Cell-fate specification is a key developmental event. The physiological function of a multicellular organism depends on the generation of the proper number and diversity of cell types. Signals from broadly expressed receptors that interact with co-evolved germline ligands control most differentiation decisions. But, adaptive immunity depends on the function of T- and B-cells, which express unique surface receptors that are created by somatic DNA rearrangement and random chain pairing. These clonal receptors help to determine which precursor lymphocytes will successfully mature. Therefore, the lineage-specific differentiation of immature CD4+CD8+ (CD4 Antigen Positive CD8 Antigen Positive) T-cells into CD4+ or CD8+ mature T-cells is regulated by clonally-expressed, somatically-generated TCRs (T-Cell Receptors) of unpredictable fine specificity. Each mature T-cell generally retains expression of the co-receptor molecule (CD4 or CD8) that has an MHC (Major Histocompatibility Complex)-binding property that matches that of its TCR (Ref. 1 & 2). Two models are proposed initially to explain the remarkable outcome-‘instruction’ of lineage choice by initial signaling events or ‘selection’ after a stochastic fate decision which limits further development to cells with coordinated TCR and co-receptor specificities. The mistake-prone instruction of lineage choice precedes a subsequent selection step that filters out most incorrect decisions (Ref. 2).
Even though these co-receptors are not expressed in the early phase, the expression of other cell surface molecules, particularly the THY1 (Thy1 T-Cell Antigen), c-Kit, CD44 (CD44 Antigen) and CD25 (CD25 Antigen), marks the developmental progression of the DN (Double Negative) population. The initial thymocyte population displays c-Kit, the receptor for stem cell growth factor, and subsequently expresses CD44, an adhesion molecule, and then CD25, the Alpha-Chain of the IL-2 Receptor. During this period, the cells proliferate but the TCR genes remain unrearranged.
Once the cells stop expressing c-Kit and markedly reduce CD44 expression, they begin to rearrange their TCR genes. The earliest stages of T-cell lineage commitment also witness expression of two transcription factors, Ik1 and GATA3 (GATA Binding Protein-3). Ik1 is responsible for the differentiation and/or survival of a common lymphoid progenitor and is defined as the earliest transcriptional checkpoint in lymphoid lineage commitment (Ref. 1 & 2). Most highly immature progenitors, comprising about 1–2% of thymocytes, are double-negative (DN) cells that express neither CD4 nor CD8. DN cells progress through stages of DN1 through DN4. DN1a and DN1b populations are enriched in early thymic progenitor cells, although they may still give rise to natural killer cells, myeloid cells and, to a limited degree, B-cells. Thymocyte precursors or lymphoid progenitors from bone marrow enter the thymus and rearrange TCR-Beta (T-Cell Receptor- Beta), TCR-Gamma and TCR-Delta genes. Somatic rearrangement of the genes encoding the TCR-Beta , TCR-Gamma and TCR-Delta chains, which is essential for TCR expression, begins in DN2 cells and is mainly completed during the DN3 stage. Also, a sustained Notch signaling throughout the transition from DN2 to DN3 itself promotes gene rearrangement at the Tcrg, Tcrd and Tcrb loci. Although sustained Notch signaling through the DN1 and DN2 stages is required for the differentiation of both Gamma/Delta and Alpha/Beta T-cells, its effects on those distinct lineages may not be equivalent. The TCR-Notch synergy and TCR signal strength models both espouse late commitment of bipotent progenitors to either the Alpha/Beta or the Gamma/Delta T-cell lineage, at least in part as a result of differences in signals emanating from pre-TCR and full TCR complexes (Ref. 3).
Contingent on Beta-selection, the cells progress to the DN4 stage where TCR-Beta chain is expressed from a productively rearranged Tcrb locus and pairs with the invariant pre-TCR-Alpha chain to form a pre-TCR (Ref. 3). Three related HMG (High Mobility Group) box transcription factors TCF1 (T-Cell Factor-1), LEF1 (Lymphoid Enhancer-Binding Factor-1), and SOX4 (SRY (Sex Determining Region-Y) Box-4) act as important regulators of thymocyte development and enhance the expansion of DN thymocytes and their differentiation into ‘Double-Positive’ (DP) cells. In addition to their structural relatedness and shared DNA-binding specificities, TCF1 and LEF1 also display largely overlapping patterns of expression during thymocyte development. Both genes are expressed in all T-cell subsets from early DN cells to mature peripheral SP (Single-Positive) T-cells. Like TCF1 and LEF1, SOX4 also plays an important role in the proliferation and maturation of DN thymocytes. SOX4 regulates the expansion and differentiation of pro-B-cells. Thymocytes that productively rearrange TCR-Beta express the product of the rearranged allele on the cell surface as part of a pre-TCR complex, which also includes the pre-T-Alpha invariant chain and CD3 components, and differentiate into DP thymocytes (Ref. 2 & 3).
Once a signal has been transmitted through the pre-TCR, it halts further Beta-chain gene rearrangement and induces expression of both CD4 and CD8 co-receptors and is called DP CD4+CD8+ cells and begin to proliferate. During this proliferative phase of DP thymocytes, TCR-Beta and TCR-Alpha chain gene rearrangement does not occur even though both the RAG1 (Recombinase Activating Gene-1) and RAG2 genes are transcriptionally active. Rearrangement of the Alpha-chain genes cannot take place at this stage because the protein, RAG2 (Recombinase Activating Protein-2) is rapidly degraded in proliferating cells. For this reason Alpha-chain gene rearrangement does not begin until the DP cells stop proliferating and RAG2 protein levels increase. This proliferative phase contributes to T-cell diversity by generating a clone of cells with a single TCR-Beta chain rearrangement. Each of the cells within this clone then rearranges different Alpha-chain genes, allowing for greater diversity (Ref. 4).
Most double-negative thymocytes thus progress down a different developmental pathway, they stop proliferating and begin to rearrange the TCR-Beta chain genes and express the Beta-Chain. Those cells of the Alpha/Beta lineage that fail to productively rearrange and express Beta-chains die. Newly synthesized, pre-TCR consists of a complex of the first receptor chain to be expressed (the Beta-Chain) complexed with a unique chain (pre-T-Alpha) and recognize its intra-thymic ligand to transmit signals through the CD3 complex. Pre-TCR signaling, coupled with signals from cytokine and possibly other receptors, promotes cell survival, proliferation, and differentiation, with DN4 cells subsequently becoming immature SP cells as they acquire small amounts of CD8. The importance of immature single-positive cells is unclear, but it seems that such cells rapidly up-regulate CD4, forming the DP (Ref. 3 & 4). These DP thymocytes are short-lived cells that rearrange their TCR-Alpha gene. The product of the rearranged TCR-Alpha allele associates with the TCR-Beta chain and CD3 components, and a TCR-Alpha/TCR-Beta complex is expressed at the cell surface. By the time that a T-cell has rearranged and begun to express the Alpha-chain of the TCR, the DP cells no longer express such early markers as CD44 and CD25. The possession of a TCR enables DP thymocytes to undergo the rigors of positive and negative selection guided by self-MHC and self-MHC plus peptide (Ref. 4).
In general, three mature T-cell populations are produced and move to the peripheral lymphoid organs. Most of the T-cells express the TCR-Alpha/TCR-Beta with either CD4 or CD8 as co-receptors. A few T-cells express the Gamma/Delta-TCR; most of these lack both CD4 and CD8 and migrate towards the periphery. Further positive selection of the DP thymocytes takes place in the cortical region of the thymus and involves the interaction of immature thymocytes with cortical epithelial cells. The TCRs tend to cluster with the MHC molecules at sites of contact. Interaction of immature CD4+CD8+ thymocytes with thymic epithelial cells, mediated by MHC-restricted TCRs, allows the cells to receive a protective signal that prevents them from undergoing cell death; cells whose receptors are not MHC restricted, and therefore not able to bind MHC molecules, would not interact with the thymic epithelial cells and consequently would not receive the protective signal, leading to their death by apoptosis (Ref. 1 & 5). During positive selection, the RAG1 and RAG2 proteins required for gene rearrangement and modification continue to be expressed. Only those whose TCR-Alpha/TCR-Beta heterodimer recognizes a self-MHC molecule are selected for survival. Consequently, the capacity to try more than one combination of TCR-Alpha/TCR-Beta chains is important because it gives the thymocyte opportunities to “retake” the test for positive selection. If by chance, the cell manages to rearrange an Alpha-chain that allows the resulting TCR-Alpha/TCR-Beta to recognize self-MHC, the cell is spared; if not, then the cell dies by apoptosis (Ref. 2 & 5).
The avidity of the TCR-Alpha/TCR-Beta for self-peptide-MHC complexes determines the fate of the DP thymocyte. Thymocytes with high avidity for self-peptide-MHC complexes are eliminated by negative selection whereas those with low avidity die by neglect in the thymic cortex (Ref. 4 & 5). The population of MHC-restricted thymocytes that survive positive selection compromises some cells with low-affinity receptors for self-antigen presented by self-MHC molecules and other cells with high-affinity receptors. The latter thymocytes undergo negative selection by an interaction with bone marrow derived Antigen Presenting Cells/APCs (dendritic cells and macrophages) in the thymic medulla. During negative selection, dendritic cells and macrophages bearing class-I and class-II MHC molecules interact with thymocytes bearing high-affinity receptors for self-antigen plus self-MHC molecules alone. The interaction involves the TCR of the thymocyte that is undergoing selection and MHC molecules on the cells, mediating negative selection, but the precise details of the process are not yet known. Cells that experience negative selection are observed to undergo death by apoptosis. Tolerance to self-antigens is thereby achieved by eliminating T-cells that are self-reactive and allowing maturation only of T-cells specific for foreign antigen plus self-MHC molecules (altered self) (Ref. 2 & 5).
Cells with intermediate avidity for self-peptide-MHC complexes survive and differentiate into mature CD8+ T-cells/Tc/Cytotoxic T-cells (if MHC Class-I restricted) or CD4+ T-cells/TH cells/T-Helper cells (if MHC class II restricted). This positive selection is followed by characteristic phenotypic changes, such as the up-regulation of CD5 or CD69 expression. At the transcriptional level, the zinc-finger transcription factor LKLF (Lung Kruppel-Like Factor) appears to be required to program and maintain the quiescent phenotype in mature SP thymocytes and T-cells. Thus, expression of the LKLF gene is developmentally activated during the transition to the mature SP stage of thymocyte development, remains elevated in resting SP T-cells, and is rapidly extinguished after the activation of these cells by TCR cross-linking (Ref. 1 & 3).
To date, lineage commitment is also studied mainly from two opposite perspectives: a ‘top-down’ approach, focused on understanding the role of TCR signaling, and a ‘bottoms-up’ approach, focused on elucidating the transcriptional control of co-receptor expression. A new ‘inside-out’ approach based on elucidating the upstream effectors and downstream targets of Th-POK (T-Helper Inducing POK Factor; also known as c-Krox/ZFP76), the master regulator of lineage commitment that might allow these processes to be mechanistically linked (Ref. 6). In the thymus, the differentiation of immature thymocytes is determined by their cis-acting genetic potential and by myriad interactions in trans, including those with epithelial and myeloid stromal cells and with other thymocytes. Given the combinatorial diversity of such influences, it is not unexpected that their collective actions produce an ‘organized chaos’ that permits several different types of T-cells to emerge from the thymus. At the CD4+CD8+ stage, thymocytes that express mature TCR-Alpha/TCR-Beta develop into one of many lineages. These lineages include conventional naive CD4+ and CD8+ T-cells, NKT (Natural Killer T-cells), Treg cells (CD4+CD25+ Regulatory T-cells), and CD8+ innate T-cells (Ref. 3).
Several signaling proteins and transcription factors are crucial for the maturation and/or function of these lineages.
- For the development of conventional naive CD4+ T-cells, GATA3 and Th-POK transcription factors are essential (Ref. 7). The expression pattern of the Th-POK gene in developing thymocytes is tightly controlled in a manner consistent with a specific role in CD4 commitment. During positive selection, Th-POK is preferentially up-modulated in MHC class II–restricted thymocytes and persists throughout their subsequent development (Ref. 6).
- Similarly, ITK (Interleukin-2 (IL-2)-Inducible T-cell Kinase) and RLK (Resting Lymphocyte Kinase) are required for the development of conventional naive CD8+ T-cells. In the presence of ITK and RLK, positive-selection–inducing TCR signals of long duration or those involving many TCRs activate Tec-kinase–dependent downstream pathways.
- The Treg cells express FoxP3 (Forkhead Box-P3) as they mature, and require FoxP3 for their function.
- NKT cells are dependent on a large number of signaling proteins and transcription factors for their development and function, including SAP (SLAM (Signaling Lymphocytic Activating Molecule)-Associated Protein), Fyn , PKC-Theta (Protein Kinase-C- Theta), IKK-Beta (Inhibitor of Nuclear Factor-KappaB (NF-KappaB)) Kinase-2), NF-KappaB, IRF1 (Interferon Regulatory Factor-1), Ets1, Elf4 (E74-Like Factor-4; also known as MEF), Runx1 (Runt-related Transcription Factor-1), Runx3, ROR-\Gamma t (Retinoic-Acid-Receptor-Related Orphan Receptor-Gammat), and TBX21 . These single-positive cells finally migrate to the periphery of the thymus.
- So far, no factors are known to be essential for the development of innate CD8+ T-cells, while dispensable for the other T-cell lineages (Ref. 7).
There are still many uncertainties about lineage-choice mechanisms. A clearer understanding of Notch activity in CD4/CD8 development is required for targeting mutations specifically to DP thymocytes (Ref. 1 & 4). The most difficult challenge remains to determine how differences in TCR affinity or in the nature of TCR interactions with classical versus non-classical MHC molecules produce distinct signaling outcomes that are responsible for inducing two different lineages of mature T-cells, conventional versus innate. Also, the postulated function of Th-POK in recruitment of chromatin remodeling factors and gene silencing shows the importance of gene silencing in lymphoid development and may play a direct role in lineage-specific co-receptor silencing. Because of its complexity, it is not surprising that there is as yet little consensus concerning the mechanism by which lineage commitment is signaled in developing DP thymocytes. At issue is the basic mechanism by which competent T-cells arise to protect the host organism as the fundamental purpose of the immune system. However, it is clear that the functional relationship of TCR and Notch signaling are vital in the selection and specification of CD4 and CD8 T-cell lineages (Ref. 6 & 7).
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