Early T cell development

The majority of work on T cell development has used the murine system as a model and consequently there is not a great wealth of information about human T cell development. However, the two pathways are believed to be similar and therefore the following description will concentrate on murine T cell development.

All mature blood cell lineages are ultimately derived from haemopoietic stem cells (HSCs). HSCs have multilineage potential and are capable of self renewal such that they can long term reconstitute bone marrow depleted by irradiation following engraftment and protect lethally irradiated mice from bone marrow failure. HSCs continuously give rise to lineage restricted precursors, whose progeny are finally committed to a single cell lineage (Rodewald, 1995).

The thymus is continuously seeded by precursor cells from the bone marrow however the degree of commitment of these precursor cells is not clear. A rare population has been identified in adult murine thymus which is characterised by expression of low levels of CD4 which has yet to initiate TCR-P rearrangement (Wu et a l, 1991b). These CD4'° cells are capable of reconstituting the T and B lymphoid components in irradiated mice (Wu et al, 1991a). Likewise a ckit^ intrathymic stem cell with reconstitution potential has been identified (Matsuzaki et al, 1993). These

observations have lead to the suggestion that this CD4^° ckit^ population may represent a committed lymphoid stem cell.

Early murine intrathymic T cell development can be considered to include 5 control points, to be discussed further below. Figure 1.6 shows a schematic representation of this pathway (Godfrey and Zlotnik, 1993). Early thymocytes have for some time been identified as double negative for CD4 and CD8 (DN). However this DN population has also been found to include some mature CD3^ cells (Fowlkes and Pardoll, 1989). Therefore early thymocytes are better defined as CD4’ CD8’ CD3‘ or triple negative (TN). The development of these TN cells can be followed by the expression of two markers: CD44 and CD25 (IL-2Ra) (Boyd and Hugo, 1991; Nikolic Zugic, 1991; Rothenberg, 1992; Shortman, 1992). The first step in this development is the induction of CD25 such that the thymocytes mature from CD44^CD25'TN to CD44 CD25 TN. This step may correspond to the first control point, that is T cell lineage commitment, since CD25^ TN cells cannot give rise to dendritic cells after in vivo transfer (Ardavin

et al, 1993) and there is no evidence for a CD25^ B cell precursor (Ehlich et a l,

1993).

The second control point involves initiation of TCR-p and TCR-y gene rearrangement and is accompanied by the transition from CD44^CD25 TN to the CD44 CD25 TN stage. This is supported by the finding that CD44^CD25^TN cells still have their TCR- P and TCR-y genes in their germline configuration whilst CD44 CD25 TN cells are capable of producing full length TCR-p transcripts (Godfrey et al, 1993).

The third control point involves the branching of the developmental pathway to

Figure 1.6: Schematic Representation of Murine T Cell Development

BONE MARROW

TN

TN

"p-Selection" .5. CD4 & CD8 Expression TCR-a Rearrangement & Expression CD44 CD25 c-kif

► DP

PERIPHERAL BLOOD

Numbered arrows indicate the 5 control points referred to in section 1.6.2. TN refers to CD4 CD8 CD3', triple negative thymocytes.

"TN" refers to the possibility that these cells may express low levels of CD3/TCR-P. DP refers to CD4 CD8\ double positive cells, ap/yô refers to T cell receptor type.

yô T cells usually have incompletely rearranged TCR-P genes (Godfrey et a l, 1993) this places the branch point at the CD44 CD25 TN stage.

Early selection for cells expressing a functional TCR-p chain constitutes the fourth control point. The role of TCR-p in early thymocyte development became clear from experiments where a TCR-p transgene was expressed in SCID or RAG deficient mice in which thymocyte development is arrested at the CD44 CD25 TN stage. Introduction of a TCR-p transgene allows progression from the CD44 CD25 TN to CD44 CD25' CD4 CD8^ or double positive (DP) stage (reviewed in Groettrup and von Boehmer, 1993). By contrast, since TCR-a gene rearrangement occurs after that of the TCR-p gene, introduction of a TCR-a transgene fails to prevent the arrest at the CD44" CD25^TN stage (Shinkai et al, 1993). This early stage TCR-P chain selection and allelic exclusion requires expression of the TCR-P protein. This was established by the study of TCR-P transgenic mice which show inhibited V-D-J rearrangement only when the transgene could express a functional protein (Krimpenfort et a l, 1989). However, it has been shown in several murine and human T cell lines that expression of the TCR-P chain is strictly dependent on expression of the TCR-a chain (Saito et al, 1987; Koning et a l, 1988; Letoumeur and Malissen, 1989). This paradox was resolved by the discovery of a 33kDa protein (gp33) which forms a disulphide linked heterodimer with TCRp on pre-T cells (Groettrup et al, 1993). This dimer is strongly

associated with CD3e and 5 such that on crosslinking of either TCRp or CD3e rapid

calcium mobilisation results. This indicates the existence of a signal transducing TCRP-protein complex on the surface of pre-T cells, in the absence of TCR-a, the

stimulation of which can block further TCR-P gene rearrangement and initiate TCR-a gene rearrangement (Groettrup and von Boehmer, 1993).

The final control point in the development of a bone marrow stem cell to a DP thymocyte involves the induction of CD4 and CD8, and TCR-a gene rearrangement and expression. CD25 expression is down regulated and CD4 and CD8 are expressed at low levels such that the thymocyte has the phenotype CD44*CD25’CD4*°CD8'°. In addition the TCR-a genes are now rearranged and expressed. This step must be

controlled by signals from the thymic microenvironment since isolated CD25 TN cells are unable to differentiate to the DP stage in vitro (Petrie et al, 1990; Suda and

Zlotnik, 1991). Some of these signals may be delivered via the TCR-p chain however TCRa expression can occur in the absence of TCR-P (Mombaerts et a l, 1992a) and CD4 and CD8 can be induced on TCR-P" SCID mouse thymocytes (Shores et al,

1990; Iwashima et a l, 1991). Therefore other factors such as cytokines and cell surface molecules are likely to be involved.