1. Generation of antibodies and T cell receptors by V(D)J recombination Lymphocyte development: generation of cells with functional and useful antigen receptors Selection of B cells and T cells based on their specificity to decrease self-reactivity Diseases resulting from defects or errors in lymphocyte development: immunodeficiencies and cancers Lecture on lymphocyte development

2. Generation of antibody & TCR diversity How do we make >10 9 different antibodies or TCRs?

3. Generation of antibody & TCR diversity How do we make >10 9 different antibodies or TCRs? Answer: Genes for antibodies and TCRs are present in pieces that can be combined in many different ways in different lymphocytes (combinatorial diversity) and diversity is further increased by adding or deleting nucleotides at the junctions (junctional diversity)

4. VDJ recombination Ordered rearrangement of gene segments Lymphocyte DNA has 1 functional (in-frame) gene of each type (usually); each cell is unique until clonal expansion and selection Expression of these functional genes follows normal rules of molecular biology C  is always first heavy chain type 100

5. Generation of Antibody Diversity -Combinatorial diversity: -H chains: 100 V H x 27 D H x 6J H = 16,200  light chains: 40 V  x 5 J  = 175  light chains: 30 V x 4 J = 120 -295 L chains x 16,200 H chains = 4.8 x 10 6 -Junctional diversity (addition or deletion of nucleotides at recombination sites, especially of H chain), estimated to add up to 3x10 7 fold to overall diversity (J and D form last loop in Ig domain structure; Ab can tolerate different amino acid lengths resulting from junctional diversity)

6. Machinery of VDJ recombination Rag1, Rag2 (“recombination activating genes”) –Only expressed in developing lymphocytes –Recognize “recombination signal sequences” adjacent to V, D and J segments in DNA –Which Ig or TCR locus is rearranged is also regulated Artemis and Non-homologous end-joining recombination machinery (DNA repair system; expressed in all cells) Uncommon forms of SCID resulting from mutations in Rag1, Rag2 or Artemis (autosomal; total 5-10% of SCID) (SCID=severe combined immunodeficiency)

7. Lymphoid malignancies resulting from errors in VDJ recombination VDJ Recombination reactions contributes to translocation leading to over-expression of a cellular growth or survival promoting gene (“oncogene”)

8. There is an ILM/Problem set on VDJ recombination and lymphocyte development on iROCKET (nothing to hand in) (answers will be posted next Monday)

9. Lymphocyte Development Lymphocyte development is designed to generate functional lymphocytes with useful antigen receptors that are not self-reactive

10. B cell development IgH rearrangement  or rearrangement B cell development requires successful VDJ recombination Heavy chain protein is required for developing cell to progress to pre-B cell stage and start rearrangements at Ig Light chain genes

11. Pre-BCR and B cell development  heavy chain combines with surrogate light chains to form pre- BCR (analogous to BCR) pre-BCR signals to cell to move to next stage of development Pre-BCR and BCR signaling are defective in X-linked agammaglobulinemia due to loss-of- function mutations in Btk: strong block in B cell development and no antibodies. Multiple protein tyrosine kinases including Btk Btk: Bruton’s tyrosine kinase

12. B cell development IgH rearrangement  or rearrangement B cell development requires successful VDJ recombination Heavy chain protein is required to progress to pre-B cell stage and start rearrangements at Ig Light chain genes Light chain protein is required to progress to B cell stage and leave the bone marrow

13. One B cell makes only one heavy chain and one light chain This property of expressing only one of two Ig alleles is called “allelic exclusion”

14. One B cell makes only one heavy chain and one light chain This property of expressing only one of two Ig alleles is called “allelic exclusion” MECHANISM OF ALLELIC EXCLUSION Once a functional heavy chain is made, the pre-BCR can assemble and send signals, this redirects the Rag1 and Rag2 proteins away from the IgH locus and toward the IgL loci Once a functional light chain is made, Rag 1 and Rag 2 expression is turned off

15. One B cell makes only one heavy chain and one light chain This property of expressing only one of two Ig alleles is called “allelic exclusion” MECHANISM OF ALLELIC EXCLUSION Once a functional heavy chain is made, the pre-BCR can assemble and send signals, this redirects the Rag1 and Rag2 proteins away from the IgH locus and toward the IgL loci Once a functional light chain is made, Rag 1 and Rag 2 expression is turned off Allelic exclusion enhances the efficiency of antibodies, since one B cell and its clonal progeny will make homogenous antibodies with two identical binding sites.

16. Central Tolerance of B cells to Self-antigens CONCEPT: Self-antigen is always present but foreign antigens are generally not present at sites of development (due to timing and/or routes of antigen trafficking); therefore developing lymphocytes that see antigen are typically seeing self-antigen

17. Central Tolerance of B cells to Self-antigens CONCEPT: Self-antigen is always present but foreign antigens are generally not present at sites of development (due to timing and/or routes of antigen trafficking); therefore developing lymphocytes that see antigen are typically seeing self-antigen Immature B cell + self-antigen: B cells can continue to rearrange IgL genes, try to change L chain and lose self-reactivity (“receptor editing”) B cells can die (“clonal deletion” or “negative selection”) B cells can become refractory to activation (“clonal anergy”)

18. KEY POINT: The effect of BCR signaling is dependent on the developmental stage Pro-B cells + pre-BCR signal: developmental progression Immature B cell + antigen: inactivation or death of the cell Mature B cell + antigen: activation of the cell (but needs additional signals--described in subsequent lectures) Central Tolerance of B cells to Self-antigens II

19. Development and central tolerance for T cells are nearly the same as for B cells

20. The thymus is a specialized organ for T cell development In DiGeorge syndrome, the thymus gland fails to develop and T cell development is impaired The thymus has several specialized cell types that contribute to T cell development, primarily by presentation of MHC + peptides for selective events (other organs are also affected)

21. Early steps in T cell development Stages in development of T cells are defined by expression of the co-receptors CD4 and CD8 Early Lymphoid progenitor

22. Early steps in T cell development Stages in development of T cells are defined by expression of the co-receptors CD4 and CD8 TCR  is rearranged first; this forms a pre- TCR together with “pre-TCR  ” pre-TCR signals to promote T cell development to the CD4+CD8+ stage, where TCR  rearranges Early Lymphoid progenitor

23. Early steps in T cell development Stages in development of T cells are defined by expression of the co-receptors CD4 and CD8 TCR  is rearranged first; this forms a pre- TCR together with “pre-T  ” pre-TCR signals to promote T cell development to the CD4+CD8+ stage, where TCR  rearranges Survival is dependent on cytokines from thymic environment, especially IL-7 (X-linked form of SCID is caused by mutations in the  c cytokine receptor chain, which is required for IL-7 response) Early Lymphoid progenitor

24. Positive Selection links the specificity of a T cell to its development

25. Positive Selection of Thymocytes Cessation of VDJ recombination at the TCR  locus Loss of expression of the “wrong” co-receptor (e.g.: positive selection with MHC II leads to retained expression of CD4 and turned off expression of CD8) Choice of the corresponding functional lineage (helper vs. cytotoxic T cell) Maturation and eventual export from the thymus to the recirculating T cell pool Receipt of a weak signal through the TCR results in:

26. Positive Selection of Thymocytes Cessation of V(D)J recombination at the TCR  locus Loss of expression of the “wrong” co-receptor (e.g.: positive selection with MHC II leads to retained expression of CD4 and turned off expression of CD8) Choice of the corresponding functional lineage (helper vs. cytotoxic T cell) Maturation and eventual export from the thymus to the recirculating T cell pool KEY POINT: Positive selection is the process that links the specificity of that cell’s TCR for MHC I vs. MHC II to expression of CD4 or CD8 AND to the functional potential of that T cell (helper vs. cytotoxic), therefore specificity and function are matched to give a useful T cell. Receipt of a weak signal through the TCR results in:

27. Positive and Negative Selection in T cell Development

28. Positive vs. Negative Selection NEGATIVE SELECTION: results from strong interaction of a self peptide/MHC complex with the TCR of a thymocyte. (Foreign antigens are not brought to the thymus by APC, so typically antigen in thymus is self) POSITIVE SELECTION: results from weak interaction of a self peptide/MHC complex with the TCR of a thymocyte. (Links the MHC specificity of the TCR to the functional potential of each T cell) Resulting T cells are more likely to be useful

29. T cell tolerance T cell tolerance is created by a combination of mechanisms, of which negative selection is only one. In the thymus, some self-reactive T cells become “regulatory T cells” rather than dying. Regulatory T cells can suppress T cell immune responses in the periphery. These cells and other mechanisms of peripheral tolerance will be discussed next week by Dr. Abbas.

30. Inherited Immunodeficiencies

31. Individual presents with abnormally severe and frequent infections, often early in life Types of infections are often an indication of what type of immunodeficiency Some of the more common immunodeficiencies are X- linked, so this is potentially important information to share with parents Genetic causes include immune-specific genes and also genes involved in purine metabolism (salvage pathway especially important for lymphocytes) Therapies include replacement therapy (B cell immunodeficiencies) and bone marrow transplantation

32. Characteristics of Inherited Immunodeficiencies “Severe combined immunodeficiency (SCID)” Figure 12-1 Abbas and Lichtman TB also

33. Defects in Lymphocyte development leading to Immunodeficiency Figure 12-2 Abbas and Lichtman

34. Immunodeficiencies resulting from defective lymphocyte activation Note: lymphocyte activation is covered in later lectures Figure 12-4 Abbas and Lichtman