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. 2019 Jul;71(7):465-478.
doi: 10.1007/s00251-019-01118-9. Epub 2019 May 23.

Conservation of molecular and cellular phenotypes of invariant NKT cells between humans and non-human primates

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Conservation of molecular and cellular phenotypes of invariant NKT cells between humans and non-human primates

Krystle K Q Yu et al. Immunogenetics. 2019 Jul.

Abstract

Invariant NKT (iNKT) cells in both humans and non-human primates are activated by the glycolipid antigen, α-galactosylceramide (α-GalCer). However, the extent to which the molecular mechanisms of antigen recognition and in vivo phenotypes of iNKT cells are conserved among primate species has not been determined. Using an evolutionary genetic approach, we found a lack of diversifying selection in CD1 genes over 45 million years of evolution, which stands in stark contrast to the history of the MHC system for presenting peptide antigens to T cells. The invariant T cell receptor (TCR)-α chain was strictly conserved across all seven primate clades. Invariant NKT cells from rhesus macaques (Macaca mulatta) bind human CD1D-α-GalCer tetramer and are activated by α-GalCer-loaded human CD1D transfectants. The dominant TCR-β chain cloned from a rhesus-derived iNKT cell line is nearly identical to that found in the human iNKT TCR, and transduction of the rhesus iNKT TCR into human Jurkat cells show that it is sufficient for binding human CD1D-α-GalCer tetramer. Finally, we used a 20-color flow cytometry panel to probe tissue phenotypes of iNKT cells in a cohort of rhesus macaques. We discovered several tissue-resident iNKT populations that have not been previously described in non-human primates but are known in humans, such as TCR-γδ iNKTs. These data reveal a diversity of iNKT cell phenotypes despite convergent evolution of the genes required for lipid antigen presentation and recognition in humans and non-human primates.

Keywords: CD1D; Non-human primate; T cell receptor; iNKT cells.

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Figures

Fig. 1
Fig. 1
Conservation of the CD1 primate locus. Primate CD1 homolog sequences were acquired from a combination of bioinformatic sources. A maximum likelihood CD1 gene tree was constructed after including two chicken homologs for rooting. Apart from CD1A in olive baboon, each paralog exists as a single gene copy in each primate species and forms a phylogenetically distinct group that generally matches the species phylogeny (bootstrap probabilities are reported as percentages for separation of each paralog group). CD1D is likely the oldest gene and shows the most conservation among primate species
Fig. 2
Fig. 2
Rhesus iNKT cells stain with human α-GalCer-loaded CD1D tetramers and are activated by human CD1D-transfected antigen-presenting cells. a The frequency of α-GalCer-specific T cells was identified as CD3+ and CD1D-α-GalCer tetramer positive (CD1D-ɑGC Tet+). Shown is representative staining from a rhesus macaque (bottom) and human PBMC (top). Tetramer-positive cells were further examined for expression of CD4 or CD8α co-receptor. b CD1D-ɑGC Tet+ cells were sorted and expanded in vitro after which the T cell lines were screened with tetramer to determine specificity for α-GalCer. c Human K562 cells that were stably transfected with human CD1D were co-incubated with iNKT cells in the presence or absence of α-GalCer. Intracellular cytokine analysis shows that cells produce IL-2, TNF, and IFN-γ in the presence of human CD1D and α-GalCer. Data are expressed as percent cytokine positive of CD3+ cells. Sorting and T cell line data are representative of one rhesus macaque, but tetramer staining of T cells and the iNKT cell line was confirmed in at least two experiments. Intracellular cytokine staining was performed twice on the iNKT cell line, and a representative graph is shown
Fig. 3
Fig. 3
The human and non-human primate iNKT cell TCR is highly conserved. a Modeling of rhesus macaque CD1D-NKT interactions based on the crystal structure of the human orthologs (PDB 2po6). α-GalCer (black) is shown loaded onto CD1D (light blue). Residues with nonsynonymous substitutions were mutated in silico with the lowest energy rotamer. Residues were subsequently colored based on expected interactions with CD1D (royal blue), nonsynonymous mutations with conserved biochemical properties (green), and nonsynonymous mutations with differing biochemical properties (red). b The TRAV10 CDR1α and TRAJ18 CDR3α gene segments that are present as a germline rearrangement in iNKT cells were examined across the genomes of ten simian primates, and sections crucial for binding are boxed. Residues important for TCR binding to CD1D and α-GalCer are shown in blue and are highly conserved. Conservative mutations are shown in green, and non-conservative substitutions in red. c The rhesus iNKT TCR was transduced into Jurkat cells and stained with human CD1D-α-GalCer or control tetramer. Data are expressed as percent tetramer-positive events of anti-mouse TCR-positive cells and are representative of at least three independent experiments
Fig. 4
Fig. 4
Tissue phenotypes of iNKT cells in rhesus macaques. PBMC and tissues from healthy rhesus macaques (Rh) were stained with human (Hu) α-GalCer-loaded CD1D tetramers (CD1D-ɑGC Tet). a Gating strategy for identifying iNKT cells from rhesus macaque blood and tissue samples proceeds from singlets to CD45+ cells (pan leukocytes) to CD16 cells to remove NK cells. Viable cells were then identified, and MR1-5-OP-RU tetramer was used to exclude MAIT cells. Finally, CD3+CD1D-αGC Tet+ cells were identified. b Frequency of iNKT cells in PBMC and associated tissues from 12 rhesus macaques following staining with CD1D-αGC tetramer staining. Data are presented as the percentage of CD1D-ɑGC tetramer-positive cells among CD3+ cells. All tissues from the same animal are identified by color. c Co-receptor usage, d CD161 expression, e CD69 expression, and f NKG2A expression among iNKT cells from rhesus macaque PBMC and tissues identified in Online Resource 3 expressed as a percentage of all CD1D-ɑGC tetramer-positive cells

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