• 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

Generation of antibody & TCR diversity

• How do we make >109 different antibodies or TCRs?

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)

VDJrecombinati

on•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

QuickTime™ and a decompressor

are needed to see this picture.

Generation of Antibody Diversity

- Combinatorial diversity:- H chains: 100 VH x 27 DH x 6JH = 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 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)

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)

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”)

QuickTime™ and a decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

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

Lymphocyte Development

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

B cell development

IgHrearrangement

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

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 BtkBtk: Bruton’s tyrosine kinase

QuickTime™ and a decompressor

are needed to see this picture.

B cell development

IgHrearrangement

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

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”

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

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.

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

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”)

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

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

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)

QuickTime™ and a decompressor

are needed to see this picture.

Early steps in T cell development

•Stages in development of T cells are defined by expression of the co-receptors CD4 and CD8

EarlyLymphoidprogenitor

QuickTime™ and a decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

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

EarlyLymphoidprogenitor

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)

EarlyLymphoidprogenitor

Positive Selection links the specificity of a T cell to its

development

QuickTime™ and a decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

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:

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:

Positive and Negative Selection in T cell Development

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

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.

Inherited Immunodeficiencies

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 (salvage pathway especially important for lymphocytes)

•Therapies include replacement therapy (B cell immunodeficiencies) and bone marrow transplantation

Characteristics of Inherited

Immunodeficiencies

“Severe combined immunodeficiency (SCID)”

Figure 12-1 Abbas and Lichtman

TB also

Defects in Lymphocyte development leading to

Immunodeficiency

Figure 12-2 Abbas and Lichtman

Immunodeficiencies resulting from defective lymphocyte

activation

Note: lymphocyte activation is covered in later lectures

Figure 12-4 Abbas and Lichtman