Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 23;11(5):e0156114.
doi: 10.1371/journal.pone.0156114. eCollection 2016.

Mouse and Human CD1d-Self-Lipid Complexes Are Recognized Differently by Murine Invariant Natural Killer T Cell Receptors

Tingxi Guo  1   2 , Kenji Chamoto  1 , Munehide Nakatsugawa  1 , Toshiki Ochi  1 , Yuki Yamashita  1 , Mark Anczurowski  1   2 , Marcus O Butler  1   2   3 , Naoto Hirano  1   2
Affiliations

Mouse and Human CD1d-Self-Lipid Complexes Are Recognized Differently by Murine Invariant Natural Killer T Cell Receptors

Tingxi Guo et al. PLoS One. .

Abstract

Invariant natural killer T (iNKT) cells recognize self-lipids presented by CD1d through characteristic TCRs, which mainly consist of the invariant Vα14-Jα18 TCRα chain and Vβ8.2, 7 or 2 TCRβ chains with hypervariable CDR3β sequences in mice. The iNKT cell-CD1d axis is conserved between humans and mice, and human CD1d reactivity of murine iNKT cells have been described. However, the detailed differences between the recognition of human and mouse CD1d bound to various self-lipids by mouse iNKT TCRs are largely unknown. In this study, we generated a de novo murine iNKT TCR repertoire with a wider range of autoreactivity compared with that of naturally occurring peripheral iNKT TCRs. Vβ8.2 mouse iNKT TCRs capable of recognizing the human CD1d-self-lipid tetramer were identified, although such clones were not detectable in the Vβ7 or Vβ2 iNKT TCR repertoire. In line with previously reports, clonotypic Vβ8.2 iNKT TCRs with unique CDR3β loops did not discriminate among lipids presented by mouse CD1d. Unexpectedly, however, these iNKT TCRs showed greater ligand selectivity toward human CD1d presenting the same lipids. Our findings demonstrated that the recognition of mouse and human CD1d-self-lipid complexes by murine iNKT TCRs is not conserved, thereby further elucidating the differences between cognate and cross-species reactivity of self-antigens by mouse iNKT TCRs.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Generating de novo mouse iNKT TCRs.
(A) Splenocytes from CD1d knockout (KO) mice of the C57BL/6 background were transduced with the full-length invariant Vα14 TCRα (Vα14i) chain, tagged with the extracellular portion of human nerve growth factor receptor (ΔNGFR), or the tag alone. The transfectants were stained with anti-NGFR mAb and unloaded or PBS-57 loaded mouse CD1d (mCD1d) tetramer. (B) EL4-based artificial APCs (aAPC) were stained with anti-mCD1d and anti-mouse CD80 mAbs (black lines). The solid gray color indicates the control. (C) Vα14i-transduced CD1d-/- splenocytes were stimulated with aAPC and then stained with anti-NGFR mAb and unloaded or PBS-57 loaded mCD1d tetramer. Mean percentages ± SD are shown in the graph. The data are representative of three independent experiments.
Fig 2
Fig 2. Staining of mouse iNKT TCR library transfectants with mouse and human CD1d tetramers.
(A) The total Vβ8.2, 7 and 2 libraries from the PBS-57 tetramer positive populations of Vα14i-transduced CD1d-/- splenocytes (unselected), aAPC-stimulated Vα14i-transduced CD1d-/- splenocytes (aAPC stimulated), and the splenocytes of a wild-type mouse (peripheral) were cloned. Each library was reconstituted in 5KC cells that stably expressed Vα14i. (B) The transfectants were stained with anti-mouse CD3 (mCD3) mAb and stained with the unloaded or PBS-57 loaded mCD1d or hCD1d tetramers. The number above the gate indicates the percentage among CD3+ transfectants. Mean percentages ± SD are shown in the graphs. The data are representative of three independent experiments.
Fig 3
Fig 3. Murine Vβ8.2 iNKT TCRs recognize mCD1d-self-lipid complexes without ligand selectivity.
(A and B) Clonotypic Vβ8.2 TCRβ chains were cloned from the unloaded mCD1d and hCD1d tetramer-positive population of the aAPC-stimulated library and reconstituted in 5KC cells expressing Vα14i. Each transfectant was stained with anti-mCD3 mAb and unloaded or PBS-57, β-GlcCer, LPC, pLPE, and eLPA loaded mCD1d tetramers. MFI values shown are based on CD3+ cells. The raw data for three representative clones are shown in A. Staining for all other transfectants are shown in S1 Fig. MFI was normalized to that of control transfectants stained with the same tetramer. (C) Spearman correlation coefficients were calculated between the indicated pairs of antigens. (D) CD3 expression of CD3+ 5KC transfectants are shown by overlaid multicolor histograms. The gray solid indicates baseline fluorescence of TCR-/- 5KC. Mean values ± SD are shown in the graphs. Data are representative of two independent experiments.
Fig 4
Fig 4. Mouse Vβ8.2 iNKT TCRs recognized only the unloaded hCD1d tetramer but not other self-lipid hCD1d tetramers.
(A and B) The same transfectants shown in Fig 3 were stained with anti-mCD3 mAb and unloaded or PBS-57-, β-GlcCer-, LPC-, pLPE-, and eLPA-loaded hCD1d tetramers. MFI values shown are based on CD3+ cells. The raw data for three representative clones are shown in A. Staining for all other transfectants are shown in S1 Fig. MFI was normalized to that of control transfectants stained with the same tetramer. Mean values ± SD are shown in the graph. (C) The Spearman correlation coefficients between the MFIs obtained by PBS-57 mCD1d and hCD1d tetramer staining (left), and unloaded mCD1d and hCD1d tetramer staining (right) are shown. The data are representative of two independent experiments.
Fig 5
Fig 5. The Jurkat 76.3E1 T cell line as a physiologically relevant host for testing hCD1d reactivity.
(A) The unselected Vβ8.2, 7, and 2 libraries were reconstituted in 3E1 cells and stained with anti-human CD3 (hCD3) mAb and unloaded (lower) or PBS-57 loaded (upper) hCD1d tetramers. (B) The unselected Vβ8.2 library transfectants were stained with anti-hCD3 mAb and unloaded, or β-GlcCer-, LPC-, pLPE-, and eLPA-loaded hCD1d tetramers. Self-lipid tetramer staining percentages were compared by one-way ANOVA followed by Bonferroni post-hoc test. ** p < 0.01, *** p < 0.001.The number above the gate indicates the percentage among the CD3+ transfectants. Mean percentages ± SD are shown in the graphs. The data are representative of two to three independent experiments.
Fig 6
Fig 6. Mouse iNKT TCRs recognize hCD1d-lipid complexes with greater ligand selectivity than mCD1d-lipid complexes.
Mouse Vβ8.2 TCRβ chains were cloned from the unloaded hCD1d tetramer-positive population of the unselected library shown in Fig 5B. Clonotypic TCRβ chains were reconstituted in 3E1 cells and stimulated with plate-bound unloaded or β-GlcCer-, LPC-, pLPE-, and eLPA-loaded hCD1d (A) or mCD1d (B) monomers. Surface CD69 expression was measured by flow cytometry after stimulation. The Spearman correlation coefficient values were calculated for the reactivity against the indicated pairs of mCD1d-lipid (C) and hCD1d-lipid (D) complexes. The data are representative of two independent experiments. (E) The Spearman correlation coefficient values pooled from repeated experiments were compared by an unpaired, two-sample t-test. ** p < 0.01. Mean values ± SD are shown in the graphs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Godfrey DI, Uldrich AP, McCluskey J, Rossjohn J, Moody DB. The burgeoning family of unconventional T cells. Nature immunology. 2015;16(11):1114–23. Epub 2015/10/21. 10.1038/ni.3298 . - DOI - PubMed
    1. Rossjohn J, Pellicci DG, Patel O, Gapin L, Godfrey DI. Recognition of CD1d-restricted antigens by natural killer T cells. Nature reviews Immunology. 2012;12(12):845–57. Epub 2012/11/17. 10.1038/nri3328 - DOI - PMC - PubMed
    1. Matsuda JL, Mallevaey T, Scott-Browne J, Gapin L. CD1d-restricted iNKT cells, the 'Swiss-Army knife' of the immune system. Current opinion in immunology. 2008;20(3):358–68. Epub 2008/05/27. 10.1016/j.coi.2008.03.018 - DOI - PMC - PubMed
    1. Salio M, Silk JD, Jones EY, Cerundolo V. Biology of CD1- and MR1-restricted T cells. Annual review of immunology. 2014;32:323–66. Epub 2014/02/07. 10.1146/annurev-immunol-032713-120243 . - DOI - PubMed
    1. Brennan PJ, Brigl M, Brenner MB. Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nature reviews Immunology. 2013;13(2):101–17. Epub 2013/01/22. 10.1038/nri3369 . - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources