Q. Describe the processes that control the development of T lymphocytes in the thymus
This essay will describe the processes which govern the development of thymocytes in the Thymus. It will attempt to detail these processes in a linear fashion, tracing the journey of a thymocyte entering the thymus, activation of notch receptors, passage through the regions of the cortex and the accompanying subjection to positive negative selection and gene rearrangement – ending with a mature thymocyte leaving the medulla.
The thymus is a bilobed, primary lymphoid organ located in the anterior mediastinum, behind the sternum and slightly above the heart. It is a critical component of the lymphatic system, tasked with receiving immature T-cells (thymocytes) from the bone marrow and developing them into active, mature T-cells. The organ is partitioned into lobules [figure 1] which are comprised of an inner medulla and a cortex encased by an outer capsule (Ritter & Crispe, 1992). These regions are critical in producing mature lymphocytes and have environments catering to these functions (Murphy et al, 2008). The consequences of a malfunctioning or inactive thymus (such as would be caused by DiGeorge syndrome) are severe, as T-cells cannot be produced by any other method and are involved in a vast array of immunological processes such as delayed allergy, homograft rejection and even the maturation of B cells (Astaldi and Lisiewicz, 1971).
The point of entry into the thymus for naïve T-cells is through blood vessels in the cortico-medullary junction [figure 1]. Upon entry, these progenitors are designated as T–cells (as opposed to a B-cell lineage) after receiving signals from stromal cells which activate the Notch1 receptor thereby activating specific genes which bestow T-cell traits (Murphy et al, 2008).
In mechanical terms, the Notch1 surface receptor present on the thymocyte encounters the Notch ligand on the thymic epithelium, a process of protelytic cleavage begins. This cleavage releases the Notch1 protein, which leaves the intracellular domain and enters the thymocytes nucleus [figure 2]. In the nucleus, Notch1 replaces repressors and enlists co-activators, creating a transcription factor complex. In the absence of these repressive factors, and the presence of transcription complex, genes required for the T-cell lineage are ‘switched on’ (Parkham, 2009). Thymic epithelial cells are also important as they provide soluble factors such as the cytokine interleukin7 and stem cell factor, both of which aid in development (Shakib et al, 2009).
Now that these thymocytes have been committed to the T lineage, and are present in the thymus, the next stage is for them to progress toward the outer cortex. After roughly one week T-cells can be differentiated from stem cells by the presence of adhesion molecule CD2 and glycoprotein CD5. At this point, the T-cells are clearly delineated as such, but do not yet display the surface markers which characterise mature T-cells (the CD3 receptor complex as well as either the CD4 or CD8 co-receptors), and have not yet undergone receptor gene rearrangement (Murphy et al, 2008). At this point, they are classed as double negative thymocytes.
During the migration through the cortex, thymocytes undergo two processes; intense proliferation in the subcapsular region, under the influence of cortical epithelial cells/mesenchymal cells (Starr et al, 2002); while concurrently rearranging genes critical for maturation into functioning, specific lymphocytes.
Genetic rearrangement is necessary to commit the T-lymphocytes to a specific T lineage as well as ensure that the properties of only one of these lineages are displayed. In the subcapsular region, the β, γ and δ regions of the gene loci begin rearranging concurrently (Murphy et al, 2008). In simple terms, the first loci to successfully rearrange and ergo produce the relevant receptor chain determines the lineage. The first outcome is that signals from the pre-TCR pre-empt γ and δ rearrangement, making the cell an α:β T-cell. The other, less likely outcome is that the γ:δ TCR halts β rearrangement and the cell becomes a γ:δ cell. In the first case, the δ locus is deleted (ensuring the traits of an αβ cell), while in the latter the γ:δ cell simply matures and enters the periphery. Those cells which cannot generate functional receptors are subject to apoptosis, their remains being phagocytosed by resident macrophages in the thymus.
Very few (~5%) thymocytes commit to the γ:δ line, while most (~95%) become α:β cells. γ:δ cells exit the thymus, entering the periphery and undergo extrathymic development, becoming, for example, intraepithelial lymphocytes in the gut (Sumen et al, 2004). α:β committed thymocytes remain in the thymus and transition from the D3 to the D4 stage then begin proliferating.
Once this proliferation has ceased, the thymocytes become single positive cells displaying the CD8 receptor and then double positive cells. The cells are termed double positive (DP) as they possess both CD4 and CD8 receptors. The double positive cells begin moving through the central area of the cortex, in the direction of the medulla. These cells will undergo a programmed cell death in the next 4 days unless positive selection – the process of ensuring cells react to self peptides and self MHC – occurs, followed by negative selection (Murphy et al, 2008). Unreactive/over reactive cells die by apoptosis and are cleared by macrophages.
A number of transcription factors control the death of DP cells, including: RORγ – a steroid transcription factor, TCF-1 and LEF-1. Collectively, these transcription factors ensure the expression of an anti-atopic factor (Bcl-XL). It is this factor which prevents programmed cell death (Starr et al, 2002).
There is also a logical reason that thymocytes become double positive before completing their maturation. Having both CD4 &CD8 allows the opportunity to recognise class 1 MHC and fail to recognise class 2 (and vice versa), surviving regardless. In this regard, the DP stage increases the chances of any single thymocyte maturing.
DP thymocytes group around cortical epithelial cells, and it is here that they are exposed to MHC molecules. Thymocytes either generate a response using one of their receptors and continue to the medulla or fail to respond and succumb to apoptosis. Surviving thymocytes become single positive for their reactive receptor (Takahama, 2006). Now that we have a population of single positive thymocytes that can react to self peptides in conjunction with self MHC, they must now undergo negative selection to ensure that they do not react too strongly to these same signals and cause autoimmune conditions (Mowat, 2011).
Single positive thymocytes are subject to negative selection while crossing over into the medulla and those which survive become functioning, mature thymocytes. At this stage, self antigens are provided by both macrophages and dendritic cells, and these self antigens tend to be those most likely to be encountered in peripheral tissue and instigate an autoimmune reaction. Stromal cells are also important as they produce proteins which can only be found in certain tissues of the body (Anderson et al, 2002/Ohashi P, 2002), meaning that thymocytes can be tested on likely sources of an autoimmune reaction found throughout the body before reaching those tissues.
T cells are unique among lymphocytes in that they are MHC restricted, i.e react to antigens only when accompanied by MHC, and this is why the positive/negative selection processes are necessary. Those few (~2%) of thymocytes which complete the gruelling selection process spend a short time in the medulla, before exiting the thymus, heading for the thymus dependant areas of the periphery (Mowat, 2011).