Definition of developmental stages

In the mouse, the embryonic thymus is seeded by stem cells from the fetal liver around days 11-12 of gestation, while the adult thymus is repopulated by stem cells from the bone marrow. The human thymus is repopulated by stem cells from fetal liver around 8 weeks of gestation, while the bone marrow cells can function as a source of stem cells from 16 weeks of gestation onwards. Mature functional T cells can be identified in the developing mouse thymus around the day of birth (days 19-20 of gestation), and in the human thymus around 20 weeks of gestation. Although it is clear that the developmental pathways of fetal and adult lymphopoiesis are not identical, they follow by and large similar successive steps. The cell surface markers currently available allow, together with a few other essential features (Table 1), an overall distinction into two stages during embryonic thymocyte development: 1) an early pro- and pre-T cells stage during which commitment to T cell development is imposed, and 2) a later phase during which committed precursors further expand and differentiate.

Early phase

This major expansion phase, during which all thymocytes express a CD3 CD4 CD8 (triple negative, TN) phenotype, marks the time that progenitors gradually lose their multipotent developmental potential, and become irreversibly committed to T cell development. The TN thymocyte subset is not homogeneous, as further subdivisions can be made on the block in mice block in deficient in mutants

pTa CD3 Ick block in mice block in deficient in mutants

pTa CD3 Ick

Figure 2 Scheme of T cell differentiation, summarizing the phases discussed in this article, and indicating the block in development observed in mice carrying the indicated mutations.

basis of size and expression of the CD44 glycoprotein, and the receptors for stem cell factor (CD117) and interleukin 2 (IL-2, CD25 (in the mouse). Such cytokine receptors represent a crucial role for these types of growth factors in early lymphoid development.

In the human thymus, the markers CD34, CD38 and CD1 mark distinct progenitor populations within the TN compartment. Importantly, the earliest progenitors in the TN population are not yet exclusively committed to the T cell lineage, and progenitors that do not express CD25 (for the mousei or GDI (for the human) can still develop into a special subset of dendritic cells, and into natural killer (NK) and B cells. At the same time, these progenitors appear to have lost the ability to develop into the myeloid or erythroid lineages. For the mouse, irrc versible commitment to T cell lineage development occurs sometime during the CD25 stage and coincides with initiation of TCR (3 chain gene rearrangements: CD25+ cells that express CD44 still have the TCR 3 chain genes in germline configuration and can still become dendritic cells, while loss of CD44 coincides with appearance of extensive rearrangements. In the human, it appears to be the CD34CD1 ' stage that marks the committed T cell precursors, while a stage corresponding to the uncommitted CD25" lymphoid progenitors in the mouse, with potential for development into dendritic cells, B cells and NK cells, is represented by the CD34+CD1 population. What is important to realize in the type of precursor-progeny studies that have led to these nominations of developmental stages (Table 1), is that they have yet to be performed at the clonal level or with clonal markers. It is still possible that the studies that have led to the concept of a multipotential lymphoid progenitor for B, NK, T and dendritic cells, are in reality due to the circumstance that the cell populations used contain a mixture of committed precursors for dendritic, B, NK and T cells. Introduction of suitable marker genes in early progenitors should help resolve this issue in future studies.

The most pertinent feature of the early FN period of thymocyte development is, from the point of T cell commitment, the beginning of rearrangements and transcription of TCR gene loci. It is not clear, however, whether these rearrangements themselves determine T cell commitment, or whether they are linked to some yet to be defined control mechanism. When rearrangements are prevented, such as in mice lacking the recombinase-activating genes (RAG), T cell development is arrested at the early pre-T cell stage (Table 1). TCR gene loci are in germline configuration among the earliest progenitors, while the proportion of cells undergoing y, 8 and (3 chain rearrangements can be seen to progressively increase following RAG gene expression. Thymocytes with rearrangements in one TCR locus tend to have rearrangements in the other loci, such that a given cell apparently undergoes synchronous rearrangements of all TCRT loci. Yet, rearrangements do not occur simultaneously for all TCR loci: completion of certain defined V-D-J and V-J rearrangements in the 8 and y loci, respectively, is achieved earlier than that in the TCR (3 loci, while the (3 chain rearrangements occur later, and V-J a chain rearrangements are not even initiated until TCR 3 chain rearrangements have been completed. This ordered appearance of rearrangements is also reflected at the level of transcription: full-length y and 8 gene mRNA can be detected first, followed by 3 gene transcripts, and a chain mRNA coming last. Interestingly, preferential usage of particular V^ and VB segments has been observed during early and late days of fetal ontogeny, fuelling suggestions that specific yS cells exhibit defined functions in early T cell differentiation.

The complete TCR 3 chain forms a heterodimeric pre-TCR complex with a nonpolymorphic glycoprotein termed 'pre-Ta' (see Figure 1), which becomes expressed at the cell surface and employs the CD3 chains for signal transduction. While the precise order of appearance of the different components of the CD3 complex (consisting of e, 8, and y chains, and CC dimers; see Figure 1) is not known, it is clear that they are available for formation of the pre-TCR complex as soon as the TCR 3 chain and the pre-Ta chain are expressed. There is some debate as to which CD3 components are expressed in the pre-TCR as compared to the mature TCR (Figure 1), but CD3£, CD3y and CD3e have been positively identified in the pre-TCR. It remains to be established whether the different CD3 chains have unique or overlapping functions in T cell development, and other nonpolymorphic proteins associated with the pre-TCR are being identified.

In the adult thymus, a TN subset can also be found; it constitutes a minor (<5%) portion of all thymocyte subsets, and precursors of all T lineage cells (both CD4^CD8+, double positive (DP), and CD4"CD8" or CD4 CD8+, single positive (SP) cells) can be found among the TN cells of both fetal and adult origin. Such TN cells have, upon i.v. transfer into irradiated recipients, the capacity to home back to the thymus where they differentiate in a single wave, indicating their limited self-renewal capacity. Both homing and precursor activity are contained within a subset of adult TN cells with a CD44+CD25 phenotype, and these adult TN cells, like their fetal counterparts, pass through a CD25 stage before they lose CD25 and CD44 and give rise to DP cells. Again, this stage marks the loss of ability to generate NK and dendritic cells, and true commitment to T cell development.

Later phase

Completion of TCRa gene rearrangements, appearance of mature T cell surface proteins such as CI)4 and CD8 at the cell surface, and upregulation of the TCR-CD3 complex at the cell surface mark the subsequent phase of thymocyte development. a3 T cell precursors in fact do not enter this stage if they have not completed their TCR 3 gene rearrangements. Small numbers of thymocytes begin to express CDS first (the so-called 'immature single positive stage', ISP in Table 1), and this is followed by appearance of CD4 expression, such that after both relative and absolute increases, the majority of thymocytes exhibit the double positive or DP (CD4 ' CDS ) phenotype. Early DP thymocytes express only low levels of the TCR-CD3 complex, but this gradually increases. The proportion of DP T cells expressing low levels of the a3 TCR-CD3 complex progressively increases, and single positive (SP) CD4 or CDS cells with notably higher levels of the a3 TCR-CD3 complex finally appear. The precise function of DP cells was for many years unclear, but it has been conclusively demonstrated that these DP cells contain precursors for the SP thymocytes. T Cell functions such as cytolysis and cytokine production can be induced through receptor-mediated activation in a subpopul-ation of SP cells, so these cells have apparently differentiated to a mature stage.

During the final stage of T cell development, selection of the T cell repertoire has to occur (see also below). Potentially harmful cells, such as autoreactive cells, must be eliminated or inactivated (a process termed 'negative selection'), and events favoring preferential differentiation of T cells capable of recognizing foreign antigens in association with self MHC products (a process termed 'positive selection') must allow for the generation of useful T cells. It has been recognized for many years that final T cell selection for export is a consequence of interactions between surface proteins on immature thymocytes and the nonlymphoid elements in the thymus. Thymic selection must logically involve the MHC gene products (class I and class II), the a3 TCR, and the CD4 and CDS molecules, as antigen recognition by the a3 TCR is facilitated by the CD4 and CDS co-receptors. These coreceptors not only increase the affinity of interactions between T cells and MHC (CD4 molecules can bind to MHC class II, and CDS molecules can bind to MHC class I), but also func tion as signaling molecules in mature T cells and immature thymocytes. Thymic selection can only begin after expression of the CD4, CD8 and otf} TCR-CD3 complex has occurred, and consequences of its effects are therefore first observed in these late stages of thymic development. The contribution of adhesion and costimulatory receptors is only beginning to be addressed, as is the identification of the signaling pathways involved. Features yet to be accounted for include the fact that the T cell repertoire is nonrandomly expressed in the two main subsets of T cells, i.e. most MHC class Il-restricted T cells are CD4+CD8~, and most MHC class ¡-restricted T cells are CD4 CD8+.

The nonlymphoid (stromal) cells that present the MHC-peptide complexes to developing T cells can be divided into two main cell populations: bone mar-row-derived cells, such as dendritic cells, and non-bone marrow-derived cells, such as epithelial cells. Many experimental models have attempted to define whether positive and negative selection can be compartmentalized, i.e. whether specialized stromal cells can determine the fate of TCR-expressing thymocytes interacting with the MHC antigens expressed on the surface of stromal cells. Both types of stromal cells can express class II and class I MHC antigens, and earlier studies established some basic rules about the role of epithelial and hematopoietic cells in T cell selection.

Hematopoietic cells are responsible for the induction of clonal deletion in potentially self-reactive DP thymocytes, as a result of presumably high-affinity interactions between such DP cells and self MHC-peptide complexes. Since hematopoietic cells can also effectively function as APCs for activation of mature T cells, induction of clonal deletion is clearly not a consequence of intrinsic properties of such APCs. Rather, signals delivered by hematopoietic cells must be perceived differently by TCR-expressing DP thymocytes than by SP mature T cells, and it is clear that the genetic make-up of DP cells poises them for death. It has long been known that DP thymocytes do not display the same set of functions as mature T cells do upon TCR-mediated signaling, and death may be as pertinent a function for immature T cells as 'conventional' hallmarks of activation, such as cytokine production and cytolysis, for mature T cells. Nevertheless, signals delivered to DP cells by epithelial cells result in a different outcome: preferential differentiation (positive selection), if their TCR fits the MHC-peptide complexes presented with a presumably lower level of affinity. It is not clear which factors determine the difference between positive and negative selection, nor have any differences between signals delivered by epithelial cells and dendritic cells been identified. Among the more likely possibilities currently studied is differential participation of costimulatory signals, and it will be a true revelation in immunobiology indeed when this fascinating puzzle is solved.

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