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. 2001 Apr 24;98(9):5158-63.
doi: 10.1073/pnas.091100398. Epub 2001 Apr 17.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes

A K Simon  1 , O Williams, J Mongkolsapaya, B Jin, X N Xu, H Walczak, G R Screaton
Affiliations

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes

A K Simon et al. Proc Natl Acad Sci U S A. .

Abstract

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

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Figures

Figure 1
Figure 1
Expression of TRAIL-R1 and -R2 and sensitivity to TRAIL of Jurkat and BJAB. Cells were stained with mAbs against TRAIL-R1 (solid line) or TRAIL-R2 (dashed line). Sensitivity to cross-linked TRAIL and polyclonal antisera to TRAIL-R1 and -R2 was tested with the use of a chromium release assay. Killing was shown to be specific by blocking with 20 μg/ml TRAIL-R2-Fc where indicated. Error bars were calculated from triplicates, this experiment was repeated at least three times.
Figure 2
Figure 2
Human thymic organ culture. Human thymocyes were cultured in the presence or absence of anti-CD3 for 18 h. (A) Staining with anti-CD4 and anti-CD8. (B) Analysis of apoptosis by annexin V staining after electronic gating on CD4+CD8+, CD8+, or CD4+ subpopulations of anti-CD3 (500 ng/ml) stimulated thymocytes (gray line) or unstimulated thymocytes (filled histograms). These results are representative of at least five different experiments.
Figure 3
Figure 3
Sensitivity to TRAIL. Total human thymocytes or peripheral blood mononuclear cells were cultured for 18 h with polyclonal TRAIL-R1 or -R2 antiserum, cross-linked TRAIL, dexamethasone, preimmune serum (preimmune), or anti-flag in the presence or absence of 100 ng/ml anti-CD3 and stained with annexin V. (A) Human peripheral lymphocytes. (B and C) Human thymocytes cultured in suspension. (D–G) Human thymus organ cultures.
Figure 4
Figure 4
Human thymocytes express TRAIL-R1 and -R2 upon activation. Thymocytes from organ cultures were cultured in the absence or presence of 100 ng/ml anti-CD3 for 18 h and then stained with mAbs to TRAIL-R1 or -R2 (gray line) or with irrelevant antibody (filled histogram). This is a representative of three separate experiments.
Figure 5
Figure 5
Effects of TRAIL inhibition on negative selection in HTOC. (A) Human thymocytes were cultured in organs for 18 h in the presence of dexamethasone (dex) or a lethal dose of anti-CD3, with or without 20 μg/ml of TRAIL-R2-Fc. Double-positive thymocytes stained positive with annexin V were considered apoptotic. This experiment was repeated at least five times with similar results. (B) HTOC was performed in the presence or absence of superantigen enterotoxin B and/or TRAIL-R2-Fc. After 24 h thymocytes were stained for Vβ2 (not shown) and Vβ17. In a further 24-h culture period, superantigen enterotoxin B was omitted to reveal potential TCR surface expression in the absence of antigen (Right column). Histogram plots are gated on CD4+CD8− thymocytes. This experiment was performed in triplicate with standard deviations of 0.2–0.4%.
Figure 6
Figure 6
Analysis of negative selection in the murine F5 model. (C) The same experiment was performed in FTOC of F5 Rag−/−, and results were analyzed after 24 h. The loss of CD4+CD8+ thymocytes is indicated as a percentage. (B) The viability of these cells was also assessed by 7-amino antinomycin D; the percentage of dead cells is indicated. (A) F5 Rag-1−/− Tap-1−/− mice were coinjected with NP68 and TRAIL-R2-Fc where indicated and killed after 24 h, and thymocytes were analyzed for CD4 and CD8.
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