The Fas antigen is involved in thymic T-cell development as a costimulatory molecule, but not in the deletion of neglected thymocytes Kazuhiro Kurasawa,

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The Fas antigen is involved in thymic T-cell development as a costimulatory molecule, but not in the deletion of neglected thymocytes Kazuhiro Kurasawa, MD, Yoshiko Hashimoto, MD, Masaaki Kasai, MD, Itsuo Iwamoto, MD, PhD Journal of Allergy and Clinical Immunology Volume 106, Issue 1, Pages S19-S31 (July 2000) DOI: 10.1067/mai.2000.106773

Fig. 1 . The generation of 4 genotypes of DO10 lpr mice. Four genotypes of F2 mice bearing DO10 transgenic TCR (DO10 lpr mice; [1] H-2d/k, lpr/+, [2] H-2d/k, lpr/lpr, [3] H-2k/k, lpr/+, and [4] H-2k/k, lpr/lpr) were generated by crossing F1 mice (DO10 TCR transgenic mice × MRL-lpr/lpr mice) bearing DO10 transgenic TCR with MRL-lpr/lpr mice. DO10 lpr mice were analyzed for DO10 transgenic TCR expression, lpr genotype, Fas expression, and H-2 haplotype. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 2 . The cell numbers of thymocytes and splenocytes in the 4 genotypes of DO10 lpr mice. The cell numbers of thymocytes and splenocytes from the 4 genotypes of DO10 lpr mice (8-10 weeks old) were counted. Data are means ± SEM for 14 mice in each group. Thymus: *P < .001, significantly different from the mean value of H-2d/k lpr/+ or H-2k/k lpr/+ mice. Spleen: *P < .05, significantly different from the mean value of H-2d/k lpr/+ mice. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 3 . Flow cytometry analysis (A ) and cell numbers (B ) of thymocyte subpopulations in the 4 genotypes of DO10 lpr mice. Thymocyte subpopulations in the 4 genotypes of DO10 lpr mice were analyzed by flow cytometry with anti–CD4-FITC and anti–CD8-PE mAb. Data are means ± SEM for 14 mice in each group. Double-positive (DP ) thymocytes: *P < .001, significantly different from the mean value of H-2d/k lpr/+ or H-2k/k lpr/+ mice. Double-negative (DN ) thymocytes: *P < .01 and **P < .001, significantly different from the mean value of H-2d/k lpr/+ and H-2k/k lpr/+ mice, respectively. CD4+ single-positive (SP ) thymocytes: *P < .05 and **P < .001, significantly different from the mean value of H-2d/k lpr/+ and H-2k/k lpr/+ mice, respectively; +P < .02, significantly different from the mean value of H-2d/k lpr/+ mice. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 4 . TCR expression on thymocyte subpopulations in the 4 genotypes of DO10 lpr mice. Thymocytes from the 4 genotypes of DO10 lpr mice were stained with biotinylated KJ1-26 (A ; DO10 transgenic TCR clonotype mAb) or biotinylated anti-TCR-αβ (B ), anti–CD4-FITC, anti–CD8-PE, and streptoavidin-Tricolor, and cells were analyzed by flow cytometry. This Figure represents 8 separate experiments. DN , Double-negative; DP , double-positive; SP , single-positive. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 5 . CD5 and CD69 expressions on CD4+CD8+ double-positive thymocytes in the 4 genotypes of DO10 lpr mice. Thymocytes from the 4 genotypes of DO10 lpr mice were stained with biotinylated anti-CD5 or biotin-ylated anti-CD69, anti–CD4-FITC, anti–CD8-PE, and streptoavidin-Tricolor; and cells were analyzed by flow cytometry. This figure represents 5 separate experiments. The mean fluorescence intensities of CD5 expression on double-positive thymocytes were 821, 513, 187, and 82 in the mice with H-2d/k lpr/+, H-2d/k lpr/lpr, H-2k/k lpr/+, and H-2k/k lpr/lpr, respectively. CD69high populations were 12.11%, 4.55%, 3.95%, and 2.48% of double-positive thymocytes in those mice, respectively. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 6 . Histologic examination of thymus from DO10 lpr mice. Hematoxylin-eosin sections of thymus from H-2k/k lpr/+ (A ) and H-2k/k lpr/lpr (B ) mice are depicted. (Original magnification, ×40.) Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 7 . DO10 transgenic TCR expression on CD4+ and CD8+ splenocytes in the 4 genotypes of DO10 lpr mice. Splenocytes from the 4 genotypes of DO10 lpr mice were stained with biotinylated KJ1-26, anti–CD4-FITC, anti–CD8-PE, and streptoavidin-Tricolor; and the cells were analyzed by flow cytometry to examine the expression levels of DO10 transgenic TCR on CD4+ and CD8+ splenocytes (A ). (B ). The cell numbers of KJ1-26–positive CD4+ and CD8+ splenocytes in the 4 genotypes of DO10 lpr mice. Data are means ± SEM for 8 mice in each group. *P < .05, significantly different from the mean value of H-2d/k lpr/+ mice. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)

Fig. 8 . Proliferative responses of splenocytes to OVA 323-339 in the 4 genotypes of DO10 lpr mice. Splenocytes (1 × 105) from the 4 genotypes of DO10 lpr mice were cultured without (open columns ) or with OVA 323-339 (1 mmol/L; solid columns ) at 37°C for 72 hours in the absence (A ) or presence (B ) of 1 × 105 irradiated BALB/c (H-2d) splenocytes as antigen-presenting cells. Cells were pulsed with 3H-thymidine for the last 16 hours, harvested, and 3H incorporation was counted. This figure represents 3 separate experiments. Journal of Allergy and Clinical Immunology 2000 106, S19-S31DOI: (10.1067/mai.2000.106773)