Lecture 10 Thymocyte selection II Thymocyte positive selection A word about signaling Demonstration in radiation bone marrow chimeras Implications for transplantation The importance of eliminating mature T cells in bone marrow transplantation Thymocyte negative selection The limitations of thymic (central) tolerance The role of ectopic antigen expression & AIRE Reminder about the existence of gd T cells

Positive and Negative Selection The thymus: Selects the useful Ignores the useless Removes the harmful Positive Selection Death by neglect Negative selection

Figure 5-12 part 1 of 2 CD4 CD8 ab TCR CD8 T cell CD4 T cell

Effects of defined mutations on T cell selection MHC class I deficient No mature CD8 cells MHC class II deficient No mature CD4 T cells TAP deficient No mature CD8 T cells CD8 deficient No killer cells CD4 deficient No helper cells CD3 deficient No T cells RAG deficient No T cells (or B cells) pTa deficient Few ab T cells No thymus (DiGeorge) No T mature cells No thymocytes (ADA-) No T mature cells

The signals from cell surface receptors that lead to positive and negative selection are believe to start with protein phosphorylations followed by poorly understood signal integration that finally leads to changes in transcription of genes determining cell fate and differentiation.

Figure 6-16

Figure 5-13

Bone marrow transplantation and radiation chimerism This technique relies on several differences between white blood cells and other cell types. White blood cells, especially lymphocytes, are very sensitive to radiation-induced or chemotherapy-induced cell death. Most host cells, including the thymic epithelium are radiation resistant. White blood cells can be reconstituted by repopulation from bone marrow stem cells. Treated recipients are depleted of leukocytes then reconstituted (rescued) with bone marrow (usually depleted of recirculating T cells) or hematopoietic stem cells that give rise to all blood cells.

lymphocytes in lymph nodes, spleen Cells of the immune system Bone marrow/fetal liver Thymus Naïve, resting lymphocytes in lymph nodes, spleen Adaptive Innate

Figure 5-10

Figure 5-12 part 1 of 2 CD4 CD8 ab TCR

Figure 5-13 What happens in a radiation chimera? Positive selection to host cortical epithelial cells Figure 5-13 Radioresistant host type Bone marrow derived Donor type Negative selection to donor and host MHC

Host type controls MHC restriction of T cell repertoire AXB A AXB B

See next slide for explanation Development of Major Histocompatibility Complex Restriction Patterns in Bone Marrow Plus Thymic Chimeras See next slide for explanation

Explanation of the chimera experiment This is the radiation chimera experiment that you will need to understand. The nomenclature is bone marrow donor -> irradiated recipient. Thus an F1 -> parent is an irradiated mouse of strain A reconstituted with bone marrow from strain (AxB)F1. Please understand this. In an F1 -> parent chimera, the F1 T cells mature in the epithelial environment of the parent that expresses only one of the two SETS of alleles. This skews the population to express TCRs that recognize the parental alleles at some moderate affinity. Although I didn’t go into the second line of this experiment, it is accurate, and actually makes a separate point. This would be the parent -> F1. If bone marrow from the A strain is used to reconstitute an irradiated (AxB)F1, then the T cells will be selected to recognize both strain A and strain B. The APCs will express only MHC from strain A (bone marrow origin), and thus the virus will be presented only on strain A. Thus, despite the fact that the T cell population has the potential to respond to either A or B, the antigen is presented only on A, and thus in the virus-response, the only the T cells restricted to virus + A will be expanded. The last two lines show that the MHC allele expressed by the thymus epithelium restricts the population to the recognition of antigen peptides presented by MHC allele of the thymus--despite the fact that, in these mice, the antigen peptides are presented by both a and b alleles.

Figure 5-10

Figure 12-31

Figure 12-41

What happens to thymic positive selection if there is an MHC incompatibility between donor and host?

Figure 5-13 What happens in a radiation chimera? Positive selection to host cortical epithelial cells Figure 5-13 Radioresistant host type Bone marrow derived Donor type Negative selection to donor and host MHC

Figure 5-11

Positive and Negative selection Figure 5.12 Allogeneic chimera experiment: A -> B Semi-allogeneic chimera experiment: A -> (AxB)F1

Molecular basis of direct allogeneic MHC recognition

The importance of eliminating Mature T cells from the bone marrow inoculum before transplant

Figure 12-33

Figure 12-34

Molding of thymocyte repertoire by self MHC + peptides Double positive thymocyte ab TCR CD4 CD8 Too strong signal death by negative selection No signal death by neglect Appropriate, weak signal through MHC+self peptide promotes survival loss of irrelevant coreceptor (CD8 or CD4) maturation to helper or killer phenotype

Self-tolerance mechanisms in the thymus (Central tolerance) Cells with high affinity to self (+MHC) are eliminated Problem: The thymus is a relatively small, sequestered organ. How can T cell tolerance be induced to self-proteins produced by other tissues? Three redundant solutions AIRE induced, "ectopic" expression of peripheral antigens in the thymus The generation of regulatory (suppressor) cells Mature T cells are subject to additional tolerance mechanisms (Peripheral tolerance)

AIRE Mutations of this gene are responsible for the clinical disorder autoimmune polyendocrinopathy syndrome type I Patients develop multiple autoimmune syndromes to endocrine organs, such as parathyroid and adrenal glands. The protein is a transcription factor that is believed to turn on many tissue specific genes IN THYMIC MEDULLARY EPITHELIAL CELLS and plays a role in negative selection.

Figure 13-9 part 1 of 2 Slide from Janeway book

Figure 13-9 part 2 of 2 Slide from Janeway book

gd T cells also develop in the thymus CD4 ~4% CD8 Developing T cells with no b chain: including gd

Figure 5-13

Figure 5-4

Figure 5-9

Summary of thymocyte development The major pathway of thymocyte development involves ab T cells. The thymus is also necessary for gd T cell development. ab T cell development starts with b gene recombination, expression, and clonal expansion. Cells then recombine TCRa and are screened for positive selection on cortical epithelial cells. Thus "self MHC" is defined by the cortical epithelial cell. Positive selection involves signals initiated with tyrosine phosphorylations that promote directed differentiation. Lymphocytes can be repopulated by bone marrow transplantation. Negative selection occurs in the thymus, where T cells reactive to many self-antigens are expressed on (or by) medullary epithelial cells and other antigen presenting cells. Negative selection can also occur at later developmental stages.