Differentiation of CD3-4-8- human fetal thymocytes in vivo: characterization of a CD3-4+8- intermediate.

Abstract

SummaryHuman thymocyte differentiation was examined by injecting fetal thymic progenitor populations into human thymic xenografts in SCID-hu mice. Thymic progenitors were fluorescently labeled with the lipophilic dye PKH2. The phenotypes of their progeny could be identified by flow cytometric analysis of cells with a very high fluorescent PKH2 signal. Intrathymic injection of purified triple negative (TN) CD3-4-8-thymocytes resulted in the sequential appearance of CD3-4+8 -, CD3-4+8 +, and CD3+4+8 + cells, with the subsequent appearance of anal1 numbers of phenotypically mature CD3 +4 + 8-and CD3 + 4-8 + ceils over a 4-d period. Sorted CD3-4+8 -thymocytes injected intrathymically rapidly differentiated to CD4+8 + cells. CD4 + 8 + fetal thymocytes in cell eycle differentiated into phenotypically mature CD3 +4+8-and CD3 +4-8 + populations, whereas nondividing CD4+8 + cells failed to differentiate after intrathymic transfer. The number of cell divisions that occurred between the injection of TN thymocytes and their progeny at different time points was estimated based on the decrease in the intensity of the PKH2 label. The average length of the cell cycle for the TN population was calculated to be 24 h. The SCID-hu model thus provides a useful tool for studying the kinetics of cell division and differentiation of human thymocytes in vivo.A major site of T lymphocyte differentiation is the thymus (for a review see reference 1). CD3-4-8-, triple negative (TN) 1 (or in some cases CD3-41~ [2]) thymic progenitors from the bone marrow (3), fetal liver, and yolk sac have been shown in mice (4, 5) and humans (6, 7) to migrate to the thymus where they undergo maturation and differentiation. In vivo studies of murine thymocytes have demonstrated differentiation of TN thymocytes to mature CD3+4+8 -and CD3 +4-8 + single positive populations via immature CD3-4-8 + followed by CD4+8 + intermediates (8, 9). The fate of most developing thymocytes is intrathymic death, either early through a failure of the TCR expressed on the cell surface to be engaged by its self-MHC, or later in maturation through a process by which self-reactive thymocytes are eliminated, termed negative selection (10-13). Before or concomitant to negative selection, CD4+8+3 l~ ceils that are specific for self-MHC undergo positive selection to develop 1Abbreviations used in this paper: APC, allophycocyanin; DP, double positive; NCS, normal calf serum; PI, propidium iodide; SA, Streptavidin; TR, Texas red.into T cells that express high levels of TCR in association with the CD4 or CD8 coreceptor, and which are capable of interacting with antigen bound to self-MHC (10, 14-16). Thymocytes that have successfully passed through positive and negative selection develop into TCR hi T calls with mature phenotypes, emigrate to the periphery, and home to peripheral lymphoid organs (17)(18)(19). The kinetics of call division and differentiation during these processes have been partially defined in murine systems (20)(21)(22)(23), and to our knowledge, have not been described...

How One Thing Led to Another

“…We later showed that the thymic outer cortex contains immature cells that differentiate into naive T cells of all T cell subsets (35,46), reaching the medulla and undergoing selection, which in our hands began after the first T cell receptor (TCR) rearrangement of β-chains and then α-chains, with positive selection preceding negative selection at distinct phases of thymic maturation (47)(48)(49)(50)(51)(52)(53)(54)(55). At each stage of maturation the TCR/CD3 complex was upregulated starting with 1 unit in the CD4 lo 8 lo ckit + IL7R + immature blasts, which if positively selected gave rise to CD4 hi 8 lo or CD4 lo 8 hi transitional intermediate (TI) cells with 3 units of TCR expression on the cell surface but went on to become the double positive (DP)-high blasts with 1 unit of TCR expression, which rearranged a second TCR α-chain for another chance at positive selection (55).…”

Thymocyte Maturation: Selection for In-Frame TCR α-Chain Rearrangement Is Followed by Selection for Shorter TCR β-Chain Complementarity-Determining Region 3

“…This division of DP thymocytes into almost equivalent numbers of CD3 neg and CD3 low is specific to man and is not found in nontransgenic laboratory mice. In man, triple-negative cells proceed to the DP CD3 neg stage through an ISP CD3 Ϫ CD4 ϩ CD8 Ϫ compartment (21). The most mature cells are SP, showing the following markers: CD3 high CD4 ϩ CD8 Ϫ and CD3 high CD4 Ϫ CD8 ϩ (reviewed in Ref.…”

Detection of a CD4+CD8−CD3− cell subpopulation during the differentiation of cord blood CD34+ cells into T cells in vitro

“…Our (Kraft et al 1993) proposed, the CD4 + CD8 -CD3 -population is a sequential intermediate between CD3 -CD4 -CD8 -and CD4 + CD8 + CD3 + . Our result support Kraft's proposal as we observed that the proportion of the CD4 + CD8 -CD3 -subset decreased with time while the proportions of the CD3 + CD4 + or CD3 + CD8 + subsets increased.…”

“…Kraft et al (Kraft et al 1993) described this CD4 + CD8 -CD3 -cell population, which represents an alternative intrathymic T-cell precursor, as a monocytic population. Gardner et al (Gardner et al 1998) also found that after two weeks of culture on a thymic monolayer, donor-derived cells maintained the classic phenotypes of cortical thymocytes (CD4 + CD8 -CD3 -and CD4 + CD8 + ) in proportions similar to those of fresh thymic samples.…”

“…The fate of most developing thymocytes is intrathymic death, either early through a failure of the surface T-cell receptor to be engaged by self MHC (positive selection) or later in a maturation process by which self-reactive thymocytes are eliminated (negative selection). Only 1-2% of total thymocytes are exported to the periphery after undergoing positive and negative selection (Deftos et al 2000;Kappler et al 1987;Kraft et al 1993;Su and Manley 2002;Swat et al 1992). CD3 -CD4 -CD8 -thymocytes result in the sequential appearance of CD3 -CD4 + CD8 -and CD3 + CD4 + CD8 + cells, and immature CD3 -CD4 + single-positive cells are thought to be mainly located in the outer cortex and to be blastic and cortisone sensitive (Guidos et al 1990).…”