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Article Navigation >  Journal of Genetics and Genomics  > 2026  > Accepted Manuscript

Engineering Tregs-mediated immune tolerance via foxp3a overexpression to evade allograft transplantation barriers in zebrafish

doi: 10.1016/j.jgg.2026.02.024
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    a. State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Hubei Hongshan Laboratory, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China;

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    b. School of Marine Biology and Fisheries, Hainan University, Haikou, Hainan 570228, China;

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    c. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

Funds:

D Program of China (2023YFD2400200), Natural Science Foundation of Hubei Province (2025AFA053), Natural Science Foundation of Wuhan (2024040701010069), Science and Technology Special Fund of Hainan Province (ZDYF2024XDNY256), and State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (2024BBSA01).

We sincerely thank Luyuan Pan and Linglu Li from the China Zebrafish Resource Center (CZRC) for their technical assistance with in vitro fertilization, Zhixian Qiao, Xiaocui Chai, Fang Zhou, Wang Guangxin, Yuan Xiao, and Zhenfei Xing from the analytical and testing center of the Institute of Hydrobiology, CAS, for their assistance with bulk-seq, confocal imaging, and sperm imaging. This work was supported by the National Natural Science Foundation of China (32025037 and 32403020), the Strategic Priority Research Program of Chinese Academy of Sciences (CAS) (XDB0730300), the STI2030—Major Projects (2023ZD0405503), the National Key R&

  • Received Date: 2025-10-13
  • Accepted Date: 2026-02-26
  • Rev Recd Date: 2026-02-25
    • Available Online: 2026-03-04
    Abstract
  • In mammals, regulatory T cells (Tregs) are widely exploited to promote immune tolerance in organ transplantation. In zebrafish, although germline stem cell (GSC) or gonadal primordium transplantation into immunodeficient hosts can accelerate gamete production, maintaining immunocompromised lines presents substantial practical challenges. To overcome this limitation, this study generates a Tg(CMV:foxp3a) zebrafish line through systemic overexpression of Forkhead box P3a (Foxp3a), the lineage-defining transcription factor of Tregs. Transcriptomic and in situ hybridization analysis reveal downregulation of the Treg negative regulator cd127 and upregulation of multiple immunosuppressive factors in the head kidney and thymus. Single-cell RNA sequencing further demonstrates a reduction in effector T and B cell populations, accompanied by an increase in quiescent T cells exhibiting resting Treg-like features. Importantly, using Tg(CMV:foxp3a) fish as hosts for subcutaneous gonadal primordium transplantation (SGPT) and intraperitoneal GSC transplantation (IGCT) markedly accelerates germ cell maturation and enables efficient establishment of stable transgenic lines. Post-transplantation analysis indicates delayed and attenuated immune activation, enhanced graft survival, and rapid induction of immunosuppressive states. Together, foxp3a overexpression reshapes the immune landscape to confer immune tolerance, providing a practical Tregs-based alternative to immunodeficient hosts for fish genome manipulation and transplantation.
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  • Allan, S.E., Alstad, A.N., Merindol, N., Crellin, N.K., Amendola, M., Bacchetta, R., Naldini, L., Roncarolo, M.G., Soudeyns, H., Levings, M.K., 2008. Generation of potent and stable human CD4+ T regulatory cells by activation-independent expression of FOXP3. Mol. Ther. 16, 194-202.
    Almeida, F.F.L., Kristoffersen, C., Taranger, G.L., Schulz, R.W., 2008. Spermatogenesis in atlantic cod (Gadus morhua): a novel model of cystic germ cell development. Biol. Reprod. 78, 27-34.
    Babel, N., Stervbo, U., Reinke, P., Volk, H.-D., 2019. The identity card of T cells-clinical utility of T-cell receptor repertoire analysis in transplantation. Transplantation 103, 1544-1555.
    Beermann, J., Piccoli, M.-T., Viereck, J., Thum, T., 2016. Non-coding RNAs in development and disease: background, mechanisms, and therapeutic approaches. Physiol. Rev. 96, 1297-1325.
    Butler, A., Hoffman, P., Smibert, P., Papalexi, E., Satija, R., 2018. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411-420.
    Cao, Z., Mao, X., Luo, L., 2019. Germline stem cells drive ovary regeneration in zebrafish. Cell Rep. 26, 1709-1717.
    Chen, Z., He, M., Wang, H., Li, X., Qin, R., Ye, D., Zhai, X., Zhu, J., Zhang, Q., Hu, P., et al., 2024. Intestinal DHA-PA-PG axis promotes digestive organ expansion by mediating usage of maternally deposited yolk lipids. Nat. Commun. 15, 9769.
    Choi, H.M.T., Schwarzkopf, M., Fornace, M.E., Acharya, A., Artavanis, G., Stegmaier, J., Cunha, A., Pierce, N.A., 2018. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, dev165753.
    Ciruna, B., Weidinger, G., Knaut, H., Thisse, B., Thisse, C., Raz, E., Schier, A.F., 2002. Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc. Natl. Acad. Sci. U. S. A. 99, 14919-14924.
    Deng, J., Xiong, F., Ren, Z., Wang, X., Wang, H., He, M., Ye, D., Sun, Y., 2025. cdk2 is essential for synapsis completion and gametogenesis in zebrafish (in press). Water Biol. Secur.
    Dikiy, S., Rudensky, A.Y., 2023. Principles of regulatory T cell function. Immunity 56, 240-255.
    Dura, B., Choi, J.-Y., Zhang, K., Damsky, W., Thakral, D., Bosenberg, M., Craft, J., Fan, R., 2018. scFTD-seq: freeze-thaw lysis based, portable approach toward highly distributed single-cell 3′ mRNA profiling. Nucleic Acids Res. 47, e16.
    Farlora, R., Hattori-Ihara, S., Takeuchi, Y., Hayashi, M., Octavera, A., Alimuddin, Yoshizaki, G., 2014. Intraperitoneal germ cell transplantation in the Nile tilapia Oreochromis niloticus. Mar. Biotechnol. 16, 309-320.
    Fontenot, J.D., Gavin, M.A., Rudensky, A.Y., 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330-336.
    Gocher-Demske, A.M., Cui, J., Szymczak-Workman, A.L., Vignali, K.M., Latini, J.N., Pieklo, G.P., Kimball, J.C., Avery, L., Cipolla, E.M., Huckestein, B.R., et al., 2023. IFNγ-induction of TH1-like regulatory T cells controls antiviral responses. Nat. Immunol. 24, 841-854.
    Grant, F.M., Yang, J., Nasrallah, R., Clarke, J., Sadiyah, F., Whiteside, S.K., Imianowski, C.J., Kuo, P., Vardaka, P., Todorov, T., et al., 2020. BACH2 drives quiescence and maintenance of resting Treg cells to promote homeostasis and cancer immunosuppression. J. Exp. Med. 217, e20190711.
    Guo, C., Liu, Q., Zong, D., Zhang, W., Zuo, Z., Yu, Q., Sha, Q., Zhu, L., Gao, X., Fang, J., et al., 2022. Single-cell transcriptome profiling and chromatin accessibility reveal an exhausted regulatory CD4+ T cell subset in systemic lupus erythematosus. Cell Rep. 41, 111606.
    Honaker, Y., Hubbard, N., Xiang, Y., Fisher, L., Hagin, D., Sommer, K., Song, Y., Yang, S.J., Lopez, C., Tappen, T., et al., 2020. Gene editing to induce FOXP3 expression in human CD4+T cells leads to a stable regulatory phenotype and function. Sci. Transl. Med. 12, eaay6422.
    Hori, S., Nomura, T., Sakaguchi, S., 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057-1061.
    Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Hioki, K., Ueyama, Y., Koyanagi, Y., Sugamura, K., Tsuji, K., et al., 2002. NOD/SCID/γcnull mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100, 3175-3182.
    Jansen, K., Cevhertas, L., Ma, S., Satitsuksanoa, P., Akdis, M., van de Veen, W., 2021. Regulatory B cells, A to Z. Allergy 76, 2699-2715.
    Jung, I.H., Chung, Y.-Y., Jung, D.E., Kim, Y.J., Kim, D.H., Kim, K.-S., Park, S.W., 2016. Impaired lymphocytes development and xenotransplantation of gastrointestinal tumor cells in Prkdc -null SCID zebrafish model. Neoplasia 18, 468-479.
    Kanamori, M., Nakatsukasa, H., Okada, M., Lu, Q., Yoshimura, A., 2016. Induced regulatory T cells: their development, stability, and applications. Trends Immunol. 37, 803-811.
    Kasheta, M., Painter, C.A., Moore, F.E., Lobbardi, R., Bryll, A., Freiman, E., Stachura, D., Rogers, A.B., Houvras, Y., Langenau, D.M., et al., 2017. Identification and characterization of T reg-like cells in zebrafish. J. Exp. Med. 214, 3519-3530.
    Kawasaki, T., Siegfried, K.R., Sakai, N., 2016. Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture. Development 143, 566-574.
    Khattri, R., Cox, T., Yasayko, S.-A., Ramsdell, F., 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4, 337-342.
    Kikuchi, K., 2020. New function of zebrafish regulatory T cells in organ regeneration. Curr. Opin. Immunol. 63, 7-13.
    Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310.
    Klawon, D.E.J., Pagane, N., Walker, M.T., Ganci, N.K., Miller, C.H., Gai, E., Rodriguez, D.M., Ryan-Payseur, B.K., Duncombe, R.K., Adams, E.J., et al., 2025. Regulatory T cells constrain T cells of shared specificity to enforce tolerance during infection. Science 387, eadk3248.
    Krummel, M.F., Allison, J.P., 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182, 459-465.
    Lauener, M., AzadPour, S., Abdossamadi, S., Parthasarathy, V., Ng, B., Ostroumov, E., Cuvelier, G.D.E., Levings, M.K., MacDonald, K.N., Kariminia, A., et al., 2023. CD56bright CD16- natural killer cells as an important regulatory mechanism in chronic graft-versus-host disease. Haematologica 108, 761-771.
    Li, X., Zhang, F., Wu, N., Ye, D., Wang, Y., Zhang, X., Sun, Y., Zhang, Y.-A., 2020. A critical role of foxp3a-positive regulatory T cells in maintaining immune homeostasis in zebrafish testis development. J. Genet. Genomics 47, 547-561.
    Liao, Y., Smyth, G.K., Shi, W., 2013. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930.
    Lv, P., Ma, D., Gao, S., Zhang, Y., Bae, Y.-K., Liang, G., Gao, S., Choi, J.-H., Kim, C.-H., Wang, L., et al., 2020. Generation of foxn1/Casper mutant zebrafish for allograft and xenograft of normal and malignant cells. Stem Cell Rep. 15, 749-760.
    Malissen, B., Gregoire, C., Malissen, M., Roncagalli, R., 2014. Integrative biology of T cell activation. Nat. Immunol. 15, 790-797.
    Menning, A., Hopken, U.E., Siegmund, K., Lipp, M., Hamann, A., Huehn, J., 2007. Distinctive role of CCR7 in migration and functional activity of naive- and effector/memory-like Treg subsets. Eur. J. Immunol. 37, 1575-1583.
    Montano-Loza, A.J., Rodriguez-Peralvarez, M.L., Pageaux, G.-P., Sanchez-Fueyo, A., Feng, S., 2023. Liver transplantation immunology: Immunosuppression, rejection, and immunomodulation. J. Hepatol. 78, 1199-1215.
    Moore, J.C., Mulligan, T.S., Yordan, N.T., Castranova, D., Pham, V.N., Tang, Q., Lobbardi, R., Anselmo, A., Liwski, R.S., Berman, J.N., et al., 2016. T cell immune deficiency in zap70 mutant zebrafish. Mol. Cell. Biol. 36, 2868-2876.
    Oravecz, A., Apostolov, A., Polak, K., Jost, B., Le Gras, S., Chan, S., Kastner, P., 2015. Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2. Nat. Commun. 6, 8823.
    Rice, J.B., White, A.G., Scarpati, L.M., Wan, G., Nelson, W.W., 2017. Long-term systemic corticosteroid exposure: a systematic literature review. Clin. Ther. 39, 2216-2229.
    Sakaguchi, S., Yamaguchi, T., Nomura, T., Ono, M., 2008. Regulatory T cells and immune tolerance. Cell 133, 775-787.
    Santamaria, J.C., Borelli, A., Irla, M., 2021. Regulatory T cell heterogeneity in the thymus: impact on their functional activities. Front. Immunol. 12, 643153.
    Satija, R., Farrell, J.A., Gennert, D., Schier, A.F., Regev, A., 2015. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495-502.
    Slanchev, K., Stebler, J., de la Cueva-Mendez, G., Raz, E., 2005. Development without germ cells: the role of the germ line in zebrafish sex differentiation. Proc. Natl. Acad. Sci. U. S. A. 102, 4074-4079.
    Stoletov, K., Montel, V., Lester, R.D., Gonias, S.L., Klemke, R., 2007. High-resolution imaging of the dynamic tumor cell-vascular interface in transparent zebrafish. Proc. Natl. Acad. Sci. U. S. A. 104, 17406-17411.
    Su, N., Ren, Z., Wang, H., Wang, C., Hao, Y., Ye, D., He, M., Sun, Y., 2025. Cyprinid Juji (Gobiocypris rarus) as a model fish to study germ cell development and gonadal differentiation. Commun. Biol. 9, 106.
    Sugimoto, K., Hui, S.P., Sheng, D.Z., Nakayama, M., Kikuchi, K., 2017. Zebrafish Foxp3 is required for the maintenance of immune tolerance. Dev. Comp. Immunol. 73, 156-162.
    Sumida, T.S., Cheru, N.T., Hafler, D.A., 2024. The regulation and differentiation of regulatory T cells and their dysfunction in autoimmune diseases. Nat. Rev. Immunol. 24, 503-517.
    Sun, L., Su, Y., Jiao, A., Wang, X., Zhang, B., 2023. T cells in health and disease. Signal Transduction Targeted Ther. 8, 235.
    Sun, Y., Zhang, B., Luo, L., Shi, D.L., Wang, H., Cui, Z., Huang, H., Cao, Y., Shu, X., Zhang, W., et al., 2019. Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9. Genome Res. 30, 118-126.
    Tang, Q., Abdelfattah, N.S., Blackburn, J.S., Moore, J.C., Martinez, S.A., Moore, F.E., Lobbardi, R., Tenente, I.M., Ignatius, M.S., Berman, J.N., et al., 2014. Optimized cell transplantation using adult rag2 mutant zebrafish. Nat. Methods 11, 821-824.
    Thiault, N., Darrigues, J., Adoue, V., Gros, M., Binet, B., Perals, C., Leobon, B., Fazilleau, N., Joffre, O.P., Robey, E.A., et al., 2015. Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors. Nat. Immunol. 16, 628-634.
    Tsuda, M., Tone, Y., Ogawa, C., Nagaoka, Y., Katsumata, M., Necula, A., Howie, D., Masuda, E., Waldmann, H., Tone, M., 2017. A bacterial artificial chromosome reporter system for expression of the human FOXP3 gene in mouse regulatory T-cells. Front. Immunol. 8, 279.
    Veiga-Parga, T., Sehrawat, S., Rouse, B.T., 2013. Role of regulatory T cells during virus infection. Immunol. Rev. 255, 182-196.
    Vignali, D.A.A., Collison, L.W., Workman, C.J., 2008. How regulatory T cells work. Nat. Rev. Immunol. 8, 523-532.
    Wang, J., Xie, W., Li, N., Li, W., Zhang, Z., Fan, N., Ouyang, Z., Zhao, Y., Lai, C., Li, H., et al., 2023a. Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation. Cell Stem Cell 30, 1235-1245.
    Wang, X., Zhu, J., Wang, H., Deng, W., Jiao, S., Wang, Y., He, M., Zhang, F., Liu, T., Hao, Y., et al., 2023b. Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors. Nat. Commun. 14, 7918.
    Wang, Y., Yang, H.-Q., Jiang, W., Fan, N.-N., Zhao, B.-T., Ou-Yang, Z., Liu, Z.-M., Zhao, Y., Yang, D.-S., Zhou, X.-Y., et al., 2015. Transgenic expression of human cytoxic T-lymphocyte associated antigen4-Immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting. Transgenic Res. 24, 199-211.
    Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B., Raz, E., 2003. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr. Biol. 13, 1429-1434.
    Wen, Y., Fang, W., Xiang, L.X., Pan, R.L., Shao, J.Z., 2011. Identification of Treg-like cells in Tetraodon: insight into the origin of regulatory T subsets during early vertebrate evolution. Cell. Mol. Life Sci. 68, 2615-2626.
    Wienholds, E., Schulte-Merker, S., Walderich, B., Plasterk, R.H.A., 2002. Target-selected inactivation of the zebrafish rag1 gene. Science 297, 99-102.
    Williams, L.M., Rudensky, A.Y., 2007. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat. Immunol. 8, 277-284.
    Xie, H., Wang, X., Jin, M., Li, L., Zhu, J., Kang, Y., Chen, Z., Sun, Y., Zhao, C., 2022. Cilia regulate meiotic recombination in zebrafish. J. Mol. Cell Biol. 14, mjac049.
    Yamada, Y., Nguyen, T.T., Impellizzieri, D., Mineura, K., Shibuya, R., Gomariz, A., Haberecker, M., Nilsson, J., Nombela-Arrieta, C., Jungraithmayr, W., et al., 2023. Biased IL-2 signals induce Foxp3-rich pulmonary lymphoid structures and facilitate long-term lung allograft acceptance in mice. Nat. Commun. 14, 1383.
    Yan, C., Brunson, D.C., Tang, Q., Do, D., Iftimia, N.A., Moore, J.C., Hayes, M.N., Welker, A.M., Garcia, E.G., Dubash, T.D., et al., 2019. Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish. Cell 177, 1903-1914.
    Yan, C., Yang, Q., Zhang, S., Millar, D.G., Alpert, E.J., Do, D., Veloso, A., Brunson, D.C., Drapkin, B.J., Stanzione, M., et al., 2021. Single-cell imaging of T cell immunotherapy responses in vivo. J. Exp. Med. 218, e20210314.
    Ye, D., Hao, Y., Zhu, J., Wang, C., Li, L., Li, Y., Tu, Y.-X., Ren, Z., Wang, H., Chen, Z., et al., 2026. Ultra-fast generation of all-female grass carp via transplantation of female germline stem cells into zebrafish (in press). Sci. China Life Sci.
    Ye, D., Liu, T., Li, Y., Wang, Y., Hu, W., Zhu, Z., Sun, Y., 2023. Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set. Front. Endocrinol. 14, 1044318.
    Yoshinaga, T.T., Kfoury Junior, J.R., Butzge, A.J., Olio, R.L., Hernandez-Blazquez, F.J., Oliveira Carreira, A.C., De Oliveira Massoco Salles Gomes, C., Bianchi, P.K.F.D.C., Tabata, Y.A., Hattori, R.S., 2021. Testicular subcutaneous allografting followed by immunosuppressive treatment promotes maintenance of spermatogonial cells in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 112, 108-115.
    Yu, L., Liebenberg, K., Shen, Y., Liu, F., Xu, Z., Hao, X., Wu, L., Zhang, W., Chan, H.L., Wei, B., et al., 2025. Tumor-derived arachidonic acid reprograms neutrophils to promote immune suppression and therapy resistance in triple-negative breast cancer. Immunity 58, 909-925.
    Zemmour, D., Zilionis, R., Kiner, E., Klein, A.M., Mathis, D., Benoist, C., 2018. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat. Immunol. 19, 291-301.
    Zhang, F., Hao, Y., Li, X., Li, Y., Ye, D., Zhang, R., Wang, X., He, M., Wang, H., Zhu, Z., et al., 2022. Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation. Sci. China Life Sci. 65, 969-987.
    Zhang, F., Li, X., He, M., Ye, D., Xiong, F., Amin, G., Zhu, Z., Sun, Y., 2020. Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells. J. Genet. Genomics 47, 37-47.
    Zhang, R., Jiao, S., He, M., Li, W., Wang, H., Ye, D., Zhu, Z., Sun, Y., 2025. An oocyte and yolk syncytial layer-derived Nanog-cyp11a1-pregnenolone axis promotes extraembryonic development. Sci. Bull. 70, 4150-4155.
    Zong, Y., Deng, K., Chong, W.P., 2024. Regulation of Treg cells by cytokine signaling and co-stimulatory molecules. Front. Immunol. 15, 1387975.
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