Apoptosis among CD45RA^−/low CD3⁺ Progeny Accompanies Differentiation of Human Multinegative Thymocytes

Abstract

Apoptotic cell death is widely believed to be the fate of negatively selected cells in the thymus. In this work we have used multiparameter flow cytometry to analyse reductions in DNA content that occur among differentiating human CD3-4-8- multinegative (MN) thymocytes as they acquire CD3/TCR during in vitro culture. DNA content was measured as the intensity of the DNA-binding dye DAPI. The position of the diploid peak was identified by comparison to chicken red blood cells and to unfractionated uncultured thymocytes which have a sharply defined diploid peak. Apoptosis, was defined as a reduction in DNA content. Apoptotic cell death occurred continuously throughout the 7 day culture period and at the latest stages of culture DNA fragmentation was apparent on gels. Although both CD3- and CD3+ progeny became apoptotic, it was more frequent among the CD3+ progeny of the MN thymocytes. Apoptotic progeny included 60-70% CD3+ cells, while progeny remaining diploid were 8-36% CD3+. Fifty five per cent of CD3+ TCR delta 1+ progeny had less than 75% of the diploid DNA content, while for CD3+ TCR delta 1- progeny only 28% were in this category. CD3+ TCR delta 1+ progeny also comprised 66% of the cycling cells at days 6-7 of culture, suggesting a pattern of rapid cell division followed by apoptotic cell death for this subset. A lack of positive selection in culture may trigger apoptosis among the TCR delta 1 progeny. In contrast, TCR alpha beta progeny arising in culture appear to be less susceptible to apoptosis, perhaps due to their lack of CD4 and CD8. The expression of CD45RA and CD45R0 isoforms were assessed on the progeny of MN thymocytes based on their DNA content. Although 30% of apoptotic progeny expressed CD45RA, it was present at relatively low density compared to that on diploid or cycling cells, 55% of which were CD45RAhi. The majority of apoptotic cells expressed neither CD45RA or CD45R0, but were CD45+, indicating the presence of an isoform not detected by our monoclonal antibodies (MoAbs). This is consistent with speculations that apoptotic cell death among thymocytes is preceded by a transition in CD45 isoform expression. These conditions may model early selective events resulting from high avidity TCR engagement that is independent of CD4 and/or CD8.

Beta1‐Integrins Control Spontaneous Adhesion and Motility of Human Progenitor Thymocytes and Regulate Differentiation‐Dependent Expression of the Receptor for Hyaluronan‐Mediated Motility

“…In vitro, MN Thy spontaneously differentiate to express RHAMM and CD3/T-cell receptor (TCR) by days 2-3 of culture [28,30,39]. To determine whether this is accompanied by the initiation of RHAMM-dependent motility, MN Thy were cultured for 2 days, harvested and analysed by time 1 49 Ϯ 16*** 40 Anti-␣ 4 28 Ϯ 36** Anti-␣ 5 23 Ϯ 15*** Non-blocking anti-␣ 4 180 Anti-RHAMM 150…”

B220 is Expressed on Apoptotic Thymocytes Induced by X-Irradiation.

“…Detection of DNA fragmentation on agarose gels: Analysis of DNA fragmentation was performed as described by Pilarski et al [12] with a slight modification. Thymocytes (1 × 10 6 ) were lysed in 20 µl of 10 mM EDTA, 50 mM Tris-HCl (pH 8.0) containing 0.5% (w/v) sodium-N-lauroylsarcosinate.…”

“…Irradiation: Female 6-week-old mice were exposed to 6.8 Gy of whole-body X-irradiation at a dose rate of 0.46Gy/ min with X-ray irradiator (Radioflex-350; Rigaku Denki Co., Ltd., Tokyo, Japan) operated at 250 kV and 12.5 mA with of 0.3 mm Cu and 0.5 mm Al filtration. Mice were subsequently killed at 0 (control), 3,6,9,12,18, 24, 36, 48, 72 and 168 hr after the irradiation, and then thymi were excised and weighted. Thymocyte suspensions were prepared by gently teasing the thymus with forceps in…”

“…Mice were subsequently killed at 0 (control), 3,6,9,12,18,24,36,48,72 and 168 hr after the irradiation, and then thymi were excised and weighted. Thymocyte suspensions were prepared by gently teasing the thymus with forceps in Hank's balanced salt solution containing 1% fetal bovine serum (HBSS-FBS).…”