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A nonredundant role for the adapter protein Shc in thymic T cell development

Nature Immunology volume 3, pages 749–755 (2002)Cite this article

Abstract

Signaling via the pre-T cell receptor (pre-TCR) regulates survival, proliferation, allelic exclusion and differentiation of thymocytes. The role played by the adapter protein Shc in T cells has remained controversial, and its role in pre-TCR signaling has not been addressed. We examined Shc function in thymic T cell development using two genetic approaches. Cre-loxP–mediated inducible expression in transgenic mice of a phosphorylation-defective mutant of Shc impaired signaling through the pre-TCR as well as subsequent proliferation and differentiation. Conditional deletion of the Shc locus in thymocytes also affected thymic maturation at the same pre-TCR developmental stage. Thus, both Shc expression and its tyrosine phosphorylation play an essential and nonredundant role in thymic T cell development.

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Figure 1: Expression and phosphorylation of Shc in immature thymocytes.
Figure 2: Impaired T cell development in lck-Cre-ShcFFF double-transgenic mice.
Figure 3: Defective pre-TCR signaling in lck-Cre-ShcFFF double-transgenic mice.
Figure 4: Loss of Shc expression in conditional Shc-deficient mice affects thymic development at the DN3 stage.

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Acknowledgements

We thank C. Wilson for the lck-Cre transgenic mouse line; D. Wiest for the pre-T cell line; M. McDuffie for the RAG-1−/− mice; the Transgenic Mouse Core Facility at the University of Virginia for their assistance; B. Neel, U. Lorenz and S. Burakoff for comments on the manuscript; and the members of the Ravichandran and Bender labs for helpful discussions. Supported by grants from the National Institutes of Health: GM-55761 to K. S. R. and CA85842 to T. P. B.

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Author notes

  1. Timothy P. Bender and Kodi S. Ravichandran: T. P. B. and K. S. R. contributed equally to this work.

Authors and Affiliations

  1. Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, 22908, VA, USA

    Li Zhang, Victoria Camerini, Timothy P. Bender & Kodi S. Ravichandran

  2. Department of Microbiology University of Virginia, Charlottesville, 22908, VA, USA

    Li Zhang, Timothy P. Bender & Kodi S. Ravichandran

  3. Department of Pediatrics, University of Virginia, Charlottesville, 22908, VA, USA

    Victoria Camerini

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  1. Li Zhang
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Correspondence to Kodi S. Ravichandran.

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Supplementary information

Web Fig. 1.

A second independent ShcFFF transgenic mouse line shows a similar defect in thymic development. (a) Increased percentage of DN thymocytes in lck-Cre-ShcFFF mice. Total thymocytes from ShcFFF and lck-Cre-ShcFFF mice were analyzed for CD4 and CD8 expression by flow cytometry. The percentages of cells in each quadrant as well as the total number of thymocytes from individual mice are shown. The data are representative of three independent analyses. (b) CD25 and CD44 surface expression in DN thymocyte subsets from ShcFFF and lck-Cre-ShcFFF mice were analyzed by flow cytometry (from the same mice in a). Sustained high expression of CD25 in DN subsets of lck-Cre-ShcFFF double-transgenic mice was also seen (as shown in Figure 3a and in the conditional Shc knockout mice). (JPG 103 kb)

Web Fig. 2.

Targeting of the Shc1 locus in ES cells. (a) Southern blot for homologous recombination. DNA from G418-resistant ES clones were digested with SphI and hybridized with probe 1. The 11-kb and 3.8-kb bands represented the wild-type and targeted Shc1 alleles, respectively. (b) Southern blot for homologous recombinant ES clones retaining the inserted loxP site in genomic Shc1 intron 1. DNA from ES clones with the homologous recombination (HR) was cut with BglII and hybridized with probe 2. The 12.3-kb band was generated from the ES clones with homologous recombination but which lost the inserted loxP site 5' of exon 1 during the homologous recombination process. The 9.8-kb band was generated from HR-ES clones which retained the inserted loxP sites during the homologous recombination process. The 10.5-kb band was generated from the wild-type alleles. (c) Southern blot for Cre-mediated deletion in ES cells. After transient transfection of pI-Cre into clone 141, DNA from the neomycin-sensitive ES clones were cut with BglII and hybridized with probe 2. The bands corresponding to WT, floxed and deleted alleles are indicated. *A nonspecific band present in all clones. (d) PCR screen for the floxed Shc1 allele in tail DNA. The forward primer used was 5'-CAGCCGGCCAACTCTAAG-3' and the reverse primer was 5'-GCCCTCGGACAGAGCAATCATGTC-3'. The bands representing the WT and floxed alleles are indicated. (JPG 38 kb)

Web Fig. 3.

Analysis of peripheral T cells from lck-Cre-ShcFFF mice. (a) Single-cell suspensions of splenocytes were stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry. Only a modest and inconsistent decrease in the numbers of T cells was found in the periphery in contrast to the significant decrease in the thymus. The negligible decrease in peripheral T cell numbers in the lck-Cre-ShcFFF mice is most likely due to the homeostatic expansion of T cells that leave the thymus and failed to delete the STOP cassette. (b) Splenic T cells from the control and the lck-Cre-ShcFFF mice were analyzed for proliferation after stimulation with anti-CD3 ± IL-2. 96-well plates were coated with 3 μg/ml of anti-CD3 or PBS control. Splenocytes (2 × 105) were plated onto each well in triplicate in the presence or absence of 50 U/ml of recombinant human IL-2. After 72 h of incubation, the cells were pulsed with 1 μCi of [3H]thymidine and 18 h later the incorporation was measured. There was no apparent difference in the proliferative capacity of the peripheral T cells between the control and the lck-Cre-ShcFFF mice. (JPG 41 kb)

Web Fig. 4.

Expression of ShcFFF does not result in increased apoptosis of DN3 thymocytes, as determined by annexin V staining. Single-cell suspension of thymocytes prepared from ShcFFF alone and lck-Cre-ShcFFF mice were analyzed by annexin V staining either fresh out of the animal (0 h) or subsequent to cell culture for the indicated times. The DN3 cells were gated and the percentage of annexin V-positive and propidium iodide-negative cells was determined. There was no significant difference between control and lck-Cre-ShcFFF thymocytes after 24 h in culture. The data presented are representative of three independent experiments. (JPG 76 kb)

Web Fig. 5.

ShcFFF does not affect γδ T cell development. (a) No apparent difference in γδ T cell numbers in the thymus between control and lck-Cre-ShcFFF mice. CD4-CD8- (DN) thymocytes from control mice (lck-Cre alone or ShcFFF alone) or lck-Cre-ShcFFF double-transgenic mice were analyzed for percentage of cells expressing TCRγ δ and CD3. The data are representative of three independent experiments. The absolute numbers of γδ T cells in the control mice were 6 × 105 ± 0.55 × 105 (n = 3) and in the lck-Cre-ShcFFF mice were 5.8 × 105 ± 0.7 × 105 (n = 3). Of note, the lck-Cre-ShcFFF mouse shown above had a severe decrease in total thymocyte numbers (7 × 106) compared to the control mouse (2 × 108). (b) Distribution of αβ and γδ T cells in the intestinal intraepithelial lymphocyte (IEL) population in the lck-Cre-ShcFFF double-transgenic mice. IEL isolated from the control mice and the lck-Cre-ShcFFF mice were analyzed for the composition of αβ and γδ TCR expressing cells with the anti-TCRαβ antibody H57-597 (BD-Pharmingen) and anti-TCRγδ antibody GL3 (Caltag). The data presented are representative of three independent control and experimental mice that were analyzed. The mean percentage of γδ T cells within the IEL compartment in the control mice was 56 ± 12 and 49 ± 7 in the lck-Cre-ShcFFF. (JPG 68 kb)

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Zhang, L., Camerini, V., Bender, T. et al. A nonredundant role for the adapter protein Shc in thymic T cell development. Nat Immunol 3, 749–755 (2002). https://doi.org/10.1038/ni820

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