Pre-TCRalpha and TCRalpha are not interchangeable partners of TCRbeta during T lymphocyte development - PubMed (original) (raw)

Pre-TCRalpha and TCRalpha are not interchangeable partners of TCRbeta during T lymphocyte development

Christine Borowski et al. J Exp Med. 2004.

Abstract

In contrast with the alphabeta T cell receptor (TCR), the pre-TCR spontaneously segregates to membrane rafts from where it signals in a cell-autonomous fashion. The disparate behaviors of these two receptors may stem either from differences inherent to the distinct developmental stages during which they are expressed, or from features intrinsic and unique to the receptor components themselves. Here, we express TCRalpha precisely at the pre-TCR checkpoint, at levels resembling those of endogenous pre-TCRalpha (pTalpha), and in the absence of endogenous pTalpha. Both in isolation and more dramatically when in competition with pTalpha, TCRalpha induced defective proliferation, survival, and differentiation of alphabeta T lymphocyte precursors, as well as impaired commitment to the alphabeta T lymphocyte lineage. Substitution of TCRalpha transmembrane and cytoplasmic domains with those of pTalpha generated a hybrid molecule possessing enhanced competitive abilities. We conclude that features intrinsic to the pre-TCR, which are absent in TCRalpha, are essential for its unique function.

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Figures

Figure 1.

Figure 1.

Generation of transgenic mice. (A) Structures of the WTpTα, TCRα, and TCRα/pTα hybrid molecules. (B) Semi-quantitative RT-PCR analysis of primer efficiency with β-actin control. Fivefold serial dilutions of the same sample of TCRα/pTα hybrid whole thymocyte cDNA were used in all samples. Lckp recognizes transcript p56_Lck_ proximal promoter sequences, mus3 recognizes WTpTα sequences, and TCRalo recognizes TCRα sequences. (C) Semi-quantitative RT-PCR analysis of transgene expression (top) in transgenic founders with β-actin controls (bottom). Fivefold serial dilutions of whole thymocyte cDNA were used. (D) Total thymocyte numbers in transgenic mice. Multiple founders of each transgenic construct were analyzed. Mice were analyzed at 3–5 wk of age.

Figure 2.

Figure 2.

Defective differentiation, cell cycle entry, and survival of thymocytes expressing TCRα and hybrid transgenes. (A) Dot plots depict the CD4 versus CD8 profile of a minimum of five 3–5-wk-old mice of each transgenic construct. Multiple founders were analyzed. (B) Percentage of sorted Gr-1−Ter-119−Dx5−CD19−TCRγδ−CD4−CD8−CD25−CD44− (DN4) thymocytes in the S/G2/M phases of the cell cycle. DNA content was analyzed by propidium iodide staining. (C) Percentage of DN4 thymocytes expressing externalized phosphatidyl serine apoptosis marker as revealed by staining with Annexin V. (D) Percentage of CD4+8+ thymocytes expressing intracellular TCRβ (shaded histogram). Overlay represents intracellular staining with isotype-matched control antibody.

Figure 3.

Figure 3.

Defective αβ T lineage commitment induced by TCR complexes containing TCRα and hybrid molecules. Proportion (A) and absolute number (B) of CD4−8− thymocytes expressing surface TCRγδ. (C) Percentage of CD4−8− thymocytes coexpressing TCRγδ and TCRαβ on the cell surface. (D) Histograms depict the CD4−8−TCRβ+ population representative of a minimum of five 3–5-wk-old mice of each transgenic construct. Multiple founders were analyzed. (E) Surface HSA expression on CD4−8−TCRβ+ thymocytes in TCRα (overlay) and WTpTα (fill) mice. (F) Surface HSA expression of CD4−8−TCRβ+ (shaded histogram) and CD4−8−TCRγδ+ (overlay) thymocytes in TCRα mice.

Figure 4.

Figure 4.

Competitive ability of TCRα, hybrid, and WTpTα-derived thymocytes. (A) Dot plots depict apparent domination of WTpTα-derived thymocytes when in direct competition with TCRα and TCRα/pTα hybrid–derived thymocytes. (B) Dot plots depict the CD4 versus CD8 surface profile of WTpTα, TCRα, and TCRα/pTα hybrid–derived thymocytes during competitive thymic reconstitution. (C) Average fold excess of WTpTα-derived thymocytes during competitive thymic reconstitution, normalized to any fold excess CD4−8−25−44+ (DN1) cells in either population. WTpTα versus _pT_α−/− competition is included as a negative control to illustrate background. Dots depict individual mice for each type of competition.

Figure 5.

Figure 5.

Enhanced performance of hybrid at pre-TCR checkpoint requires intact pTα cytoplasmic domain. Percent of CD4−8− thymocytes expressing CD25+CD44− (DN3) surface phenotype in each competing population. Three individual mice are shown for each competition.

Figure 6.

Figure 6.

Influence of the extracellular region of TCRα on the behavior of the hybrid molecule. TCRβ levels on the surface of the 58α−β+ T cell hybridoma are influenced by the second extracellular domain TCRα. 58α−β+ hybridoma cells were infected with retroviruses encoding WTpTα, TCRα, or TCRα/pTα hybrid molecules, followed by an internal ribosomal entry site and enhanced green fluorescent protein (EGFP). Infected cells were identified by EGFP expression. Shaded histograms represent TCRβ surface staining on cells infected with retroviruses expressing WTpTα, TCRα, or TCRα/pTα hybrid molecules. The overlay represents TCRβ staining on cells infected with a control retrovirus expressing EGFP alone. The 58α−β+ T cell hybridoma is a variant of the DO-11.10.7 mouse T cell hybridoma that does not express functional TCRβ or TCRα chains (28).

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