The Immune System:

Antigen – A molecule that can bind specifically to an antigen receptor, such as an antibody.

B cell – A lymphocyte that produces antibodies. Development occurs primarily in bone marrow. B cells emerging from the marrow undergo further differentiation in the bloodstream and peripheral lymphoid organs. The Immune System:

B cell receptor (BCR) – The receptor for antigen expressed on the surface of B lymphocytes. The BCR has the same structure and specificity of the antibody that will be produced by the same B cell after its activation by antigen. The Immune System:

T cell receptor (TCR) – The antigen receptor on T lymphocytes. It is clonally expressed and binds to a complex of MHC class I or class II protein and antigen-derived peptide.

Antibody response – An immune response that is mediated primarily by antibodies. It is defined as immunity that can be transferred from one organism to another by serum antibody. Primary and secondary response to antigen Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen

Clonal selection – The theory proposes that each lymphocyte expresses a single antigen receptor specificity and that only those lymphocytes that bind to a given antigen are stimulated to proliferate and to function in eliminating that antigen.

Thus, the antigen "selects" the lymphocytes to be activated. Clonal selection was originally a theory, but it is now an established principle in immunology. Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen

The B cell and T cell repertoires include BCRs and TCRs with a variety of specificities. Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen

Each B cell expresses a unique BCR and each T cell expresses a unique TCR.

A broad repertoire of BCRs/antibodies and TCRs exists at any time in an organism.

Antigen binding to a BCR or TCR triggers the clonal proliferation of that receptor-bearing B or T cell. Immunoglobulin (Ig) Molecule:

An antibody (immunoglobulin) consists of a tetramer of two identical light (L) chains and two identical heavy (H) chains. There are two families of L chains (Igλ and Igκ) and a single family of IgH chains.

Each chain has an N- terminal variable (V) region and a C- terminal constant (C) An antibody (immunoglobulin, or Ig) region. The V region molecule is a heterodimer consisting recognizes the antigen and the C region of two identical heavy chains and two mediates the effector identical light chains. responses. Ig Genes are Assembled from Discrete DNA Segments in developing B Lymphocytes V(D)J recombination

Unique mechanism of genetic recombination occurring only in B and T lymphocytes during their early stages of maturation.

Occurs in primary lymphoid organs (bone marrow for B cells and thymus for T cells).

It is a random process.

Results in novel amino acid sequences in the antigen-binding regions of Igs and TCRs that allow for the recognition of antigens from nearly all pathogens, altered self cells (e.g. cancer cells) and self cells (e.g. autoimmunity). Ig gene locations:

Human Ig heavy chain locus: chromosome 14.

Human Ig kappa light chain: chromosome 2

Human Ig lambda chain: chromosome 22 Multigene organization of germline Ig genes Somatic Recombination:

Occurs in the somatic cells and not the germline cells.

Germ-line DNA in progenitor B cells provides H and L chain genes directing the synthesis of Heavy and Light chains respectively. They have V, (D) and J segments in un-rearranged form.

H chain (chromosome 14) locus has 3 regions – V, D, and J L chain (chromosome 2 for κ and 22 for λ) locus has 2 regions – V and J (no “D”)

VDJ rearrangement occurs first during the maturation of B cells.

VDJ rearrangement on ‘H’ chain occurs in pro-B cells to produce the Heavy chain.

VJ rearrangement on ‘L’ chain occurs in precursor B cells to produce the Light chain.

After all the re-arrangements, the B cells are classed as immature B cells. Stages of B cell differentiation in bone marrow are defined by Ig gene rearrangements

B CELL STAGE Stem cell Early pro-B Late pro-B Large pre-B

IgH GENE Germline CONFIGURATION DH to JH VH to DHJH VHDHJH

Pre-B cell receptor expressed

Ig light chain gene has not yet rearranged Ig Genes are Assembled from Discrete DNA Segments in developing B Lymphocytes

V and C regions are separately encoded by V(D)J gene segments and C gene segments, respectively.

A gene coding for a whole Ig chain is generated by somatic recombination of V(D)J genes (variable, diversity, and joining genes in the H chain; variable and joining genes in the L chain) giving rise to V domains, to be expressed together with a given C gene (C domain). H Chains Are Assembled first by Two Sequential Recombination Events

The units for H chain recombination are a VH gene, a D

segment, and a JH-CH gene segment.

The first recombination joins D to JH-CH. The second recombination joins VH to DJH-CH to yield VH-DJH-CH.

The CH segment consists of four exons. VDJ Recombination: V, D, J flanking sequences

Sequencing up and down stream of V, D and J elements. Conserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement that depended upon the locus

Vl 7 23 9 9 12 7 Jl

Vk 7 12 9 9 23 7 Jk

9 12 7 D 7 12 9

VH 7 23 9 9 23 7 JH Recombination signal sequences (RSS)

HEPTAMER - Always contiguous with NONAMER - Separated from coding sequence the heptamer by a 12 or 23 9 12 7 D 7 12 9 nucleotide spacer

VH 7 23 9 9 23 7 JH

√ √ 9 12 7 D 7 12 9

VH 7 23 9 9 23 7 JH

12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS The length of the spacer region corresponds to approximately one (12 basepairs) or two turns (23 basepairs) of the DNA helix. Following what is known as the 12/23 Rule V(D)J DNA Recombination Uses RSS and Occurs by Deletion or Inversion

Recombination occurs by double-strand DNA breaks (DSBs) at the heptamers of two RSSs with different spacers: 12/23 rule.

Breakage and recombination at RSSs generate VJC sequences.

Adapted from D. B. Roth, Nat. Rev. Immunol. 3 (2003): 656-666. RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments

The recombination activating gene (RAG) proteins are necessary and sufficient for the cleavage reaction.

RAG1 recognizes the nonamer consensus sequences for recombination. RAG2 binds to RAG1 and cleaves DNA at the heptamer. The reaction resembles a transposition. Severe Combined ImmunoDeficiency (SCID)

A syndrome that stems from mutations in different genes that result in B and/or T cell deficiency.

X-linked SCID is due to IL-2R γ chain gene mutations; autosomal recessive SCID can be due to RAG1/RAG2 mutations, Artemis gene mutations, Jak3 gene mutations, ADA gene mutations, IL-7R α-chain mutations, CD3 δ or ε mutations, or CD45 gene mutations. Recombination Generates Diversity

4 The human IgH locus can generate in excess of 10 VHDJH sequences. Imprecision of joining and insertion of unencoded nucleotides 6 further increases VHDJH diversity to 10 sequences. 4 Recombined VHDJH-CH can be paired with in excess of 10 recombined VκJκ-Cκ or VλJλ-Cλ chains.

The human and mouse Igk families consist of Vk gene segments and five Jk segments The lambda family consists of Vl gene linked to a single Ck gene segment. segments and a small number of Jl-Cl gene segments. Allelic Exclusion Is Triggered by Productive Rearrangements

V(D)J gene rearrangement is productive if it leads to expression of a protein.

A productive V(D)J gene rearrangement prevents any further rearrangement of the same kind from occurring, whereas a nonproductive rearrangement does not.

Allelic exclusion applies separately to L chains (only one VκJκ or VλJλ may be productively rearranged) and to VHDJH chains (one H chain is productively rearranged). B cells have several chances to successfully rearrange Ig genes

Early Pro B Late Pro B Pre B Immature B V -DJ k on first DH-JH YES H H YES YES IgMk On first On first chromosome NO chromosome chromosome YES NO NO YES YES k on second Y chromosome B DH-JH VH-DJH NO On second On second l on first YES IgMl chromosome chromosome chromosome NO NO YES NO Y l on second B B chromosome NO Igs comprise five classes according to the type of CH chain:

Immunoglobulin type and functions are determined by the H chain. Primary and secondary response to antigen

Ig H chain Switching Class Switching Is Effected by DNA Recombination (Class Switch DNA Recombination, CSR)

Class switching is effected by a recombination between S regions that deletes the DNA between the upstream CH region gene cluster and the

downstream CH region gene cluster that is the Class switching of CH genes. target of recombination. Somatic Hypermutation (SHM) Generates Additional Diversity

Somatic hypermutation (SHM) introduces somatic mutations in the antigen-binding V(D)J sequence. Such mutations occur mostly as substitutions of individual bases. Occurs in mature B cells (making Ig molecules.

Somatic mutation occurs in the region surrounding the V segment and extends over the recombined V(D)J segment. The 4 mechanisms in which B cells generate receptor diversity:

1.Somatic recombination: 1.VDJ recombination for heavy chain 2.VJ recombination (No “D” segment) for light chain

2.Pairing of various light chains with heavy chains.

3.Junctional diversity (nucleotide addition or removal).

4.Somatic hypermutation (single point mutations in antibody antigen-binding V region). Primary and secondary response to antigen

Ig H chain Switching

Affinity Maturation The T-cell receptor (TcR) is a heterodimer.

TcR heterodimers are either: – ab (alpha•beta) – gd (gamma•delta)

There are two types of T-cells: TH & TC. Can they have the same TcR? (Yes.) BcR versus TcR structure: TcR (αβ)-Peptide (P)- MHC (α1-3) Interaction:

TcR

Peptide

MHC The T Cell Receptor for Antigen (TcR) Is Related to the BCR T cells use a mechanism of V(D)J recombination similar to that of B cells to express either of two types of TcR. TcRαβ is found on more than 95% and TCRγδ on less than 5% of T lymphocytes in the adult.

The human TcR locus contains interspersed and segments. The TcR locus contains many V gene segments spread over ~500 kb that lie ~280 kb upstream of the two D-J-C clusters. Organisation of TcR genes

L & V x70-80 J x 61 C

TcR a

L & V x52 Db1 Jb1 x 6 Cb1 Db2 Jb2 x 7 Cb2

TcR b

TcR genes segmented into V, (D), J & C elements (VARIABLE, DIVERSITY, JOINING & CONSTANT) Closely resemble Ig genes (a~IgL and b~IgH)

This example shows the mouse TcR locus TcR Gene rearrangents (BM and Thymus): Generation of diversity in the TcR

COMBINATORIAL DIVERSITY Multiple germline segments

In the human TcR Variable (V) segments: ~70a, 52b Diversity (D) segments: 0a, 2b Joining (J) segments: 61a, 13b The need to pair a and b chains to form a binding site doubles the potential for diversity

JUNCTIONAL DIVERSITY Addition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements

SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR T cell co-receptor molecules

Lck PTK TcR Lck PTK TcR

CD8 a3 CD4 b2 b a

MHC Class I MHC Class II

CD4 and CD8 can increase the sensitivity of T cells to peptide antigen MHC complexes by ~100 fold TcR-CD3 complex

a TcR b

CD3 CD3 The CD3g, d, e or z (zeta) chains e d g e are required for cell surface expression of the TcR-CD3 complex and signalling through the TcR

+ - - + + - -

The intracytoplasmic region of the TcR chain is too short z z to transduce a signal

Signalling is initiated by aggregation of TcR by MHC-peptide complexes on APC