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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.

Suzuki H, Shinkai Y, Granger LG, Alt FW, Love PE, Singer A - J. Exp. Med. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA.

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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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).
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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).

Mentions: To assess the possibility that DP thymocytes spontaneously terminated CD8 coreceptor synthesis even in the absence of TCR–CD3 signals, we examined DP thymocytes from TCRα° mice by the coreceptor reexpression assay. TCRα° thymocytes cannot express conventional αβ TCR complexes and, consequently, cannot differentiate beyond the DP stage of development (21). To enrich for DP thymocytes that might have committed to the CD4 or CD8 T cell lineages, we electronically sorted for CD4+8lo and CD4lo8+ transitional cell populations and utilized the coreceptor reexpression assay to determine the coreceptor molecules they were actively synthesizing (Fig. 1, A and B). In the coreceptor reexpression assay, preexisting surface CD4 and CD8 coreceptor molecules are removed from the sorted cells by treatment with low doses of pronase, and the stripped cells then placed into single cell suspension cultures for 14 h. Metabolic activity of cultured cells is inhibited at 4°C, so that cell surface coreceptor reexpression does not occur (Fig. 1 A and B, middle columns). However, cells cultured at 37°C do reexpress the CD4 and/or CD8 coreceptor molecules that they are actively synthesizing. Indeed, we have previously demonstrated that coreceptor reexpression in this assay requires active coreceptor transcription and protein synthesis (8). Interestingly, virtually all CD4+8lo sorted cells from TCRα° DP thymocytes reexpressed both CD4 and CD8 coreceptors and so reappeared as DP cells (Fig. 1 A, top). Identical results were obtained with CD4lo8+ sorted cells from TCRα° mice (Fig. 1 B, top). That is, none of the DP thymocytes present in TCRα° mice had selectively terminated either CD4 or CD8 coreceptor synthesis, indicating that none had undergone lineage commitment. Thus, these results indicated that lineage commitment did not occur spontaneously in immature DP thymocytes but might be dependent upon signals transduced by surface TCR–CD3 complexes.


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.

Suzuki H, Shinkai Y, Granger LG, Alt FW, Love PE, Singer A - J. Exp. Med. (1997)

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).
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Related In: Results  -  Collection

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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).
Mentions: To assess the possibility that DP thymocytes spontaneously terminated CD8 coreceptor synthesis even in the absence of TCR–CD3 signals, we examined DP thymocytes from TCRα° mice by the coreceptor reexpression assay. TCRα° thymocytes cannot express conventional αβ TCR complexes and, consequently, cannot differentiate beyond the DP stage of development (21). To enrich for DP thymocytes that might have committed to the CD4 or CD8 T cell lineages, we electronically sorted for CD4+8lo and CD4lo8+ transitional cell populations and utilized the coreceptor reexpression assay to determine the coreceptor molecules they were actively synthesizing (Fig. 1, A and B). In the coreceptor reexpression assay, preexisting surface CD4 and CD8 coreceptor molecules are removed from the sorted cells by treatment with low doses of pronase, and the stripped cells then placed into single cell suspension cultures for 14 h. Metabolic activity of cultured cells is inhibited at 4°C, so that cell surface coreceptor reexpression does not occur (Fig. 1 A and B, middle columns). However, cells cultured at 37°C do reexpress the CD4 and/or CD8 coreceptor molecules that they are actively synthesizing. Indeed, we have previously demonstrated that coreceptor reexpression in this assay requires active coreceptor transcription and protein synthesis (8). Interestingly, virtually all CD4+8lo sorted cells from TCRα° DP thymocytes reexpressed both CD4 and CD8 coreceptors and so reappeared as DP cells (Fig. 1 A, top). Identical results were obtained with CD4lo8+ sorted cells from TCRα° mice (Fig. 1 B, top). That is, none of the DP thymocytes present in TCRα° mice had selectively terminated either CD4 or CD8 coreceptor synthesis, indicating that none had undergone lineage commitment. Thus, these results indicated that lineage commitment did not occur spontaneously in immature DP thymocytes but might be dependent upon signals transduced by surface TCR–CD3 complexes.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA.

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.

Show MeSH
Related in: MedlinePlus