Commitment of immature CD4+8+ thymocytes to the CD4 lineage requires CD3 signaling but does not require expression of clonotypic T cell receptor (TCR) chains - PubMed (original) (raw)

Commitment of immature CD4+8+ thymocytes to the CD4 lineage requires CD3 signaling but does not require expression of clonotypic T cell receptor (TCR) chains

H Suzuki et al. J Exp Med. 1997.

Abstract

As a consequence of positive selection in the thymus, immature CD4(+)8(+) double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4(+) or CD8(+) T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)-CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or zeta chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.

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Figures

Figure 1

Figure 1

Lineage commitment in sorted CD4+8lo and CD4lo8+ thymocytes from experimental mice. Purified populations of CD4+8lo thymocytes (A) and CD4lo8+ thymocytes (B) were obtained by electronic cell sorting according to the indicated sorting gates superimposed on the starting thymocyte populations (left columns). Sorted thymocyte populations were stripped of surface coreceptor molecules by treatment with low doses of pronase, after which they were placed in suspension cultures at 4°C (middle panels) or 37°C (right panels) for 12–16 h and restained for CD4 and CD8 surface expression. The coreceptor reexpression assay detects the coreceptor molecules that individual thymocytes synthesized during the 37°C culture, and is dependent upon new transcription and new protein synthesis (8). Sorted thymocytes that reexpress both CD4 and CD8 coreceptor proteins are lineage-uncommitted cells; those reexpressing only CD4 are CD4 committed; and those reexpressing only CD8 are CD8 committed. Cells cultured at 4°C do not reexpress surface coreceptor molecules so that their CD4–CD8 histograms reflect whatever coreceptor molecules that potentially remain after pronase treatment (middle columns). As we have previously described (8), the anti-CD4 mAb used to prepare thymocytes for cell sorting minimally interferes with stripping of surface CD4 molecules by pronase, resulting in a small number of residual CD4 molecules remaining on the cell surface. Consequently, to highlight changes in coreceptor reexpression during 37°C cultures, histogram boxes were drawn based on the 4°C profiles of each sorted and pronase-stripped cell population. The frequency of cells in each box is indicated. The number of the thymocytes obtained in these experimental mice were the following: TCRα° (9 × 107 cells), γ-irradiated RAG2° (4 × 107 cells), anti-CD3ε mAb–injected RAG2° (1.1 × 108 cells), anti-CD3ε mAb–injected RAG°TCRζ° (8 × 107 cells), and anti-Tac mAb–injected RAG°–TTζ (4 × 107 cells).

Figure 1

Figure 1

Lineage commitment in sorted CD4+8lo and CD4lo8+ thymocytes from experimental mice. Purified populations of CD4+8lo thymocytes (A) and CD4lo8+ thymocytes (B) were obtained by electronic cell sorting according to the indicated sorting gates superimposed on the starting thymocyte populations (left columns). Sorted thymocyte populations were stripped of surface coreceptor molecules by treatment with low doses of pronase, after which they were placed in suspension cultures at 4°C (middle panels) or 37°C (right panels) for 12–16 h and restained for CD4 and CD8 surface expression. The coreceptor reexpression assay detects the coreceptor molecules that individual thymocytes synthesized during the 37°C culture, and is dependent upon new transcription and new protein synthesis (8). Sorted thymocytes that reexpress both CD4 and CD8 coreceptor proteins are lineage-uncommitted cells; those reexpressing only CD4 are CD4 committed; and those reexpressing only CD8 are CD8 committed. Cells cultured at 4°C do not reexpress surface coreceptor molecules so that their CD4–CD8 histograms reflect whatever coreceptor molecules that potentially remain after pronase treatment (middle columns). As we have previously described (8), the anti-CD4 mAb used to prepare thymocytes for cell sorting minimally interferes with stripping of surface CD4 molecules by pronase, resulting in a small number of residual CD4 molecules remaining on the cell surface. Consequently, to highlight changes in coreceptor reexpression during 37°C cultures, histogram boxes were drawn based on the 4°C profiles of each sorted and pronase-stripped cell population. The frequency of cells in each box is indicated. The number of the thymocytes obtained in these experimental mice were the following: TCRα° (9 × 107 cells), γ-irradiated RAG2° (4 × 107 cells), anti-CD3ε mAb–injected RAG2° (1.1 × 108 cells), anti-CD3ε mAb–injected RAG°TCRζ° (8 × 107 cells), and anti-Tac mAb–injected RAG°–TTζ (4 × 107 cells).

Figure 2

Figure 2

CD5 surface expression on thymocytes from experimental mice. Thymocytes from the indicated experimental mice were stained with anti-CD5 mAb (solid line) or an irrelevant antibody (shaded curve). CD5 expression on normal B6 thymocytes stained at the same time as a positive control is also shown for comparison (dotted line).

Figure 3

Figure 3

Correlation between induction of CD4 commitment and surface CD5 upregulation. RAG° mice received one intraperitoneal injection of either 250 μg or 10 μg of affinity-purified anti-CD3ε mAb. 8 d later, thymocytes were sorted according to the indicated gates and assessed for lineage commitment by the coreceptor reexpression assay (left panels). Unsorted thymocytes were placed in culture for 12 h at 4°C, at which temperature thymocyte phenotype is stable, and then assessed for surface CD5 expression (right panels). Solid line indicates CD5 expression on experimental thymocytes. Shaded curve represents negative control staining of experimental thymocytes with an irrelevant mAb. The dotted line represents CD5 expression on normal B6 thymocytes stained at the same time as a positive control and is shown for comparison. The number of thymocytes obtained on day 8 were 1–1.5 × 108 cells and 3–4 × 107 cells with the injection of 250 μg and 10 μg, respectively.

Figure 4

Figure 4

CD8-committed thymocytes do not appear in RAG2° thymi even after two injections of anti-CD3 mAb. RAG2° mice were injected with 250 μg of affinity-purified anti-CD3ε mAb on both day 0 and 8. On day 12, thymocytes were harvested, sorted into CD4+8lo and CD4lo8+ populations, and assessed by the coreceptor reexpression assay for appearance of CD4-committed and CD8-committed cells. Numbers in each box represent the frequency of thymocytes falling in that box.

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References

    1. Teh HS, Kisielow P, Scott B, Kishi H, Uematsu Y, Bluthmann H, von Boehmer H. Thymic major histocompatibility complex antigens and the alpha beta T-cell receptor determine the CD4/CD8 phenotype of T cells. Nature (Lond) 1988;335:229–233. - PubMed
    1. Robey EA, Fowlkes BJ, Gordon JW, Kioussiss D, von Boehmer H, Ramsdell F, Axel R. Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage. Cell. 1991;64:99–107. - PubMed
    1. Chan SH, Benoist CB, Mathis D. A challenge to the instructive model of positive selection. Immunol Rev. 1993;135:119–131. - PubMed
    1. Chan SH, Cosgrove D, Waltzinger C, Benoist C, Mathis D. Another view of the selective model of thymocyte selection. Cell. 1993;73:225–236. - PubMed
    1. Davis CB, Killeen N, Crooks MEC, Raulet D, Littman DR. Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes. Cell. 1993;73:237–247. - PubMed

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