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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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Thymic subsets defined by TCR levels and CD69  (A) and phenotypic characterization of coreceptor expression in  each subset (B). Total thymocytes were stained for TCR,  CD69, CD4 and CD8 and analyzed by four-color flow cytometry. Percentages are given in the  insets, numbers in italics in B indicate the percentages in relation  to total thymocytes. The arrow  in A indicates the relatively  empty space between R2 and  R5, suggesting that passing  through the CD69pos gates R3  and R4 is obligatory for all cells.
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Figure 1: Thymic subsets defined by TCR levels and CD69 (A) and phenotypic characterization of coreceptor expression in each subset (B). Total thymocytes were stained for TCR, CD69, CD4 and CD8 and analyzed by four-color flow cytometry. Percentages are given in the insets, numbers in italics in B indicate the percentages in relation to total thymocytes. The arrow in A indicates the relatively empty space between R2 and R5, suggesting that passing through the CD69pos gates R3 and R4 is obligatory for all cells.

Mentions: A number of experiments have suggested that, after positive selection, CD4 SP thymocytes develop more rapidly than CD8 SP thymocytes (6–12). With one exception (7) these studies employed previously manipulated DP cells or DP subpopulations placed in reaggregate thymic organ cultures or transferred intrathymically into syngeneic mice, so that an influence of experimental manipulations on developmental kinetics was not excluded. To detect recently generated SP thymocytes in vivo, we stained thymic cell suspensions for CD4, CD8, TCR, and CD69 expression. The early activation marker CD69 is expressed transiently on activated thymocytes and discriminates cells which have recently received a selection signal from pre-selection and late post-selection thymocytes. The stepwise increase in the level of TCR expression served as a marker for the maturation sequence. As shown in Fig. 1 A, this protocol defines five sequential developmental stages: R1→ TCRnegCD69neg, R2→ TCRintCD69neg, R3→ TCRintCD69pos, R4→ TCRhi CD69pos, and R5→ TCRhiCD69neg. The obvious gap between R2 and R5 (see arrow) is consistent with the notion that transient CD69 expression (R3,R4) is obligatory for all thymocytes reaching R5. Fig. 1 B shows the expression of the coreceptors CD4 and CD8 in each of these populations. DP thymocytes account for most of the cells in R1 and R2 and decrease stepwise to less than 10% in R5. CD4 SP thymocytes are first observed in the TCRintCD69pos population (R3), whereas significant numbers of CD8 SP cells appear one stage later, i.e., in the TCRhiCD69pos population (R4). The ratios of CD4 SP to CD8 SP thymocytes decrease stepwise from more than 10 to ∼1.5 with ongoing maturation (Table 1). These data demonstrate that in vivo CD4 SP cells appear amongst cell populations which are at an earlier developmental stage than the populations containing CD8 SP cells.


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)

Thymic subsets defined by TCR levels and CD69  (A) and phenotypic characterization of coreceptor expression in  each subset (B). Total thymocytes were stained for TCR,  CD69, CD4 and CD8 and analyzed by four-color flow cytometry. Percentages are given in the  insets, numbers in italics in B indicate the percentages in relation  to total thymocytes. The arrow  in A indicates the relatively  empty space between R2 and  R5, suggesting that passing  through the CD69pos gates R3  and R4 is obligatory for all cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196127&req=5

Figure 1: Thymic subsets defined by TCR levels and CD69 (A) and phenotypic characterization of coreceptor expression in each subset (B). Total thymocytes were stained for TCR, CD69, CD4 and CD8 and analyzed by four-color flow cytometry. Percentages are given in the insets, numbers in italics in B indicate the percentages in relation to total thymocytes. The arrow in A indicates the relatively empty space between R2 and R5, suggesting that passing through the CD69pos gates R3 and R4 is obligatory for all cells.
Mentions: A number of experiments have suggested that, after positive selection, CD4 SP thymocytes develop more rapidly than CD8 SP thymocytes (6–12). With one exception (7) these studies employed previously manipulated DP cells or DP subpopulations placed in reaggregate thymic organ cultures or transferred intrathymically into syngeneic mice, so that an influence of experimental manipulations on developmental kinetics was not excluded. To detect recently generated SP thymocytes in vivo, we stained thymic cell suspensions for CD4, CD8, TCR, and CD69 expression. The early activation marker CD69 is expressed transiently on activated thymocytes and discriminates cells which have recently received a selection signal from pre-selection and late post-selection thymocytes. The stepwise increase in the level of TCR expression served as a marker for the maturation sequence. As shown in Fig. 1 A, this protocol defines five sequential developmental stages: R1→ TCRnegCD69neg, R2→ TCRintCD69neg, R3→ TCRintCD69pos, R4→ TCRhi CD69pos, and R5→ TCRhiCD69neg. The obvious gap between R2 and R5 (see arrow) is consistent with the notion that transient CD69 expression (R3,R4) is obligatory for all thymocytes reaching R5. Fig. 1 B shows the expression of the coreceptors CD4 and CD8 in each of these populations. DP thymocytes account for most of the cells in R1 and R2 and decrease stepwise to less than 10% in R5. CD4 SP thymocytes are first observed in the TCRintCD69pos population (R3), whereas significant numbers of CD8 SP cells appear one stage later, i.e., in the TCRhiCD69pos population (R4). The ratios of CD4 SP to CD8 SP thymocytes decrease stepwise from more than 10 to ∼1.5 with ongoing maturation (Table 1). These data demonstrate that in vivo CD4 SP cells appear amongst cell populations which are at an earlier developmental stage than the populations containing CD8 SP cells.

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