XB-ART-47988Proc Natl Acad Sci U S A. August 27, 2013; 110 (35): 14342-7.
Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians.
PubMed ID: 23940320
PMC ID: PMC3761575
Article link: Proc Natl Acad Sci U S A.
Grant support: R24-AI-059830 NIAID NIH HHS , R25-GM064133 NIGMS NIH HHS , R24 AI059830 NIAID NIH HHS , R25 GM064133 NIGMS NIH HHS
Genes referenced: cyp26a1 myh4 myh6 tnfrsf10b
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|Fig. 1. Identification of XNC10-T+ T-cell populations in the spleen of adult Xenopus. (A) Flow cytometry of live spleen leukocytes isolated from adult Xenopus and stained with XNC10-T and CD8, CD5, or MHC class II-specific mAbs. (B) Percent XNC10-Tbright+ (3.5% ± 3.5), XNC10-Tdim+ (2.5% ± 2.6), and CD8+ cells (15.76% ± 3.9) of total live spleen leukocytes (n = 10 adult outbred Xenopus; mean and SD for each population are shown). (C) Immunofluorescence using XNC10-T on adult Xenopus spleen leukocytes is shown in red; nuclei (blue) were stained with DAPI. (Magnification: 40×.) Arrows indicate XNC10-T+ cells. (D) Gene expression profiles of sorted cells from spleen leukocytes. (E and F) TCRα and -β variable segment (V) repertoire (listed by numbers) in sorted populations from inbreed J-strain X. laevis. TCR Vα, n = 35 clones sequences from each population; TCR Vβ, n = 26, 30, and 35 clones, respectively. Data shown are representative of three independent experiments. (G) Nucleotides and amino acids of the invariant CDR3α sequence. Va6 nucleotides are underlined, Jα1.43 nucleotides are bold, and the CDR3α region is boxed.|
|Fig. 2. Identification of XNC10-T+ cells in the spleens of early developmental stage 53 tadpoles (3 wk). Flow cytometry of spleen leukocytes pooled from either (A) four individuals or (B) one adult and double stained with XNC10-T and CD8 mAb. Data shown are representative of three different experiments.|
|Fig. 3. XNC10shRNA Tg Xenopus tadpoles have reduced expression of iVα6. Expression of (A) XNC10 or (B) iVα6 in Tg developmental stage 53/54 tadpoles (white box) or age-matched dejellied controls (black box). Results are normalized to an endodgenous control and presented as fold change in expression compared with expression of XNC10 or iVα6 in the skin of Tg tadpoles. All results are presented as mean ± SE (n = 4). *P < 0.05 and **P < 0.005 denote significant differences (Student t test). (C) F0 Tg tadpoles (stage 53, n = 12) or control larvae (stage 53, n = 15) were i.p. infected with 1 × 104 pfu FV3, and survival was monitored daily. Active FV3 infection was verified postmortem by RT-PCR using viral polymerase II on kidney-derived genomic DNA. OB, outbred animals; RQ, relative quantification.|
|Fig. 4. XNC10shRNA Tg animals have fewer XNC10-T+ cells. (A) Representative image of GFP expression in F0 Tg adult frogs (<10 mo). (B) Flow cytometry analysis of spleen from Tg animal and age-matched dejellied control using XNC10-T and CD8 mAb. Percent XNC10-T+, XNC10-Tdim+, and CD8+ cells are shown in the plot.|
|Fig. 5. TCRα repertoire in stage 50 tadpoles is limited with several dominant invariant TCR α-chains. (A) CD8+, CD8dim+, and CD8− populations were sorted from spleens of stage 50 inbred J-strain tadpoles (n = 167); potential TCRα repertoire in each population was determined using 5′RACE-PCR and next generation 454 pyrosequencing. The frequency of different Vα families based on >85% nucleotide identity for (B) CD8+, (C) CD8dim, and (D) CD8− is show with n = total reads. The frequency and number of Vα6, Vα22, Vα23, Vα40, Vα41, and Vα45 are indicated in each population. For CD8+, each slice represents a unique Vα family starting with Vα1 and moving clockwise to Vα90. The total CD8+ Vα family repertoire is summarized in Table S6. (C and D) Each of the dominant Vα family clusters was reanalyzed with a >95% nucleotide identity, and Jα gene segment use within each cluster is indicated. Undef indicates no match with >95% sequence identity to any previously identified Jα. FSC-A, forward scatter.|