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Quantitative impact of thymic clonal deletion on the T cell repertoire.

van Meerwijk JP, Marguerat S, Lees RK, Germain RN, Fowlkes BJ, MacDonald HR - J. Exp. Med. (1997)

Bottom Line: Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus.The quantitative impact of negative selection on the potentially available repertoire is currently unknown.To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of Lausanne, Epalinges, Switzerland.

ABSTRACT
Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.

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Increase in CD4SP thymocytes in MHC II°→ MHC I° chimeras is not due to (A) proliferation or (B) recirculation of peripheral T  lymphocytes. (A) Cell cycle analysis (PI incorporation) was performed on  ethanol-fixed total thymocytes and electronically sorted CD4+CD8−  TCRhigh cells (purity ⩾95%). Representative results are shown. The statistics represent mean percentage cells in S+G2/M phase ± SD from the  indicated number of experiments. (B) Four-color flow cytometry was  performed using anti-CD4, anti-CD8, and anti-TCR antibodies combined with anti-CD44, anti-HSA, or anti-CD69. The CD44, HSA, and  CD69 histograms are of electronically gated CD4+CD8−TCRhigh cells.  Representative results are shown. The statistics represent mean percentage ± SD from the indicated number of experiments.
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Figure 3: Increase in CD4SP thymocytes in MHC II°→ MHC I° chimeras is not due to (A) proliferation or (B) recirculation of peripheral T lymphocytes. (A) Cell cycle analysis (PI incorporation) was performed on ethanol-fixed total thymocytes and electronically sorted CD4+CD8− TCRhigh cells (purity ⩾95%). Representative results are shown. The statistics represent mean percentage cells in S+G2/M phase ± SD from the indicated number of experiments. (B) Four-color flow cytometry was performed using anti-CD4, anti-CD8, and anti-TCR antibodies combined with anti-CD44, anti-HSA, or anti-CD69. The CD44, HSA, and CD69 histograms are of electronically gated CD4+CD8−TCRhigh cells. Representative results are shown. The statistics represent mean percentage ± SD from the indicated number of experiments.

Mentions: Several explanations other than the lack of negative selection could account for the increased number of CD4SP cells in MHC II°→ MHC I° chimeras. However, proliferation of mature CD4SP does not account for the increase as cell cycle analysis reveals no augmentation in the number of CD4SP cells in S+G2/M phase (Fig. 3 A). A change in their maturation/export rate could result in an increased retention of CD4SP cells in the thymus. CD69 is a molecule transiently expressed during thymocyte development: in vivo, it is first expressed on CD4+CD8+ cells that have undergone MHC-mediated activation and is downmodulated only when the thymocytes have already reached the SP stage (39–41, 29). The normal ratio of mature thymocytes from the chimeras expressing high versus low levels of CD69 therefore is an indication of normal thymic maturation (Fig. 3 B). Expression of HSA on CD4SP thymocytes is also downmodulated during their final maturation (42, 43). Again, the normal ratio of HSAhigh versus HSAlow CD4SP cells in the chimeras indicates normal maturation. Taken together, the data on the expression of CD69 and HSA suggest normal thymocyte development and render the possibility of prolonged retention of CD4SP cells in the thymus unlikely. Another possibility would be the recirculation into the thymus of mature peripheral T cells (44–46). Peripheral T lymphocytes do not express significant levels of HSA (42, 43) and the lack of an increased percentage of HSA− CD4SP thymocytes (Fig. 3 B) thus is inconsistent with a recirculation model. Reentry into the adult thymus of peripheral T cells is known to be restricted to activated cells (44–46). These cells may be expected to express high levels of the memory/activation marker CD44 (47). No evidence for an increase in CD44high CD4SP thymocytes was found in the chimeras (Fig. 3 B). Collectively, these data strongly suggest that the increased number of CD4SP cells in chimeras lacking MHC class II on hematopoietic elements is not due to proliferation, to increased retention of thymocytes in the thymus, or to recirculation of peripheral T lymphocytes.


Quantitative impact of thymic clonal deletion on the T cell repertoire.

van Meerwijk JP, Marguerat S, Lees RK, Germain RN, Fowlkes BJ, MacDonald HR - J. Exp. Med. (1997)

Increase in CD4SP thymocytes in MHC II°→ MHC I° chimeras is not due to (A) proliferation or (B) recirculation of peripheral T  lymphocytes. (A) Cell cycle analysis (PI incorporation) was performed on  ethanol-fixed total thymocytes and electronically sorted CD4+CD8−  TCRhigh cells (purity ⩾95%). Representative results are shown. The statistics represent mean percentage cells in S+G2/M phase ± SD from the  indicated number of experiments. (B) Four-color flow cytometry was  performed using anti-CD4, anti-CD8, and anti-TCR antibodies combined with anti-CD44, anti-HSA, or anti-CD69. The CD44, HSA, and  CD69 histograms are of electronically gated CD4+CD8−TCRhigh cells.  Representative results are shown. The statistics represent mean percentage ± SD from the indicated number of experiments.
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Related In: Results  -  Collection

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Figure 3: Increase in CD4SP thymocytes in MHC II°→ MHC I° chimeras is not due to (A) proliferation or (B) recirculation of peripheral T lymphocytes. (A) Cell cycle analysis (PI incorporation) was performed on ethanol-fixed total thymocytes and electronically sorted CD4+CD8− TCRhigh cells (purity ⩾95%). Representative results are shown. The statistics represent mean percentage cells in S+G2/M phase ± SD from the indicated number of experiments. (B) Four-color flow cytometry was performed using anti-CD4, anti-CD8, and anti-TCR antibodies combined with anti-CD44, anti-HSA, or anti-CD69. The CD44, HSA, and CD69 histograms are of electronically gated CD4+CD8−TCRhigh cells. Representative results are shown. The statistics represent mean percentage ± SD from the indicated number of experiments.
Mentions: Several explanations other than the lack of negative selection could account for the increased number of CD4SP cells in MHC II°→ MHC I° chimeras. However, proliferation of mature CD4SP does not account for the increase as cell cycle analysis reveals no augmentation in the number of CD4SP cells in S+G2/M phase (Fig. 3 A). A change in their maturation/export rate could result in an increased retention of CD4SP cells in the thymus. CD69 is a molecule transiently expressed during thymocyte development: in vivo, it is first expressed on CD4+CD8+ cells that have undergone MHC-mediated activation and is downmodulated only when the thymocytes have already reached the SP stage (39–41, 29). The normal ratio of mature thymocytes from the chimeras expressing high versus low levels of CD69 therefore is an indication of normal thymic maturation (Fig. 3 B). Expression of HSA on CD4SP thymocytes is also downmodulated during their final maturation (42, 43). Again, the normal ratio of HSAhigh versus HSAlow CD4SP cells in the chimeras indicates normal maturation. Taken together, the data on the expression of CD69 and HSA suggest normal thymocyte development and render the possibility of prolonged retention of CD4SP cells in the thymus unlikely. Another possibility would be the recirculation into the thymus of mature peripheral T cells (44–46). Peripheral T lymphocytes do not express significant levels of HSA (42, 43) and the lack of an increased percentage of HSA− CD4SP thymocytes (Fig. 3 B) thus is inconsistent with a recirculation model. Reentry into the adult thymus of peripheral T cells is known to be restricted to activated cells (44–46). These cells may be expected to express high levels of the memory/activation marker CD44 (47). No evidence for an increase in CD44high CD4SP thymocytes was found in the chimeras (Fig. 3 B). Collectively, these data strongly suggest that the increased number of CD4SP cells in chimeras lacking MHC class II on hematopoietic elements is not due to proliferation, to increased retention of thymocytes in the thymus, or to recirculation of peripheral T lymphocytes.

Bottom Line: Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus.The quantitative impact of negative selection on the potentially available repertoire is currently unknown.To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of Lausanne, Epalinges, Switzerland.

ABSTRACT
Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.

Show MeSH
Related in: MedlinePlus