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. 1999 Oct 18;190(8):1093-102.
doi: 10.1084/jem.190.8.1093.

Allelic exclusion of the T cell receptor beta locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adaptor protein

I Aifantis  1 , V I Pivniouk, F Gärtner, J Feinberg, W Swat, F W Alt, H von Boehmer, R S Geha
Affiliations

Allelic exclusion of the T cell receptor beta locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adaptor protein

I Aifantis et al. J Exp Med. .

Abstract

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

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Figures

Figure 1
Figure 1
Pre-TCR expression on the surface of the SCB29 cell line and comparison of the labeling intensity between streptavidin–PBXL-3 and streptavidin–FITC. SCB29 cells were initially incubated with biotinylated 2F5 (pTα) and H57 (pan-TCR-α/β) antibodies (thick line) or isotype-matched controls (thin line), and surface expression was revealed by streptavidin conjugated to the secondary reagents. Similar results were obtained in two other experiments.
Figure 1
Figure 1
Pre-TCR expression on the surface of the SCB29 cell line and comparison of the labeling intensity between streptavidin–PBXL-3 and streptavidin–FITC. SCB29 cells were initially incubated with biotinylated 2F5 (pTα) and H57 (pan-TCR-α/β) antibodies (thick line) or isotype-matched controls (thin line), and surface expression was revealed by streptavidin conjugated to the secondary reagents. Similar results were obtained in two other experiments.
Figure 2
Figure 2
Expression of the pre-TCR complex on the surfaces of CD25+CD44− CD4− CD8− DN thymocytes from RAG-2−/−, pTα−/−, TCR-α−/−, and SLP-76−/− mice. CD4+ and CD8+ thymocytes and NK cells were depleted with Dynabeads, and DN cells were stained with CD25, CD44, 2.4G2 (Fc-block), H57 (TCR-β), or 2F5 (pTα) antibodies. Analysis of gated CD25+ CD44− cells is shown. Results are representative of three mice in each group.
Figure 3
Figure 3
Intracellular TCR-β expression in CD4−CD8− DN cells from WT (B6), pTα−/−, and SLP-76−/− mice. DN cells were surface stained for CD25 and subsequently cytoplasmically labeled with TCR-β antibodies. Results are representative of three mice in each group.
Figure 3
Figure 3
Intracellular TCR-β expression in CD4−CD8− DN cells from WT (B6), pTα−/−, and SLP-76−/− mice. DN cells were surface stained for CD25 and subsequently cytoplasmically labeled with TCR-β antibodies. Results are representative of three mice in each group.
Figure 3
Figure 3
Intracellular TCR-β expression in CD4−CD8− DN cells from WT (B6), pTα−/−, and SLP-76−/− mice. DN cells were surface stained for CD25 and subsequently cytoplasmically labeled with TCR-β antibodies. Results are representative of three mice in each group.
Figure 4
Figure 4
Flow cytometric analysis of thymocytes from TCR-transgenic SLP-76−/− and SLP-76+/− littermates. Surface expression on thymocytes of (A) CD3 (anti-CD3∈–PE) versus TCR-β (anti–TCR-β–FITC), (B) CD4 (anti-CD4–FITC) versus CD8 (anti-CD8–PE), and (C) CD44 and CD25 on DN thymocytes from SLP-76−/− and SLP-76+/− littermates. Cells were triple stained with anti-CD44–FITC, anti-CD25–PE, and a cocktail of biotin-conjugated mAbs to CD3, CD4, CD8, B220, Mac-1, and Gr-1, followed by streptavidin–Cy-Chrome. Analysis was performed on gated Cy-Chrome–negative cells. The percentage of cells found in each quadrant is indicated. In all FACS™ analyses, results from SLP-76+/− and WT mice were similar. Therefore, only data of SLP-76+/− mice is shown. Results are representative of four mice examined in each group.
Figure 5
Figure 5
Thymocytes from TCR-α/β–transgenic SLP-76−/− mice fail to suppress V to DJ rearrangements in the endogenous TCR-β locus. 20 ng of genomic DNA isolated from DN thymocytes of SLP-76+/−, SLP-76+/−-tg, SLP-76−/−, and SLP-76−/−-tg was amplified with primers that specifically detect rearrangements among Vβ5, Vβ8, Vβ10, and DJβ2. Equivalent loading was confirmed by amplification of a fragment of the Cμ gene (IgM). The PCR products were separated on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized with radiolabeled oligonucleotide probes specific for Jβ2.7 and Cμ. For each Vβ, there are six bands that correspond to rearrangements to the DJβ2 segments DJβ2.1 through DJβ2.7 (Jβ2.6 is a pseudogene and is not rearranged). Similar results were obtained in two other experiments.
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