The Src family tyrosine kinase Fyn regulates natural killer T cell development - PubMed (original) (raw)

The Src family tyrosine kinase Fyn regulates natural killer T cell development

P Gadue et al. J Exp Med. 1999.

Abstract

T lymphocytes express two Src tyrosine kinases, Lck and Fyn. While thymocyte and T cell subsets are largely normal in fyn(-/-) mice, animals lacking Lck have impaired T cell development. Here, it is shown that Fyn is required for the rapid burst of interleukin (IL)-4 and IL-13 synthesis, which occurs promptly after T cell receptor activation. The lack of cytokine induction in fyn mutant mice is due to a block in natural killer (NK) T cell development. Studies using bone marrow chimeras indicate that the defect behaves in a cell-autonomous manner, and the lack of NK T cells is probably not caused by inappropriate microenvironmental cues. Both NK T cells and conventional T cells express similar levels of Lck, implying that Fyn and Lck have distinct roles in regulating NK T cell ontogeny. The fyn mutation defines the first signaling molecule that is selectively required for NK T cell, but not for T lymphocyte or NK cell development.

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Figures

Figure 1

Figure 1

Rapid induction of IL-4 or IL-13 RNAs after CD3 cross-linking fails to occur in fyn mutants. (A) Splenocytes from fyn mutant (FYN) or wild-type (WT) mice were stimulated with 5 μg/ml anti-CD3 (2C11) and 1 ng/ml PMA or 1 ng/ml PMA and 500 nM ionomycin for 2 h in vitro. RNA was harvested, and an RNase protection assay specific for IL-4 and IL-13 was performed. L32 RNA expression was also determined as a loading control (−STIM). (B) Wild-type and fyn − / − mice were injected intravenously with PBS or 5 μg of anti-CD3 antibody. The spleen was harvested 1.5 h later, and IL-4/IL-13 RNA induction was measured by RNase protection assay. Shown are representative data from three separate experiments.

Figure 3

Figure 3

The fyn mutation may function in a cell-autonomous manner during NK T cell development. Irradiated mice were injected with a mix of wild-type 129Sv (NK1.1_−_; Ly9.1+) and wild-type or mutant C57Bl/6 (NK1.1+; Ly9.1−) derived bone marrow. (A) Reconstitution of NK T cells in the thymus by the different bone marrows was assessed by flow cytometry as in Fig. 2. NK T cells developed from wild-type (Wt) and β2M mutant bone marrow, but not fyn mutant bone marrow. (B) The relative contribution of 129Sv- and C57Bl/6-derived lymphoid cells in the thymus was determined by comparing Ly9.1 expression on CD4+ thymocytes by flow cytometry. All the radiation chimeras contain a mix of CD4+ thymocytes derived from the two strains. The data presented are representative of two independent experiments (chimeras: wild-type, n = 7; Fyn, n = 7; β2M, n = 4).

Figure 2

Figure 2

Fyn and Lck are both required for proper development of NK1.1+TCR-β+ cells. Thymocytes, splenocytes, and liver lymphocytes were stained with HSA, TCR-β, and NK1.1. After gating on HSAlow cells, two-color TCR-β vs. NK1.1 plots were constructed. The percentage of cells positive for both NK1.1 and TCR (NK T cells) in the circled gate is displayed in the upper right quadrant of each plot. The data shown are representative from 10 C57Bl/6 (B6, Wt), 12 Fyn (129 × B6 hybrid or B6), 4 Lck (B6), and 5 β2M (B6) mice analyzed in 5 separate experiments.

Figure 4

Figure 4

Both conventional T cells and NK T cells express similar levels of Lck and Fyn. (A) Thymocytes were cultured from C57Bl/6 mice to enrich for NK T cells. The cultures were sorted by FACS® into NK1.1+TCR-β+ (NK T cell) and NK1.1−TCR-β+ (T cell) populations. (B) These populations as well as brain extract from a fyn mutant mouse as a negative control were then assayed for Fyn and Lck expression by Western blot analysis.

Figure 4

Figure 4

Both conventional T cells and NK T cells express similar levels of Lck and Fyn. (A) Thymocytes were cultured from C57Bl/6 mice to enrich for NK T cells. The cultures were sorted by FACS® into NK1.1+TCR-β+ (NK T cell) and NK1.1−TCR-β+ (T cell) populations. (B) These populations as well as brain extract from a fyn mutant mouse as a negative control were then assayed for Fyn and Lck expression by Western blot analysis.

Figure 5

Figure 5

Quantitative RT-PCR for the canonical Vα14Jα281 rearrangement used by NK T cells. RNA was harvested from thymocytes and splenocytes, then subjected to quantitative RT-PCR analysis. The data presented have been normalized for HPRT expression (wild-type [Wt], n = 5; Fyn, n = 5; β2M, n = 4; Lck, n = 4).

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