EphB6-null mutation results in compromised T cell function
Hongyu Luo, … , Johanne Tremblay, Jiangping Wu
Hongyu Luo, … , Johanne Tremblay, Jiangping Wu
Published December 15, 2004
Citation Information: J Clin Invest. 2004;114(12):1762-1773. https://doi.org/10.1172/JCI21846.
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Categories: Article Immunology

EphB6-null mutation results in compromised T cell function

Abstract

So far, there is very limited knowledge about the role of Eph kinases, the largest family of receptor tyrosine kinases, in the immune system. Here, using EphB6–/– mice, we demonstrated that in vitro and in vivo T cell responses such as lymphokine secretion, proliferation, and the development of delayed-type skin hypersensitivity and experimental autoimmune encephalitis in EphB6–/– mice were compromised. On the other hand, humoral immune responses, such as serum levels of different Ig isotypes and IgG response to tetanus toxoid, were normal in these mice. Mechanistically, we showed that EphB6 migrated to the aggregated TCRs and rafts after TCR activation. Further downstream, in the absence of EphB6, ZAP-70 activation, LAT phosphorylation, the association of PLCγ1 with SLP-76, and p44/42 MAPK activation were diminished. Thus, we have shown that EphB6 is pivotal in T cell function.

Authors

Hongyu Luo, Guang Yu, Johanne Tremblay, Jiangping Wu

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Figure 1

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Generation of EphB6–/– mice. (A) Illustration of the targeting construct...
Generation of EphB6–/– mice. (A) Illustration of the targeting construct, primers, and probes. Primer pairs TK13/TK10 and TK12/TK11 were used to retrieve genomic sequences with PCR for the targeting construct. Primer pair Neo1/TK15 was applied to identify a 1.2-kb fragment derived from the targeted allele with PCR, and primer pair PTK3/TK15 served to identify a 1.6-kb fragment derived from the WT allele. The region of the Southern probe is shown as a thick bar; the null-mutated allele was detected as a 6.6-kb band and the WT allele as a 10.0-kb band. Primer pairs are shown as arrows. K, KpnI; Bg, BGIII; N, NcoI; Cla, ClaI; Xb, XbaI; Xh, XhoI; RV, EcoRV; H, HindIII. (B) Genotyping of EphB6–/–, EphB6+/–, and EphB6+/+ mice by Southern blotting and PCR with tail DNA. (C) Lack of EphB6 protein expression on EphB6–/– thymocytes. EphB6+/+ (dotted line) and EphB6–/– (solid line) thymocytes were stained with goat anti–mouse EphB6, followed by PE-conjugated donkey anti-goat IgG. The shaded area represents the isotypic control (goat IgG). (D and E) EphB6 expression in thymocytes and lymph node and spleen T cells from EphB6–/– mice or their WT littermates. These cells were stained with Quantum Red–labeled anti-CD4 and PE-labeled anti-CD8 Abs. Cells from WT mice were also stained with goat anti-EphB6 Ab followed by Alexa Fluor 488–labeled donkey anti-goat IgG. For cells from EphB6–/– mice (left columns), EphB6 promoter–driven expression of GFP was measured at 488 nm without staining. The percentage in gated regions represents values after deduction of background fluorescence (shaded areas; unstained EphB6+/+ cells in the left columns, and goat IgG in the right columns). All experiments were performed at least 3 times and were reproducible; representative results are shown.