFor hematoxylin and eosin (H\u0026amp;E) staining, tissues were fixed in 4% PFA and subjected to paraffin embedding. Paraffin-embedded tissues were sectioned at 3-μm thickness, and sections were processed for H\u0026amp;E staining.\u003c/p\u003e\n\u003cp\u003eThe following primary antibodies were used for staining analyses in the current study, anti-CD3 (Servicebio, Wuhan, China, GB13014), anti-CD4 (Servicebio, GB13064-2), and anti-CD8 (Servicebio, GB114196).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of humanized mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow, liver, thymus (BLT)-humanized mouse model\u0026nbsp;was constructed according to a previously published\u0026nbsp;protocol\u003csup\u003e44\u003c/sup\u003e.\u0026nbsp;Normal aborted fetuses were obtained from Shanghai First Maternity and Infant Hospital or Jing'an District Hospital of Traditional Chinese Medicine with agreement of the donors and approval of related ethical review and informed consent documents. All the procedures were approved by the Ethics Committee of School of Medicine, Tongji University, and complied with the fundamental guidelines for the proper conduct of Interim Measures for the Administration of Human Genetic Resources and related activities in academic research institutions under the jurisdiction of the Chinese Ministry of Health\u003csup\u003e52\u003c/sup\u003e.\u0026nbsp;HLA typing (HLA-A, -B and -Cw) of human foetus was performed by the\u0026nbsp;Shanghai Tissuebank Biotechnology Co., Ltd. M-NSG mice were exposed to 1Gy irradiation right before renal subcapsular transplantation of foetal liver and thymus tissues at the right side.\u0026nbsp;The thymus/liver transplanted M-NSG mice were then tail vein injected with 5.0×10\u003csup\u003e5\u003c/sup\u003e huCD34\u003csup\u003e+\u003c/sup\u003e hemopoietic stem cells (HSCs) enriched with\u0026nbsp;magnetic beads (Meltenyi, Bergisch Gladbach, Germany, 130-056-701)\u0026nbsp;from the liver of the same foetal donor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTeratoma formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse embryonic stem cells (mESCs) or human embryonic stem cells (hESCs) were injected subcutaneously over the scapula in recipient mice as indicated at a dosage of 1.0 × 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells for mESCs and 2.0 × 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells for hESCs per injection site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were carried out with Prism. Differences between groups were evaluated by Student’s \u003cem\u003et\u003c/em\u003e-test, One-way ANOVA or Log-rank test as indicated in each figure legend. Data are mean ± SEM. ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe code used in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Lin Ma at School of Medicine, Tongji University for her kind assistance and advice.\u0026nbsp;\u003cbr\u003eAborted foetuses were obtained from Shanghai First Maternity and Infant Hospital and Jing\u0026rsquo;an District Hospital of Traditional Chinese Medicine. This work is supported by grants from the National Natural Science Foundation of China (82025020, 82230072 and 32270874), the National Key Research and Development\u0026nbsp;\u003cbr\u003eProgram of China (2021YFA1100400 and 2021YFC2701400), the Science and Technology Commission of Shanghai Municipality (21140902300 and 22ZR1464000), Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (ZJ2018-ZD-004), Medical Research Project\u0026nbsp;\u003cbr\u003eof Jing\u0026apos;an District, Shanghai (2023ZX03)，Shanghai Municipal Health Commission (202240011) and Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai. This work is also sponsored by Shanghai Blue Cross Brain Hospital Co., Ltd., and Shanghai Tongji University Education Development Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L. and X.Z. conceived and initiated the project. Y.L., H.F. and X.Z. designed the experiments. Y.L., H.F., K.L., Y.Z. and Y.F. conducted the experiments. R.L. performed the single-cell RNA sequencing and TCR sequencing analysis.\u0026nbsp;X.J.Z. supports the construction of BLT-humanized mice.\u0026nbsp;Y.L., H.F. and K.L. carried out the data analysis. Y.L. and Y.T. designed conceptual figures. Y.L., Y.T. and X.Z. prepared the manuscript with input from all authors. L.L. and X.Z. supervised the project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material available at \u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Xiaoqing Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permission information\u0026nbsp;\u003c/strong\u003eis available at http://www.nature.com/reprints\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGlobal Observatory on Donation and Transplantation (GODT). Organ donation and transplantation activities 2021 report. \u003cem\u003eG. O. D. T.\u003c/em\u003e https://www.transplant-observatory.org/2021-global-report-5 (2021).\u003c/li\u003e\n \u003cli\u003eStewart, D., Hasz, R. \u0026amp; Lonze, B. Beyond donation to organ utilization in the USA. \u003cem\u003eCurr. Opin. Organ. Transplant\u003c/em\u003e. \u003cstrong\u003e28\u003c/strong\u003e, 197\u0026ndash;206 (2023).\u003c/li\u003e\n \u003cli\u003eOrgan Procurement and Transplantation Network (OPTN) \u0026amp; Scientific Registry of Transplant Recipients (SRTR). OPTN/SRTR 2021 Annual Data Report. \u003cem\u003eU.S. Department of Health and Human Services, Health Resources and Services Administration\u003c/em\u003e http://srtr.transplant.hrsa.gov/annual_reports/Default.aspx (2023).\u003c/li\u003e\n \u003cli\u003eKwong A. J. et al.\u0026nbsp;OPTN/SRTR 2021 Annual Data Report: Liver.\u0026nbsp;\u003cem\u003eAm. J. Transplant\u003c/em\u003e. \u003cstrong\u003e23\u003c/strong\u003e, S178\u0026ndash;S263 (2023).\u003c/li\u003e\n \u003cli\u003eMontgomery, R. 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J\u003c/em\u003e. https://doi.org/10.15252/embj.2020107277 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"557cc9b6-d917-4bdc-88c3-d54d5315db95","identifier":"10.13039/501100001809","name":"National Natural Science Foundation of China","awardNumber":"82025020","order_by":0},{"identity":"00aed4ee-e0b3-40c9-ab39-091d6dcab558","identifier":"10.13039/501100001809","name":"National Natural Science Foundation of China","awardNumber":" 82230072","order_by":1},{"identity":"5b987a12-f84d-4b55-a84f-7894d501058a","identifier":"10.13039/501100001809","name":"National Natural Science Foundation of China","awardNumber":"32270874","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Shanghai East Hospital","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Allotransplantation, Immunotolerance, MHC, Thymic vaccination","lastPublishedDoi":"10.21203/rs.3.rs-4080522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4080522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOrgan transplantation is the last-resort option to treat organ failure. However, less than 10% of patients benefit from this only option due to lack of major histocompatibility complex (MHC)-matched donor organs and 25-80% of donated organs could not find MHC-matched recipients. T cell allorecognition is the principal mechanism for allogeneic graft rejection. We herein present a “donor MHC-specific thymus vaccination” (DMTV) strategy to induce T cell tolerance to both autologous and allogeneic donor MHC. Allogeneic MHC molecules were expressed in the recipient thymus through adeno-associated virus infection, which led to stable expression of allogeneic MHC together with the autologous MHC in the engineered thymus. During local T cell education, those T cells recognizing either autologous MHC or allogeneic MHC were equally depleted. We constructed C57BL/6-MHC and BALB/c-MHC dual immunocompatible mice via thymus vaccination of C57BL/6-MHC into the BALB/c thymus and observed long-term tolerance after transplantation of C57BL/6 skin and C57BL/6 mouse embryonic stem cells into the vaccinated BALB/c mice. We also validated our DMTV strategy in a bone marrow, liver, thymus (BLT)-humanized mouse model for immunocompatible allotransplantation of human embryonic stem cells. Our study suggests that DMTV is a potent avenue to introduce a donor compatible immune system in recipients, which overcomes the clinical dilemma over the extreme shortage of MHC-matched donor organs for treating patients with end-stage organ failure.\u003c/p\u003e","manuscriptTitle":"Donor MHC-specific Thymus Vaccination for Immunocompatible Allotransplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 16:19:30","doi":"10.21203/rs.3.rs-4080522/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2040709b-9e05-4968-9881-c6c412cb7812","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T16:19:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-13 16:19:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4080522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4080522","identity":"rs-4080522","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}