T cell Development

Basic Principles of T Cell Development Each cell randomly rearranges a specific TCR. The presence of the pMHC epitope, stage of development and “costimulation” determine the cell’s fate. Functional Immunity & Tolerance result from the sum of a large population of many sequential pMHC epitope specific cellular responses.

Following development into mature, antigen-responsive T cells, these T cells emerge from the thymus and migrate to secondary lymphoid tissues, where they interact with antigen, antigen-presenting cells, and other lymphocytes:

The importance of the thymus in T cell development is demonstrated by inherited immune deficiencies: people that do not have a thymus (DiGeorge’s syndrome, aka Thymic Aplasia) do not develop functional T cells. DiGeorge’s syndrome results from a developmental defect – the failure of the third and fourth pharyngeal pouches to develop, which results not just in thymic defects, but also in absent parathyroids and in aortic arch defects. Thymectomy early in life reduces the ability to produce T cells. Thymectomy later in life does not markedly impair T cell number. In fact, the thymus decreases in size with age. However, the thymus can still produce new T cells up to middle-age, especially in situations where there is loss of T cells (HIV/AIDS).

While in the thymus, immature T cells, or thymocytes, undergo several changes that allow them to develop into mature T cells, ready for contact with antigen. Thymocytes interact with thymic epithelial cells and various other cells while in the thymus.

The thymus is composed of several lobes, each of which has cortical and medullary regions:

The cortex contains immature thymocytes in close contact with thymic epithelial cells. Medullary areas contain more mature thymocytes, epithelial cells, and dendritic cells and macrophages

During thymic differentiation, the great majority of thymocytes die by apoptosis, and are ingested by macrophages. Only a small minority of these T cell progenitors make it out as mature T cells

Thymic development occurs in two phases: 1)production of T cell receptors for antigen, by rearrangement of the TCR genes 2)selection of T cells that can interact effectively with self-MHC

Changes in the expression of cell-surface molecules accompany the thymic differentiation of T cells: –entering thymocytes are TCR, CD3, CD4, and CD8- negative –as thymocytes mature, and undergo rearrangement of their TCR genes to generate a functional TCR, they begin to express CD3, CD4, and CD8 –mature T cells ready to go to the periphery are TCR/CD3+, and either CD4 or CD8 positive

First phase of thymic development: rearrangement of TCR genes to produce a functional TCR Progenitor T cells enter the thymus (sub-capsular region of the outer cortex). These cells do not have rearranged TCR genes and lack expression of characteristic T cell surface molecules. Interaction with thymic stromal cells induces these progenitor T cells to proliferate. These immature thymocytes do not yet express CD4 or CD8, molecules that are expressed by mature T cells: double-negative thymocytes.

There are two types of T cell receptors:  and   TCR T cells are the most abundant, by far: (or  &  chain)

Unlike B cells, in which the genes that encode the BCR rearrange in a set order, the TCR , , and  genes start to rearrange at about the same time. If a productive  or  rearrangement occurs first, the T cell is committed to that lineage, and stops further rearrangement of the  TCR gene.

However, if  is rearranged first, then the T cell continues to proliferate, and undergoes further rearrangements. This results either in rearranged  TCR gene, yielding an  TCR lineage cell, or rearranging  and  genes, resulting in a  TCR cell.

Rearrangements that lead to an  T cell begin the rearrangement of the  TCR gene. The first step is D-J joining, followed by VDJ rearrangement. Expression of  chain stops further  chain rearrangements.

 chain is then expressed on the surface of the thymocyte in association with a surrogate  chain (pT  ). Following this, there is rearrangement of the  TCR gene, resulting in a functional  chain, and in the expression of surface TCR, in association with other T cell-associated cell surface molecules.

During this process, a cell that makes an unproductive  chain rearrangement can try again until gets a good  chain, or it exhausts its possibilities:

Thymocytes that have a functional  rearrangement, and express  or  + the surrogate  chain (pT  ) are induced to express both CD4 and CD8 simultaneously – these are called double-positive cells. Immature T cells that do not undergo a productive rearrangement die by apoptosis.

Second phase of thymic development: selection of T cells that can interact with self MHC and antigen This applies only to  TCR-bearing cells (>95% of T cells).  T cells are not restricted to interactions with MHC class I or class II molecules This phase of T cell development consists of two steps: –positive selection (TCR that can interact with self-MHC) –negative selection (eliminate self-reactive cells that are stimulated by MHC + self)

Transgenic TCRs to Self Antigens Clonal Deletion in Thymus Double TCR transgenic mice (both TCR  and  ) using a TCR from a CTL clone specific for male antigen (H-Y/H-2D b ). Female mice –most mature T cells expressed transgene TCR –normal proportion of CD4+ and CD8+ –30% of CD8+ cells specific for male H-Y. Male mice –large decrease (90%) in thymocytes (including CD4+8+) –down regulation of CD8+ among peripheral T cells –No H-Y reactive cells found; no autoimmunity P. Kisielow, H. Blüthmann, U. D. Staerz, M. Steinmetz, & H. von Boehmer. Nature 333: (1988)

C57BL/6 control male HY TCR transgenic Female HY TCR transgenic male Expression of TCR Tg in females with H-2D b results in development of CD8+T (increased CD8/CD4) Expression of TCR tg and autoantigen in males results in deletion of DP thymocytes and blocks development of Tg+ CD8 SP Harold von Boehmer HY TCR transgenic mice

In Rag-/-HY TCR transgenics all thymocytes express the TCR tg. More dramatic skewing to CD8 in females and deletion in males Female HY TCR tg CD4 0.6%; 5.8%; 1.2x x x x %; 91%; 75%; 26x %; 2x10 6 5%; 1.5x10 6 9%; 2.7x10 6 CD4 CD8 Male HY TCR tg

CD4/CD8 lineage commitment

Positive selection refers to the selection of thymocytes that are able to bind to, and interact with, self-MHC molecules In positive selection developing thymocytes continue to live if they bind MHC well enough to receive a signal through their TCR. This signal is mediated by the interactions of these cells with MHC-expressing thymic cortical epithelial cells. The ~95% of thymocytes that do not receive this signal undergo apoptosis. Positive Selection

Positive selection takes place in the cortex of the thymus lobules:

These CD4+ CD8+ TCR+ thymocytes interact with thymic epithelial cells that express both MHC class I and MHC class II molecules, complexed with self-peptides. Thymocytes that bind MHC survive; those that don’t bind to self-MHC die. TCR  chain rearrangements can continue during positive selection, allowing cells to explore alternative  chains for MHC binding. Once a T cell is positively selected, TCR rearrangement stops.

Topi KO per MHC di classe II e transgenici per MHC classe II espressa esclusivamente a livello della regione corticale del timo sotto il controllo di un promotore specifico per i cheratinociti Selezione positiva di tutte le cellule T che riconoscono complessi MHC + peptide anche quelle fortemente autoreattive In assenza di presentazione dell’Ag a livello della midollare si ha selezione positiva incontrastata

Negative selection refers to the elimination of those thymocytes that bind to self-MHC molecules + self with high affinity. In negative selection developing thymocytes die if they bind MHC + self peptides too well (strongly enough so that they would be activated by this interaction, via signaling through their TCR). Negative Selection

Thymocytes undergo negative selection in the medullary region:

There, they interact with antigen-presenting cells (dendritic cells, macrophages) that express self-antigens + MHC class I or MHC class II molecules. Thymocytes that bind to self + MHC too strongly are eliminated as possibly self-reactive cells, and undergo apoptosis. If self-reactive T cells were allowed to exit the thymus, such cells would mediate autoimmune disease.

Thymic Selection

Effects of peptides acting as agonists, antagonists or neither on thymic selection

Receptor editing