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

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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.

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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

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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).

Author information

Authors and Affiliations

  1. College of Life Sciences, Zhejiang University, Hangzhou, 310058, People’s Republic of China

    Yi Wen, Wei Fang, Li-Xin Xiang, Ruo-Lang Pan & Jian-Zhong Shao

  2. Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Zhejiang University, Hangzhou, People’s Republic of China

    Yi Wen, Wei Fang, Li-Xin Xiang, Ruo-Lang Pan & Jian-Zhong Shao

  3. Key Laboratory of Animal Epidemic Etiology and Immunology Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou, People’s Republic of China

    Yi Wen, Wei Fang, Li-Xin Xiang, Ruo-Lang Pan & Jian-Zhong Shao

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  1. Yi Wen
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Correspondence to Jian-Zhong Shao.

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Supplementary Table (DOC 54 kb)

18_2010_574_MOESM2_ESM.tif

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

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

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

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

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

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

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

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

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

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)

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Wen, Y., Fang, W., Xiang, LX. et al. 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 (2011). https://doi.org/10.1007/s00018-010-0574-5

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