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Asynchronous coreceptor downregulation after positive thymic selection: prolonged maintenance of the double positive state in CD8 lineage differentiation due to sustained biosynthesis of the CD4 coreceptor.

Barthlott T, Kohler H, Eichmann K - J. Exp. Med. (1997)

Bottom Line: To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells.The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells.Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunobiologie, Freiburg, Germany.

ABSTRACT
In several experimental systems analyzing the generation of single positive (SP) thymocytes from double positive (DP) thymocytes, CD4 SP cells have been shown to appear before CD8 SP cells. This apparent temporal asymmetry in the maturation of CD4 SP and CD8 SP thymocytes could either be due to divergent molecular differentiation programs of the two T cell lineages, or merely to slower degradation kinetics of the CD4 protein. To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells. We show that these TCRintCD69pos thymocytes represent a transitory stage in the mainstream alphabeta T cell lineage. The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells. Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

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Proteolytic stripping and requirements for coreceptor reexpression on TCRintCD69pos and on TCRhiCD69pos thymocyte populations. (A) TCRintCD69pos and TCRhiCD69pos thymocytes were purified  by cell sorting. (B) Purified cell populations were treated with PBS or  trypsin, and immediately analysed for CD4/CD8 expression. (C) PBStreated cells were incubated overnight in medium alone; trypsin-treated  cells were incubated overnight in medium alone, or in the presence of  ActD or CHX. Thereafter, cells were stained for CD4/CD8 expression.  Percentages are given in the insets. Fluorescence intensities in B and C  differ somewhat since analyses have been performed on different flow cytometers.
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Figure 3: Proteolytic stripping and requirements for coreceptor reexpression on TCRintCD69pos and on TCRhiCD69pos thymocyte populations. (A) TCRintCD69pos and TCRhiCD69pos thymocytes were purified by cell sorting. (B) Purified cell populations were treated with PBS or trypsin, and immediately analysed for CD4/CD8 expression. (C) PBStreated cells were incubated overnight in medium alone; trypsin-treated cells were incubated overnight in medium alone, or in the presence of ActD or CHX. Thereafter, cells were stained for CD4/CD8 expression. Percentages are given in the insets. Fluorescence intensities in B and C differ somewhat since analyses have been performed on different flow cytometers.

Mentions: The failure to detect CD8 SP thymocytes in the most recently selected TCRintCD69pos thymocyte subset could be due a slow degradation of the CD4 protein, in contrast to a rapid disappearance of the CD8 protein from the cell surface after activation. As a consequence, the CD4 protein would still be present on the cell surface although the CD4 gene is turned off, thus letting CD8 SP thymocytes appear as DP cells for some time. To examine this possibility, sorted TCRint CD69pos thymocytes (Fig. 3 A) were treated with trypsin/ EDTA in order to strip the coreceptors from the cell surface (Fig. 3 B). After overnight culture, reexpression of CD4 and CD8 was analyzed. Cells that have terminated CD4 synthesis in the DP subset of the TCRintCD69pos thymocytes should become detectable as an increase in CD8 SP cells after trypsinization. As shown in Fig. 3 C, the number of CD8 SP cells after trypsinization and overnight incubation was only slightly greater than that in PBStreated control thymocytes and did not exceed 1/20 of the number of CD4 SP cells regenerated after trypsinization. The level of CD4 reexpressed on trypsin-treated cells was not reduced compared to that on PBS-treated cells. Nevertheless, it was possible that the cells had sufficiently rich intracellular stores of CD4 protein to achieve complete redeposition of CD4 on the cell surface. To examine this possibility, overnight incubations after trypsin treatment were done in the presence of actinomycin D (ActD) or of cycloheximide (CHX) to inhibit transcription or translation, respectively. Whereas CHX completely blocked reexpression of both coreceptors, reappearance of low levels of CD4 on some of the cells was seen in the presence of ActD. The results suggest that coreceptor reexpression in this assay is completely dependent on translation, whereas some mRNA stores allow a limited reexpression of CD4. However, the data exclude a prolonged maintenance of CD4 surface expression after turnoff of CD4 biosynthesis.


Asynchronous coreceptor downregulation after positive thymic selection: prolonged maintenance of the double positive state in CD8 lineage differentiation due to sustained biosynthesis of the CD4 coreceptor.

Barthlott T, Kohler H, Eichmann K - J. Exp. Med. (1997)

Proteolytic stripping and requirements for coreceptor reexpression on TCRintCD69pos and on TCRhiCD69pos thymocyte populations. (A) TCRintCD69pos and TCRhiCD69pos thymocytes were purified  by cell sorting. (B) Purified cell populations were treated with PBS or  trypsin, and immediately analysed for CD4/CD8 expression. (C) PBStreated cells were incubated overnight in medium alone; trypsin-treated  cells were incubated overnight in medium alone, or in the presence of  ActD or CHX. Thereafter, cells were stained for CD4/CD8 expression.  Percentages are given in the insets. Fluorescence intensities in B and C  differ somewhat since analyses have been performed on different flow cytometers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196127&req=5

Figure 3: Proteolytic stripping and requirements for coreceptor reexpression on TCRintCD69pos and on TCRhiCD69pos thymocyte populations. (A) TCRintCD69pos and TCRhiCD69pos thymocytes were purified by cell sorting. (B) Purified cell populations were treated with PBS or trypsin, and immediately analysed for CD4/CD8 expression. (C) PBStreated cells were incubated overnight in medium alone; trypsin-treated cells were incubated overnight in medium alone, or in the presence of ActD or CHX. Thereafter, cells were stained for CD4/CD8 expression. Percentages are given in the insets. Fluorescence intensities in B and C differ somewhat since analyses have been performed on different flow cytometers.
Mentions: The failure to detect CD8 SP thymocytes in the most recently selected TCRintCD69pos thymocyte subset could be due a slow degradation of the CD4 protein, in contrast to a rapid disappearance of the CD8 protein from the cell surface after activation. As a consequence, the CD4 protein would still be present on the cell surface although the CD4 gene is turned off, thus letting CD8 SP thymocytes appear as DP cells for some time. To examine this possibility, sorted TCRint CD69pos thymocytes (Fig. 3 A) were treated with trypsin/ EDTA in order to strip the coreceptors from the cell surface (Fig. 3 B). After overnight culture, reexpression of CD4 and CD8 was analyzed. Cells that have terminated CD4 synthesis in the DP subset of the TCRintCD69pos thymocytes should become detectable as an increase in CD8 SP cells after trypsinization. As shown in Fig. 3 C, the number of CD8 SP cells after trypsinization and overnight incubation was only slightly greater than that in PBStreated control thymocytes and did not exceed 1/20 of the number of CD4 SP cells regenerated after trypsinization. The level of CD4 reexpressed on trypsin-treated cells was not reduced compared to that on PBS-treated cells. Nevertheless, it was possible that the cells had sufficiently rich intracellular stores of CD4 protein to achieve complete redeposition of CD4 on the cell surface. To examine this possibility, overnight incubations after trypsin treatment were done in the presence of actinomycin D (ActD) or of cycloheximide (CHX) to inhibit transcription or translation, respectively. Whereas CHX completely blocked reexpression of both coreceptors, reappearance of low levels of CD4 on some of the cells was seen in the presence of ActD. The results suggest that coreceptor reexpression in this assay is completely dependent on translation, whereas some mRNA stores allow a limited reexpression of CD4. However, the data exclude a prolonged maintenance of CD4 surface expression after turnoff of CD4 biosynthesis.

Bottom Line: To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells.The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells.Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunobiologie, Freiburg, Germany.

ABSTRACT
In several experimental systems analyzing the generation of single positive (SP) thymocytes from double positive (DP) thymocytes, CD4 SP cells have been shown to appear before CD8 SP cells. This apparent temporal asymmetry in the maturation of CD4 SP and CD8 SP thymocytes could either be due to divergent molecular differentiation programs of the two T cell lineages, or merely to slower degradation kinetics of the CD4 protein. To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells. We show that these TCRintCD69pos thymocytes represent a transitory stage in the mainstream alphabeta T cell lineage. The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells. Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

Show MeSH