1. 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

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

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

4. 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

5. 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.

6. Figure 6-16

7. Figure 5-13

8. 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.

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

10. Figure 5-10

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

12. 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

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

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

15. 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.

16. Figure 5-10

17. Figure 12-31

18. Figure 12-41

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

20. 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

21. Figure 5-11

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

23. Molecular basis of direct allogeneic MHC recognition

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

25. Figure 12-33

26. Figure 12-34

27. 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

28. 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)

29. 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.

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

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

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

33. Figure 5-13

34. Figure 5-4

35. Figure 5-9

36. 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.