Generation of antibodies and T cell receptors by V(D)J recombination

1. Lecture on lymphocyte development • 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

2. Generation of antibody & TCR diversity • How do we make >109 different antibodies or TCRs?

3. Generation of antibody & TCR diversity • How do we make >109 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. VDJrecombination 100 • 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 Cm is always first heavy chain type

5. Generation of Antibody Diversity • Combinatorial diversity: • H chains: 100 VH x 27 DH x 6JH = 16,200 • k light chains: 40 Vk x 5 Jk = 175 • l light chains: 30 Vl x 4 Jl = 120 • 295 L chains x 16,200 H chains = 4.8 x 106 • Junctional diversity (addition or deletion of nucleotides at recombination sites, especially of H chain), estimated to add up to 3x107 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 this Friday)

9. Lymphocyte Development Lymphocyte development is designed to generate functional lymphocytes with useful antigen receptors that are not self-reactive Much of what happens during lymphocyte development is designed to improve the efficiency of adaptive immunity

10. B cell development IgH rearrangement k or l 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 • m 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 k or l 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”) (multiple mechanisms probably reflect the demands of efficiency and the importance of tolerance to self-antigens)

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

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

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

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

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

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

24. Positive Selection of Thymocytes Receipt of a weak signal through the TCR results in: • Cessation of VDJ recombination at the TCR a 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

25. Positive Selection of Thymocytes Receipt of a weak signal through the TCR results in: • Cessation of V(D)J recombination at the TCR a 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.

26. Positive and Negative Selection in T cell Development

27. 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 (again: efficiency)

28. 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.

29. Inherited Immunodeficiencies

30. Inherited Immunodeficiencies • 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 • Therapies include replacement therapy (B cell immunodeficiencies; ADA deficiency) and bone marrow transplantation

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

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

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