T Cell Development in the Thymus David Straus

Lecture objectives T cell life cycle: development in the thymus, circulation as mature, naïve cells, activation in response to antigen, differentiation into memory cells. Change in composition of the T cell pool with age. Generation of diverse antigen receptors on T cells through sequential rearrangement of ß and a gene segments. Shaping of the repertoire through positive and negative selection. Relation of DiGeorge, Omenn and Autoimmune Polyglandular syndromes to thymic development. Why matching of HLA types is important for restoration of immune function following bone marrow transplantation. Generation of distinct T cell lineages: CD4, CD8, T reg as well as g/d T cells.

Adaptive immunity relies on having a huge pool of lymphocytes, each with unique antigen recognition capabilities. These clones can be expanded and go on to generate a memory population. memory death

Antigen receptors on T cells only recognize antigen when it is processed and bound to MHC molecules on the surface of other cells. This allows T cells to specifically target their immune function to cells which have encountered pathogens.

A variety of distinct T cell lineages provide immune functions gd T cells gd cytokines ? Treg Treg cells T

T cell precursors migrate from the bone marrow to the thymus where they mature. Mature T cells exit the thymus, enter the circulation, and travel through secondary lymphoid organs.

Figure 1-11 Mature T cells circulate through the blood and lymphatics. Processed antigen, bound to MHC on antigen presenting cells, is encountered in the lymph nodes where T cell activation takes place. Activated T cells provide effector function either in the lymph nodes, or after migration to the site of infection.

Circulating T cell pool The pool of circulating T cells is composed of thymus-derived naïve T cells, and memory T cells generated following stimulation with antigen. The size of the pool is regulated by competition for cytokine and receptor signals. M Thymus proliferation Circulating T cell pool

Cellular organization of the thymus

Epithelial cells form a dense network surrounding the developing thymocytes

DiGeorge Syndrome Genetic disease associated with chromosomal rearrangements which effect development– in severe form thymus fails to develop. T cells cannot develop SCID (Severe Combined Immunodeficiency Disease) - no T or B cell function

Thymic atrophy alters composition of the T cell pool Thymus is most active in young children and atrophies markedly with age. Thymic output is substantially diminished by age 50. T cell pool in older adults relies on homeostatic proliferation of existing T cells which favors memory T cells.

Memory T cells have a greater capacity for homeostatic proliferation, and so contribute a greater fraction of the T cell pool as thymic output is lost. This results in a restriction of the T cell repertoire in older adults Child

How is the T cell pool restored following lymphoablative conditions (e How is the T cell pool restored following lymphoablative conditions (e.g. chemotherapy, acquired immunodeficiency)? -In children, restoration of the T cell pool primarily by thymic production. -In older adults, restoration accomplished primarily by expansion of remaining lymphocytes - a limited repertoire.

Thymocyte development progenitor initial repertoire selected repertoire multiple lineages

A pool of T cells with diverse antigen specificity is generated by rearrangement of gene segments coding for the T cell antigen receptor (TCR) a b TCR

Rearrangement of gene segments encoding the T cell receptor  subunit occurs prior to alpha gene rearrangement

Following successful ß gene rearrangement, ß is expressed on the cell surface with pre-T alpha (pTa) as part of the Pre-TCR. This blocks further ß gene rearrangement (allelic exclusion).

Pre-TCR expression promotes a gene rearrangement Pre-TCR expression promotes a gene rearrangement. When a is successfully rearranged and expressed, it replaces pTa, and forms the mature ab TCR on the cell surface.

Rearrangement summary TCR b gene rearrangement occurs first – successful b rearrangement leads to cell surface expression with a surrogate a -chain (pTa). The resulting pre-TCR signals cause b rearrangement to stop (allelic exclusion) and promotes further development - proliferation and the start of a chain rearrangement. With successful a chain rearrangement, pTa is replaced and the mature ab TCR is expressed on the cell surface.

Omenn Syndrome Recombinase enzyme, encoded by RAG genes, mediates gene rearrangements necessary for antigen receptor expression during development. Omenn syndrome is a result of inherited RAG deficiency which causes a block in development. Patients have a SCID (Severe Combined Immunodeficiency Syndrome) phenotype. RAG function is required for both T and B cell development.

Question: An infant has recurrent infections, including infections with ‘opportunistic’ pathogens. This is consistent with Severe Combined Immunodeficiency (SCID). You propose either Omenn syndrome, or DiGeorge syndrome as a possible cause. How could you tell the difference between these two?

Flow cytometry can be used to determine the representation of specific cell populations (such as T and B cells) in complex mixtures (such as peripheral blood). Figure A-17 part 1 of 2 Antibodies with an attached fluorophore will emit light at a characteristic wavelength when stimulated with the appropriate light.

Flow cytometry allows the rapid determination of the amount of antibody labeling, and the type of fluorescence(color of fluorophore), associated with individual cells.

The DNA rearrangements that take place during lymphoid development can generate chromosomal translocations which give rise to malignancies.

Thymocyte development progenitor Initial repertoire selected repertoire

Positive selection T cell function demands that the antigen receptor recognize MHC- antigen complex. Receptors which cannot bind MHC are not useful. Thymocyte development requires that newly expressed  TCR engage MHC on thymic epithelium well enough to send a survival signal. Failure of newly expressed  TCR to engage MHC results in thymocyte death.

Positive selection: Recognition of MHC-peptide on thymic epithelial cells provides a survival signal for developing thymocytes. Rearrangement of a-chain gene stops after a positive selection signal is generated. (This is also the stage of CD4 and CD8 lineage commitment which will be discussed later)

Bone marrow transplantation demonstrates the importance of HLA (MHC) recognition in positive selection and T cell function. Immune function can be restored to immunodeficient patients (e.g those with an inherited deficiency, or treated with radiation and chemotherapy) by bone marrow transplants. Transfer of hematopoietic stem cells in bone marrow provides progenitors for entire immune system including T & B cells and antigen presenting cells.

Restoration of immune function following bone marrow transplantation requires matching of donor and recipient HLA types. T cell development requires recognition of HLA in the thymus, and T cell activation requires recognition of HLA in the periphery. Figure 5-11

Question: A patient receives a bone marrow transplant following ablative chemotherapy. However immune function does not appear to be restored. A re-test of the donor shows that in fact very few HLA alleles are shared between donor and recipient. What would an analysis of peripheral blood show in terms of T and B lymphocytes numbers?

Negative selection To avoid autoimmunity, cells are removed which express receptors that bind strongly to MHC-self antigen. Thymocytes expressing an  TCRs which engage MHC-self antigen with high affinity undergo apoptosis (induced to die). Negative selection of developing thymocytes is known as “central tolerance” – the basic mechanism for inducing tolerance to self. (Mechanisms for inducing tolerance in circulating, mature, T cells are known as “peripheral tolerance”).

Thymic maturation: moderate strength TCR signals are required for continued development Thymic stromal cell CD4+8+ thymocyte Negative selection - death Positive selection - Maturation to CD4+ or CD8+ No selection - death

Positive and negative selection are mediated by different types of cells in the thymus. Figure 5-13 A thymocyte which is initially positively selected, may subsequently undergo negative selection.

Autoimmune Polyglandular Syndrome (APS) demonstrates the importance of negative selection in the thymus Removal of self-reactive T cells during thymic development depends upon the expression of a large variety of self-antigens in the thymus. Individuals with APS have an autosomal recessive mutation which effects activity of a transciption factor controlling expression of 200 -1200 genes in the thymic medulla. Autoimmune responses against organ-specific antigens develop with age. Endocrine glands appear to be particularly targeted. Anderson, et al paper discusses mouse model for APS.

Development of functionally distinct T cell lineages gd TCR CD4-8- ab TCR CD4+8+ ab TCR, CD4+ ab TCR, CD8+ TCR- T reg (ab TCR, CD4+)

Mature T cells express either CD4 or CD8 Mature T cells express either CD4 or CD8. These cell surface proteins are “co-receptors” which help the TCR bind either to MHC class I or to MHC class II. Expression of CD4 is associated with helper T cells, and CD8 is associated with cytolytic T cells.

CD4 and CD8 lineages are generated during thymocyte development. TCR+ thymocytes initially express both CD4 and CD8. During positive selection expression becomes limited to either CD4 or CD8 The choice of lineage corresponds to the ability of the  TCR on an individual thymocyte to recognize either MHC class I or MHC class II. CD4 CD8

T reg cell lineage T reg cells block autoimmune activity of other T cells through a variety of mechanisms. T regs develop in thymus as ab TCR, CD4+ cells with high affinity for self-antigen/MHC. It remains unclear how Treg development is distinguished from negative selection. Treg lineage T cells express the FoxP3 transcription factor and IL-2 receptor a Defective FoxP3 alleles are associated with IPEX (Immune dysfunction, polyendocrinopathy/enteropathy, X-linked): male infants exhibit diarrhea, dermatitis, diabetes & thyroiditis.

g/d T cell lineage g/d cells express an alternate form of the TCR. g/d and a/b T cell development is exclusionary. g/d T cells represent a few % of total mature T cells and they have a relatively restricted repertoire. Resident in epithelial sites - skin and mucosa, as well as in circulation.

The g/d TCR has a very similar structure to the a/b TCR The g/d TCR has a very similar structure to the a/b TCR. It is generated by rearrangement of gene segments and the antigen-binding subunits pair with CD3 subunits for expression on the cell surface.

ab and gd T cells develop from the same precursor population ab and gd T cells develop from the same precursor population. Successful rearrangement and expression of gd receptor genes leads to commitment to that lineage and blocks ab T cell development.

Thymocyte development summary progenitor Initial repertoire MHC-selected repertoire TCR a/b, TCR g/d TCR a/b, CD4+ TCR a/b, CD8+ T reg

Lecture objectives T cell life cycle: development in the thymus, circulation as mature, naïve cells, activation in response to antigen, differentiation into memory cells. Generation of diverse antigen receptors on T cells through sequential rearrangement of ß and a gene segments. Shaping of the repertoire through positive and negative selection. Relation of DiGeorge, Omenn and Autoimmune Polyglandular syndromes to thymic development. Why matching of HLA types is important for restoration of immune function following bone marrow transplantation. Generation of distinct T cell lineages: CD4, CD8, T reg and g/d T cells.