Quantitative impact of thymic clonal deletion on the T cell repertoire - PubMed (original) (raw)

Quantitative impact of thymic clonal deletion on the T cell repertoire

J P van Meerwijk et al. J Exp Med. 1997.

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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Figures

Figure 1

Figure 1

Positive selection depends on expression of MHC molecules on radioresistant thymic epithelial cells. (A) Thymi from wild-type→ wild-type, MHC I°II°→ I°II°, and wild-type→ I°II° chimeras were analyzed by flow cytometry 6 wk after grafting. Thymocytes were stained with anti-CD8, anti-TCR, and anti-CD4 antibodies and analyzed on a FACScan® using LYSYS II software. Contour plots are 75% logarithmic and TCR histograms are from total thymus and from electronically gated cells using the gates indicated in the figure. (B) Percentages of CD4SP (CD4+CD8− TCRhigh) and CD8SP cells (CD4−CD8+TCRhigh) in thymi from the indicated chimeras were determined and depicted as percentage of an age- and sex-matched nonchimeric control mouse ± SD.

Figure 1

Figure 1

Positive selection depends on expression of MHC molecules on radioresistant thymic epithelial cells. (A) Thymi from wild-type→ wild-type, MHC I°II°→ I°II°, and wild-type→ I°II° chimeras were analyzed by flow cytometry 6 wk after grafting. Thymocytes were stained with anti-CD8, anti-TCR, and anti-CD4 antibodies and analyzed on a FACScan® using LYSYS II software. Contour plots are 75% logarithmic and TCR histograms are from total thymus and from electronically gated cells using the gates indicated in the figure. (B) Percentages of CD4SP (CD4+CD8− TCRhigh) and CD8SP cells (CD4−CD8+TCRhigh) in thymi from the indicated chimeras were determined and depicted as percentage of an age- and sex-matched nonchimeric control mouse ± SD.

Figure 2

Figure 2

Increased CD4SP (CD4+CD8−TCRhigh) thymocytes in chimeras lacking MHC class II expression on hematopoietic elements. Groups of sex- and age-matched chimeras were analyzed on the same day 6–8 wk after engraftment. Flow cytometry was performed using antiTCR, anti-CD4, and anti-CD8 antibodies. In each experiment, the ratio of CD4SP cells in the indicated groups was calculated. Error bars indicate SD. The increased ratio of CD4SP cells in MHC II°→ MHC I° versus MHC II+→ MHC I° chimeras is statistically significant as assessed by the Student's t test (P <0.0001), whereas the ratio of CD4SP thymocytes in MHC I°→ MHC I° versus MHC I+→ MHC I° chimeras is not significantly increased (P = 0.02).

Figure 4

Figure 4

Accelerated kinetics of generation of CD4SP and CD8SP thymocytes in wild-type hosts lacking MHC class II or I, respectively, on hematopoietic elements. 6-wk-old wt→ wt (n = 3, day 9; n = 6, days 10– 13), MHC I°II°→ wt (n = 3 each day), and MHC I°→ wt (n = 3) chimeras were sublethally irradiated (720 rads) and analyzed on day 9–13 as in Fig. 2. Data represent average percentage of CD4+CD8−TCRhigh and CD4−CD8+TCRhigh thymocytes ± SD.

Figure 3

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.

Figure 3

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.

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