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Cellular and Molecular Life Sciences

, Volume 68, Issue 15, pp 2615–2626 | Cite as

Identification of Treg-like cells in Tetraodon: insight into the origin of regulatory T subsets during early vertebrate evolution

  • Yi Wen
  • Wei Fang
  • Li-Xin Xiang
  • Ruo-Lang Pan
  • Jian-Zhong ShaoEmail author
Research Article

Abstract

CD4+CD25+Foxp3+ regulatory T cells (Treg cells) are critical for the maintenance of peripheral tolerance, and the suppression of autoimmune diseases and even tumors. Although Treg cells are well characterized in humans, little is known regarding their existence or occurrence in ancient vertebrates. In the present study, we report on the molecular and functional characterization of a Treg-like subset with the phenotype CD4-2+CD25-like+Foxp3-like+ from a pufferfish (Tetraodon nigroviridis) model. Functional studies showed that depletion of this subset produced an enhanced mixed lymphocyte reaction (MLR) and nonspecific cytotoxic cell (NCC) activity in vitro, as well as inflammation of the intestine in vivo. The data presented here will not only enrich the knowledge of fish immunology but will also be beneficial for a better cross-species understanding of the evolutionary history of the Treg family and Treg-mediated regulatory networks in cellular immunity.

Keywords

Foxp3 CD4 CD25 Treg cells Fish Comparative immunology 

Abbreviations

MLR

Mixed lymphocyte reaction

NCC

Nonspecific cytotoxic cell

IBD

Inflammatory bowel disease

ISH

In situ hybridization

DIG

Digoxigenin

LDH

Lactate dehydrogenase

TM

Trans-membrane

CYT

Cytoplasmic tail

Notes

Acknowledgments

This work was supported by grants from the National Basic Research Program of China (973) (2006CB101805), Hi-Tech Research and Development Program of China (863) (2008AA09Z409), the National Natural Science Foundation of China (30871936, 31072234), and the Science and Technology Foundation of Zhejiang Province (2006C12038, 2006C23045, 2006C12005, 2007C12011).

Supplementary material

18_2010_574_MOESM1_ESM.doc (54 kb)
Supplementary Table (DOC 54 kb)
18_2010_574_MOESM2_ESM.tif (210 kb)
Tetraodon foxp3-like sequences, gene organization and chromosomal synteny analyses. (A) The cDNA sequence (1411 bp) of Tetraodon foxp3-like gene (GenBank accession no. GU592499), with decoded 413 amino acid (shown beneath), showing the full length ORF (1242 bp) and 3’ UTR with a polyA tail. The zinc finger domain is underlined, the forkhead domain is in bold, and the asterisk reveals a stop codon. (B) Comparison of foxp3 gene organizations between fish and human. The Tetraodon foxp3-like gene consisted of twelve exons and eleven introns, in which the fifth and sixth exons encoded the zinc finger domain, the seventh and eighth exons encoded the leu zipper, and the last four exons encoded the forkhead domain. The rectangles represent exons and lines between them indicate introns. The numbers above rectangles or below lines represent the corresponding lengths. (C) Chromosomal syntenic analysis of Foxp3 genes among different species, showing partial syntenic relationship between fish Foxp3 and Xenopus and human Foxps genes, although it slightly divers from species, possibly suggesting the different recombination events occurred among them, in which the Foxp3 in Xenopus seems to be at a transition stage (TIFF 210 kb)
18_2010_574_MOESM3_ESM.tif (554 kb)
Multiple alignment analysis (A) and phylogenetic analysis (B) of Foxp3 orthologs among different species. In the alignment, residues shaded in black are completely conserved across all species aligned, and the residues shaded in gray are similar with respect to side chains. The dashes in the amino acid sequences indicate gaps introduced to maximize alignment. The result showed that foxp3 proteins were well conserved at both zinc finger domains in the middle and forkhead domains at C-terminals, from fish to mammals. The phylogenetic tree was constructed by the neighbor-joining method, and the numbers at branch nodes indicate percent bootstrap confidence values derived from 2000 replications. GenBank accession numbers for these amino acid sequences are as follows: human foxp3 (ABQ15210), cat foxp3 (ABN79272), mouse foxp3 (CAM25950), Xenopus foxp3 (BAG12188) and zebrafish foxp3 (FJ906821); human foxp1 (Q9H334), mouse foxp1 (P58462), Xenopus foxp1 (Q5W1J5), zebrafish foxp1b (Q2LE08), Tetraodon foxp1b (FJ358692); human foxp2 (O15409), mouse foxp2 (P58463), Xenopus foxp2 (Q4VYS1), medaka foxp2 (B1NY82), zebrafish foxp2 (Q4JNX5); human foxp4 (Q8IVH2), mouse foxp4 (Q9DBY0), and Xenopus foxp4 (Q4VYR7). Phylogenetic analysis showed the orthology of fish foxp3 genes with others from different species (TIFF 554 kb)
18_2010_574_MOESM4_ESM.tif (1.5 mb)
Tetraodon CD4-2 (GenBank accession no. EF601918) and CD4-4 (GenBank accession no. EF601919) sequences, gene organization and chromosomal synteny analyses. (A) Nucleotide and predicted amino acid sequences of CD4-2. It spanned a 930 bp ORF encoding a polypeptide of 309 amino acids with a 24 amino acid signal peptide. The mature CD4-2 protein with a predicted molecular mass of 31.4 kDa and an expected isoelectric point of 9.38 consisted of a 210 amino acid extracellular region including two Ig domains, a 23 amino acid hydrophobic trans-membrane (TM) region, and a 52 amino acid cytoplasmic tail (CYT), comprising one N-glycosylation site at the position 175-177. The N-glycosylation site is underlined, the conserved p56lck site is in bold, and the asterisk reveals a stop codon. (B) Nucleotide and predicted amino acid sequences of CD4-4. It spanned a 1401 bp ORF encoding a polypeptide of 466 amino acids with a 22 amino acid signal peptide. The mature CD4-4 protein with a predicted molecular mass of 48.9 kDa and an expected isoelectric point of 9.20 consisted of a 390 amino acid extracellular region including four Ig domains, a 23 amino acid hydrophobic trans-membrane region, and a 31 amino acid cytoplasmic tail, comprising three N-glycosylation sites at positions 288-290, 342-344 and 395-397. The N-glycosylation sites are underlined, the conserved p56lck site is in bold, and the asterisk reveals a stop codon. (C) The comparison of CD4 gene organization in Tetraodon and humans. The Tetraodon CD4s shared similarities in genomic organization. The CD4-2 gene consisted of 8 exons and 7 introns, and the CD4-4 gene consisted of nine exons and eight introns. Each domain was encoded by a single exon, except that there were two D1 exons, D1A and D1B. The rectangles represent exons and lines between them indicate introns. The numbers above rectangles or below lines represent the corresponding lengths. Essential domains of Tetraodon CD4s are shown in simplified form in Sig, signal peptide; D, IG domain; H, hinge region; TM, transmembrane region; Int, intracellular region. (D) Chromosomal syntenic analysis of CD4 genes among different species, showing partial syntenic relationship among Tetraodon, chicken, human and mouse CD4 genes. Tetraodon CD4-2s are located on chromosome 8 with two copies while the adjacent CD4-4 has only one locus (TIFF 1519 kb)
18_2010_574_MOESM5_ESM.tif (242 kb)
Phylogenetic tree of CD4 molecules. This tree was constructed by the neighbor-joining method, and the numbers at branch nodes indicate percent bootstrap confidence values derived from 2000 replications. GenBank accession numbers for these amino acid sequences are as follows: catfish CD4-4 (DQ435302), zebrafish CD4-4 (EF601917), trout CD4-4 (AAY42070), fugu CD4-4 (BAD37153), Tetraodon CD4-4 (EF601919), fugu CD4-2 (predicted), Tetraodon CD4-2 (EF601918), trout CD4-2 (AY772711), catfish CD4-2 (DQ435301), zebrafish CD4-2 (EF601915), Xenopus CD4 (predicted), chicken CD4 (ABA55042), mouse CD4 (P06334), pig CD4 (AAT52342), and human CD4 (CAA60883) (TIFF 242 kb)
18_2010_574_MOESM6_ESM.tif (454 kb)
Tetraodon CD25-like sequences (GenBank accession no. EF143577), gene organization and structural characterization. The CD25-like gene spanned 4251 bp, and consisted of eight exons and seven introns. The CD25-like cDNA was composed of 2061 bp, including a 21 bp 5’ UTR, a 717 bp ORF and a 1323 bp 3’ UTR. The deciphered CD25-like amino acid sequence was a trans-membrane protein with 238 amino acids, a molecular weight of ~25.6 kDa, and a theoretical isoelectric point of 8.28. It contained a N-terminal extra-cellular region (position 1-180) with a 24 amino acid signal peptide, one sushi domain (position 34-99), a Pro/Thr rich domain (position 106-146), a trans-membrane domain (position 181-203), and a C-terminal intra-cellular domain (position 204-238). (A) Nucleotide and predicted amino acid sequences of CD25-like ORF. The sushi domain is in bold, and the asterisk reveals a stop codon. (B) The gene organizations of Tetraodon CD25-like. White and black rectangles represent UTRs (untranslated regions) and CDSs (coding sequences), respectively, while lines between them indicate introns. The numbers above rectangles or below lines represent the corresponding lengths. (C) Essential domains of CD25-like shown in simplified (TIFF 453 kb)
18_2010_574_MOESM7_ESM.tif (75 kb)
Chromosome syntenies of CD25 (IL-2Rα)/15Rα subfamily among vertebrates, including human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), the Western clawed frog (Xenopus tropicalis) and some teleost species (Tetraodon, Tetraodon nigroviridis; fugu, Takifugu rubripes; stickleback, Gasterosteus aculeatus; medaka, Oryzias latipes and zebrafish, Danio rerio). Genes are indicated by the rectangles with annotations, genomic positions and transcription orientations (TIFF 75 kb)
18_2010_574_MOESM8_ESM.tif (59 kb)
Phylogenetic tree of CD25 (IL-2Rα)/15Rα chains. These trees were constructed by the neighbor-joining method, based on the multiple alignment of the extracted sushi domains with main responsibility for cytokine binding (the first sushi of IL-2Rα or the only sushi of IL-15Rα). The numbers at branch nodes indicate percent bootstrap confidence values derived from 2000 replications. GenBank accession numbers for these amino acid sequences or genome data are as follows: IL-2Rα (CD25): human (NM000417), mouse (NM008367), chick (AF143806), Xenopus (EL724547); IL-15Rα: human (NM002189), mouse (NM008358), chick (AI980376), Xenopus (scaffold_11:3548294-3548476); teleost CD25-like (IL-2Rα/15Rα): fugu (CA846124); stickleback (EF513159), medaka (DK176609), trout (DQ381970), salmon (EG807345), zebrafish (EF143578) (TIFF 58 kb)
18_2010_574_MOESM9_ESM.tif (775 kb)
Recombinant proteins and antibody reactivity. The fusion CD4-2, CD4-4 and CD25-like recombinant proteins were purified using Ni-NTA resin and detected on SDS-PAGE as the expected 31, 45 and 28 kDa bands, respectively. (A) Purified fusion CD4-2 and CD25-like consisting of extracellular regions with 6×His, and protein marker were exhibited by SDS-PAGE (lane 1, 2 and M). Western blot analysis of anti-CD4-2 antibody to recombinant CD4-2 and CD25-like (lane 3 and 4), and anti-CD25-like antibody to CD4-2 and CD25-like (lane 5 and 6), which were then incubated with secondary HRP-conjugated Abs, and detected by ECL plus (Amersham Biosciences); B. Purified fusion CD4-2 and CD4-4 consisting of extracellular regions with 6×His, and protein marker were exhibited by SDS-PAGE (lane 1, 2 and M). Western blot analysis of anti-CD4-2 antibody to recombinant CD4-2 and CD4-4 (lane 3 and 4), and anti-CD4-4 antibody to CD4-2 and CD4-4 (lane 5 and 6), which were then incubated with secondary Abs, and detected by ECL plus as described above (TIFF 775 kb)
18_2010_574_MOESM10_ESM.tif (1.3 mb)
Distribution of Tetraodon transcriptional factor foxp3-like. Foxp3-like mRNAs were detected in several immune related tissues of healthy Tetraodon, including peripheral leucocytes (pl), spleen (sp), kidney (ki), gut (gu), and gill (gi) by RT-PCR (A). Quantitative analyses of foxp3-like relative to β-actin in corresponding tissues by real-time RT-PCR (B). ISH detection for foxp3-like probes (C): i, paraffin section of kidney with foxp3-like probes; ii, paraffin section of kidney with hybridization solution; iii, leucocytes with foxp3-like probes; iv, leucocytes with hybridization solution. Arrows indicate foxp3-like positive signals (TIFF 1335 kb)
18_2010_574_MOESM11_ESM.tif (54 kb)
A diagram of the hypothetical evolution of IL-15Rα and IL-2Rα gene family among vertebrate species. Gene tandem duplication had been involved in the emergence and divergence of IL-15Rα and IL-2Rα in tetrapods only, but not in teleost species (TIFF 53 kb)

References

  1. 1.
    Sakaguchi S (2004) Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22:531–562PubMedCrossRefGoogle Scholar
  2. 2.
    Von Herrath MG, Harrison LC (2003) Antigen-induced regulatory T cells in autoimmunity. Nat Rev Immunol 3:223–232CrossRefGoogle Scholar
  3. 3.
    Khazaie Boehmer K, Von Boehmer H (2006) The impact of CD4+ CD25+ Treg on tumor specific CD8+ T cell cytotoxicity and cancer. Semin Cancer Biol 16:124–136CrossRefGoogle Scholar
  4. 4.
    Thornton AM, Shevach EM (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 188:287–296PubMedCrossRefGoogle Scholar
  5. 5.
    Thornton AM, Shevach EM (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol 164:183–190PubMedGoogle Scholar
  6. 6.
    Guerin LR, Prins JR, Robertson SA (2009) Regulatory T-cells and immune tolerance in pregnancy: a new target for infertility treatment? Hum Reprod Update 15:517–535PubMedCrossRefGoogle Scholar
  7. 7.
    Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164PubMedGoogle Scholar
  8. 8.
    Asseman C, Fowler S, Powrie F (2000) Control of experimental inflammatory bowel disease by regulatory T cells. Am J Respir Crit Care Med 162:S185–S189PubMedGoogle Scholar
  9. 9.
    Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336PubMedCrossRefGoogle Scholar
  10. 10.
    Zheng Y, Rudensky AY (2007) Foxp3 in control of the regulatory T cell lineage. Nat Immunol 8:457–462PubMedCrossRefGoogle Scholar
  11. 11.
    Benton MJ (1990) Phylogeny of the major tetrapod groups: morphological data and divergence dates. J Mol Evol 30:409–424PubMedCrossRefGoogle Scholar
  12. 12.
    Zwollo P, Cole S, Bromage E, Kaattari S (2005) B cell heterogeneity in the teleost kidney: evidence for a maturation gradient from anterior to posterior kidney. J Immunol 174:6608–6616PubMedGoogle Scholar
  13. 13.
    Levraud JP, Boudinot P (2009) The immune system of teleost fish. Med Sci (Paris) 25:405–411CrossRefGoogle Scholar
  14. 14.
    Matsuo MY, Asakawa S, Shimizu N, Kimura H, Nonaka M (2002) Nucleotide sequence of the MHC class I genomic region of a teleost, the medaka (Oryzias latipes). Immunogenetics 53:930–940PubMedCrossRefGoogle Scholar
  15. 15.
    Solem ST, Stenvik J (2006) Antibody repertoire development in teleosts—a review with emphasis on salmonids and Gadus morhua L. Dev Comp Immunol 30:57–76PubMedCrossRefGoogle Scholar
  16. 16.
    Laird DJ, De Tomaso AW, Cooper MD, Weissman IL (2000) 50 million years of chordate evolution: seeking the origins of adaptive immunity. Proc Natl Acad Sci USA 97:6924–6926PubMedCrossRefGoogle Scholar
  17. 17.
    Langenau DM, Zon LI (2005) The zebrafish: a new model of T cell and thymic development. Nat Rev Immunol 5:307–317PubMedCrossRefGoogle Scholar
  18. 18.
    Araki K, Akatsu K, Suetake H, Kikuchi K, Suzuki Y (2008) Characterization of CD8+ leukocytes in fugu (Takifugu rubripes) with antiserum against fugu CD8alpha. Dev Comp Immunol 32:850–858PubMedCrossRefGoogle Scholar
  19. 19.
    Wen Y, Shao JZ, Xiang LX, Fang W (2006) Cloning, characterization and expression analysis of two Tetraodon nigroviridis interleukin-16 isoform genes. Comp Biochem Physiol B Biochem Mol Biol 144:159–166PubMedCrossRefGoogle Scholar
  20. 20.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  21. 21.
    Xiang LX, Peng B, Dong WR, Yang ZF, Shao JZ (2008) Lipopolysaccharide induces apoptosis in Carassius auratus lymphocytes, a possible role in pathogenesis of bacterial infection in fish. Dev Comp Immunol 32:992–1001PubMedCrossRefGoogle Scholar
  22. 22.
    Arck PC, Merali F, Chaouat G, Clark DA (1996) Inhibition of immunoprotective CD8+ T cells as a basis for stress-triggered substance P-mediated abortion in mice. Cell Immunol 171:226–230PubMedGoogle Scholar
  23. 23.
    Godfrey WR, Ge YG, Spoden DJ, Levine BL, June CH, Blazar BR, Porter SB (2004) In vitro-expanded human CD4(+)CD25(+) T regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood 104:453–461PubMedCrossRefGoogle Scholar
  24. 24.
    Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, Kelly TE, Saulsbury FT, Chance PF, Ochs HD (2001) The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27:20–21PubMedCrossRefGoogle Scholar
  25. 25.
    Quintana FJ, Iglesias AH, Farez MF, Caccamo M, Burns EJ, Kassam N, Oukka M, Weiner HL (2010) Adaptive autoimmunity and Foxp3-based immunoregulation in zebrafish. PLoS One 5:e9478PubMedCrossRefGoogle Scholar
  26. 26.
    Mitra S, Alnabulsi A, Secombes CJ, Bird S (2010) Identification and characterization of the transcription factors involved in T cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio. FEBS J 277:128–147PubMedCrossRefGoogle Scholar
  27. 27.
    Edholm ES, Stafford JL, Quiniou SM, Waldbieser G, Miller NW, Bengten E, Wilson M (2007) Channel catfish, Ictalurus punctatus, CD4-like molecules. Dev Comp Immunol 31:172–187PubMedCrossRefGoogle Scholar
  28. 28.
    Laing KJ, Zou JJ, Purcell MK, Phillips R, Secombes CJ, Hansen JD (2006) Evolution of the CD4 family: teleost fish possess two divergent forms of CD4 in addition to lymphocyte activation gene-3. J Immunol 177:3939–3951PubMedGoogle Scholar
  29. 29.
    Dijkstra JM, Somamoto T, Moore L, Hordvik I, Ototake M, Fischer U (2006) Identification and characterization of a second CD4-like gene in teleost fish. Mol Immunol 43:410–419PubMedCrossRefGoogle Scholar
  30. 30.
    Suetake H, Araki K, Suzuki Y (2004) Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4. Immunogenetics 56:368–374PubMedCrossRefGoogle Scholar
  31. 31.
    Buonocore F, Randelli E, Casani D, Guerra L, Picchietti S, Costantini S, Facchiano AM, Zou J, Secombes CJ, Scapigliati G (2008) A CD4 homologue in sea bass (Dicentrarchus labrax): molecular characterization and structural analysis. Mol Immunol 45:3168–3177PubMedCrossRefGoogle Scholar
  32. 32.
    Doyle C, Strominger JL (1987) Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature 330:256–259PubMedCrossRefGoogle Scholar
  33. 33.
    Liu Y, Cruikshank WW, O′loughlin T, O′reilly P, Center DM, Kornfeld H (1999) Identification of a CD4 domain required for interleukin-16 binding and lymphocyte activation. J Biol Chem 274:23387–23395PubMedCrossRefGoogle Scholar
  34. 34.
    Innes JB, Kuntz MM, Kim YT, Weksler ME (1979) Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A. J Clin Invest 64:1608–1613PubMedCrossRefGoogle Scholar
  35. 35.
    Giri JG, Kumaki S, Ahdieh M, Friend DJ, Loomis A, Shanebeck K, Dubose R, Cosman D, Park LS, Anderson DM (1995) Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO J 14:3654–3663PubMedGoogle Scholar
  36. 36.
    Anderson DM, Kumaki S, Ahdieh M, Bertles J, Tometsko M, Loomis A, Giri J, Copeland NG, Gilbert DJ, Jenkins NA, Valentine V, Shapiro DN, Morris SW, Park LS, Cosman D (1995) Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes. J Biol Chem 270:29862–29869PubMedCrossRefGoogle Scholar
  37. 37.
    Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, Mcgrady G, Wahl SM (2003) Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198:1875–1886PubMedCrossRefGoogle Scholar
  38. 38.
    Watanabe N, Wang YH, Lee HK, Ito T, Wang YH, Cao W, Liu YJ (2005) Hassall’s corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature 436:1181–1185PubMedCrossRefGoogle Scholar
  39. 39.
    Bouma G, Strober W (2003) The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 3:521–533PubMedCrossRefGoogle Scholar
  40. 40.
    Boden EK, Snapper SB (2008) Regulatory T cells in inflammatory bowel disease. Curr Opin Gastroenterol 24:733–741PubMedCrossRefGoogle Scholar
  41. 41.
    Groux H, O′garra A, Bigler M, Rouleau M, Antonenko S, De Vries JE, Roncarolo MG (1997) A CD4+ T cell subset inhibits antigen-specific T cell responses and prevents colitis. Nature 389:737–742PubMedCrossRefGoogle Scholar
  42. 42.
    Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W (1993) Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263–274PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Yi Wen
    • 1
    • 2
    • 3
  • Wei Fang
    • 1
    • 2
    • 3
  • Li-Xin Xiang
    • 1
    • 2
    • 3
  • Ruo-Lang Pan
    • 1
    • 2
    • 3
  • Jian-Zhong Shao
    • 1
    • 2
    • 3
    Email author
  1. 1.College of Life SciencesZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.Key Laboratory for Cell and Gene Engineering of Zhejiang ProvinceZhejiang UniversityHangzhouPeople’s Republic of China
  3. 3.Key Laboratory of Animal Epidemic Etiology and Immunology Prevention of Ministry of AgricultureZhejiang UniversityHangzhouPeople’s Republic of China

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