Mutation of the Traj18 gene segment using TALENs to generate Natural Killer T cell deficient mice

Abstract

Invariant Natural Killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKT cells express T cell antigen receptors (TCRs) comprising a unique TCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain Trbv TCRβ chains, these TCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT deficiency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specificity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-deficient mouse strain using transcription activator-like effector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confirm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional T cells, regulatory T cells, and type Ib NKT cells is normal. This new mouse strain will serve as a new model of iNKT cell deficiency to facilitate our understanding of iNKT biology.

Figure 1. Summary of TALEN design and test of TALEN function.

(A) Schematic of positions of forward and reverse TALENs and the DNA sequences they target. The blue box indicates the coding region of Traj18. (B) Flow cytometry of HEK293 cells after transfection with the Traj18-targeting TALENs and the reporter plasmids.

Figure 2. Traj18 Sequences of founder mice.

Summary of the genotype of each founder. The Traj18 coding region is indicated in blue text with the translated amino acid sequence below. Boxes indicate the sequence targeted by the TALENs. Mutations in the founders were detected by PCR direct sequencing. Dashed lines indicate deletions while red letters indicate insertions.

Figure 3. PCR analysis of the frequency of use of genes encoding Jα for productive, in-frame, rearrangements involving gene segments of the Trav11 family in sorted CD69⁻ double-positive (CD4⁺CD8⁺) thymocytes from C57BL/6, Jα18(-10), and Jα18(neo) mice.

Order of gene segments along horizontal axes (left to right) is similar to their 5′ to 3′ organization in the mouse genome. Rearrangements for were amplified by PCR with a V-specific primer and a C-specific reverse primer (above plots), followed by high-throughput sequencing with the Ion Torrent platform. Sequence analysis was performed with in-house software, and gene identity was assigned on the basis of sequence alignment with published sequences (International ImMunoGeneTics Information System).

Figure 4. Jα18(-10) KO mice lack iNKT cells but display normal conventional T cell development.

(A) Thymocytes from 6–8 week old C57BL/6, Jα18(-10), and Jα18(neo) were stained for indicated markers to characterize specific stages of T-cell development (data representative of n ≥ 3). (B) Total thymic numbers (C) regulatory T cell frequencies (left) and regulatory T cell numbers (right) are summarized. Data shown represent mean ± SEM for each strain (minimum of 3 mice per strain). (D) Cells from thymus, spleen, and liver of 6–8 week old C57BL/6, Jα18(-10), and Jα18(neo) were stained with CD1d tetramer loaded with the α-GC analog PBS57 (PBS57-CD1d) and TCRβ to assess iNKT cells by flow cytometry (data representative of n ≥ 3). Percentage indicated is out of all T cells in indicated organ and representative of the mean of each strain (minimum of 3 mice per strain). (E) Summary of the data shown in (D) With percentage (left) and number (right) of iNKT cells in thymus, spleen, and liver of 6–8 week old C57BL/6, Jα18(-10), and Jα18(neo) mice. Data shown represent mean ± SEM for each strain (minimum of 3 mice per strain). *P < 0.05; **P < 0.01;***P < 0.001; ns, not significant.

Figure 5. Type Ib NKT cells in the Jα18(-10) thymus.

(A) Thymocytes from 6–8 week old C57BL/6 and Jα18(-10) were stained with CD1d tetramer loaded with the α-GC analog PBS57 (PBS57-CD1d) or α-GlcCer and analyzed by flow cytometry (data representative of n ≥ 3). (B) α-GlcCer/CD1d tetramer reactive thymocytes were enriched by MACS beads from C57BL/6 and pooled (3–5 mice per strain) Jα18(-10) and CD1d-/- thymi. Enriched cells were stained with TCRβ and α-GlcCer/CD1d tetramer and assessed by flow cytometry. (C) Expression of CD4, CD44, and CD69 in enriched populations from C57BL/6 (black) and Jα18(-10) (red) thymi were compared to the staining control population (DN, TCRβ⁻; gray filled). Dead cells were excluded from analysis using a viability dye. (D) Enriched cells from (A) were stained with intracellular PLZF, T-bet and RORγt to identify NKT1, NKT2 and NKT17 subsets. Dead cells were excluded from analysis.

Figure 6. Jα18(-10) mice do not respond to αGC challenge in vivo.

C57BL/6 and Jα18(-10) mice received 2 μg of αGC intravenously. Serum was collected at 2 and 18 hours after injection. IL-4 and IFN-γ was quantified by enzyme-linked immunosorbent assay. Data shown represent mean ± SEM for each group (minimum of 3 mice per group). **P < 0.01; ***P < 0.001.