immunogenetic. tonegawa’s demonstration 1976—used restriction enzymes and dna probes to show...

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Immunogenetic

Tonegawa’s demonstration• 1976—used restriction enzymes and DNA probes to show that

germ cell DNA contained several smaller DNA segments compared to DNA taken from developed lymphocytes (myeloma cells)

Bone marrow stromal cells drive Pro-B cell proliferation and maturation.

Important molecules and interactions:SCF-cKitIL-7-IL-7R

Antibody Diversity• How do we acquire so many

different types of antibodies?

– Somatic recombination

– Junctional diversity

– hypermutations

Summary of Splices

• Light chain– V-JC joining by DNA splice (lambda)– V-J joining by DNA splice (kappa)– VJ-C intron removal (RNA splice)

• Heavy chain• D-J joining by DNA splice• V-DJ joining by DNA splice• VDJ-C intron removal by RNA splice• IgM membrane bound 3rd C domain to soluble

3rd C domain by RNA splice• Class switching by DNA splice

Genes for immunoglobulin proteins are found on different chromosomes

Heavy chain rearrangement

Kappa light chain rearrangement

Lymphocyte-specific and ubiquitous enzymes are

required• RAG-1 and RAG-2 are

lymphocyte-specific– Fibroblasts transfected with

RAG-1 + RAG-2 undergo somatic recombination of Ig genes

– RAG-KO mice have no B or T cells

– RAG is active in G0 and G1 mitotic phase and is off in the proliferative status.

– RAG digest DNA in un-precise pattern.

Recombination occurs at specific sites

• Recombination signal sequences (RSS) occur adjacent to coding sequences in V, D, and J segments– Heptamer-spacer-nonamer– 12/23 rule

12/23 rule for gene recombination

• See gene recombination animation on CD

Marker of cells that have undergone V(D)J recombination

Recombination signal sequences (RSS)

12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

V 7 23 9

D7129 7 12 9

J7239

HEPTAMER - Always contiguous with coding sequence

NONAMER - Separated fromthe heptamer by a 12 or 23

nucleotide spacer

V 7 23 9

D7129 7 12 9

J7239

√ √

23-mer

12-mer

Loop of interveningDNA is excised

• Heptamers and nonamers

align back-to-back

• The shape generated by the

RSS’s acts as a target for

recombinases

7

9

97

V1 V2 V3 V4

V8V7

V6V5

V9 D J

V1 DJ

V2

V3

V4

V8

V7

V6

V5

V9

• An appropriate shape can not be formed if two 23-mer flanked elements

attempted to join (i.e. the 12-23 rule)

Molecular explanation of the 12-23 rule

V D J712

9

723

9

7 12 97239

V D J

Imprecise and random events that occur when the DNA breaks and rejoins allows new nucleotides to be inserted or lost from the sequence at and around the coding joint.

Junctional diversity

Mini-circle of DNA is permanently lost from the genome

Signal jointCoding joint

Linkage of C gene to VDJ at the m-RNA level.

Allelic Exclusion• These diversity mechanisms

often generate non-functional Ig genes: genes that contain stop codons or don't stay in the proper reading frame. The developing B cells use a mechanism called "allelic exclusion", in which each B cell makes only 1 active L chain and 1 active H chain. The cell tries each copy of the L genes and each copy of the H genes in turn:

– If an active chain is made, no further DNA splicing occurs.

– However, if a non-functional Ig is made, the cell then tries the next L or H gene.

– This process continues until an active product is made from both H and L, or until all genes have been tried (in which case the cell dies).

Multiple gene segments increase Ig diversity

Combinatorial diversity:

Heavy chains40 x 25 x 6 = 6000

Light chains40 x 5 = 200 30 x 4 = 120

Total possible:320 x 6000 = 1.9x106

Junctional diversity•Nucleotide deletion can also occur

•Occurs in HV3 (CDR3) region

•What problem could these events cause??

N nucleotide addition at joining segments: the addition of random bases

Randomness in joining process helps generate diversity by creating hypervariable of antigen binding site

V TCAG U D JAT

TA

U Endonuclease cleaves single strand at random sites in V and D segment

V TC~GAAG D JAT

TA~TAThe nucleotides that flip out, become part of the complementary DNA strand

Generation of the palindromic sequence

In terms of G to C and T to A pairing, the ‘new’ nucleotides are palindromic.The nucleotides GA and TA were not in the genomic sequence and introduce diversity of sequence at the V to D join.

V TCAG U D JAT

TA

U

Regions to be joined are juxtaposed

The nicked strand ‘flips’ out

Junctional Diversity – N nucleotide additions

V TC~GAAG D JAT

TA~TA

Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA

CACTCCTTA

TTCTTGCAA

V TC~GAAG D JAT

TA~TA

CACACCTTA

TTCTTGCAA Complementary bases anneal

V D JDNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands

TC~GAAG

ATTA~TA

CACACCTTA

TTCTTGCAA

D JTA~TAExonucleases nibble back free endsV TC~GACACACCTTA

TTCTTGCAA

V TCDTA

GTT AT AT

AG C

Imprecise joining generates diversity

V D JTCGACGTTATATAGCTGCAATATA

Junctional Diversity

TTTTTTTTTTTTTTT

Germline-encoded nucleotides

Palindromic (P) nucleotides - not in the germline

Non-template (N) encoded nucleotides - not in the germline

Creates an essentially random sequence between the V region, D region and J region in beta chains and the V region and J region in alpha chains.

Additional Diversity Mechanisms• In addition to the DNA splicing,

other variants in the antibody molecules are generated by two mechanisms:

• First, the DNA splices do not occur at a precise point: they can vary by several bases, which can lead to the addition or deletion of 1 or 2 amino acids at each splice point. – These variations can lead to

different specificities of the antibodies.

– The enzymes that do the DNA splicing (RAG1 and RAG2) produce double stranded breaks in the DNA, which is repaired imprecisely.

P and N region nucleotide alteration adds to diversity of V region

• During recombination some nucleotide bases are cut from or add to the coding regions (p nucleotides)

• Up to 15 or so randomly inserted nucleotide bases are added at the cut sites of the V, D and J regions (n nucleotides).

• TdT (terminal deoxynucleotidyl transferase) a unique enzyme found only in lymphocytes (20 N added)

• Since these bases are random, the amino acid sequence generated by these bases will also be random

B lymphocyte development

IgM and IgD are coexpressed in mature

naïve B cells

Alternative RNA processing generates transmembrane or

secreted Ig

Synthesis, assembly and secretion of

immunoglobulins

Combination of heavy and light chains adds final diversity of variable region

• 8262 possible heavy chain combinations

• 320 light chain combinations

• Over 2 million combinations

• P and N nucleotide additions and subtractions multiply this by 104

• Possible combinations over 1010

IL-7 has critical role in the proliferative activity of B and T lymphocytes precursor.

Somatic hypermutation adds even more variability

• B cell multiplication introduces additional opportunities for alterations to rearranged VJ or VDJ segments

• These regions are extremely susceptible to mutation compared to “regular” DNA, about one base in 600 is altered per two generations of dividing (expanding) lymphocyte population

Further Ig diversity arises through affinity maturation

Affinity maturation is due to somatic hypermutation

SilentNeutralDeleteriousPositive

Somatic Hypermutation• Second, there is a “somatic

hypermutation” mechanism by which random base change mutations occur in the V regions in B cells.

– This mechanism doesn't work in other cells and doesn't affect other genes: only a region of about 1.5 kb is affected.

– It only occurs as the B cell is maturing: after it has been stimulated to divide by an antigen, somatic hypermutation occurs to modify the antigen binding region.

– Those cells that bind the antigen most tightly survive and divide more than the others. This process is called “affinity maturation”.

– It is triggered by the enzyme “activation-induced cytidine deaminase” (AID), which deaminates cytidine to uracil. This base mismatch can be incorrectly repaired by several different mechanisms to generate mutations.

Isotype switching

• Irreversible

• Only occurs after a given B cell has encountered antigen

• Mechanism not fully understood– Requires AID– Requires DNA repair enzymes– Requires external signals (helper T cells)

Isotype switching occurs in activated B cells

Class Switching• Heavy chains fall into 5 classes,

based on their C regions. • Each H gene has C regions for

all 5 classes arranged on the chromosome, with the IgM C region nearest to the V regions.

• There are several different C regions for some of the classes.

• IgM is the initial Ab made by each B cell.

• However, after a while the B cell switches to a different class.

• This is done using a third DNA splice, in which the DNA between the VDJ and the constant region for the new class is spliced out.

Class switching among constant regions: generation of IgG, IgA and IgE with same antigenic determinants—idiotypes

7 means of generating antibody diversity

Location of variability occurs within CDR regions of V domains (antigen binding sites)

Mature B cells undergo Positive and negative selection in the bone marrow and then migrate to the peripheral blood. Negative selection carried out with membrane bound self antigen for immature expressing IgM+ B cells and resulted in apoptosis. In some cases receptor editing (mainly for k gene) resulted in changes in the specificity of B cells.

Stages in B-cell maturation in the bone marrow

From Pro- to Pre-B cell transition events

B cell development

T cell development

T Cells These are some of the General characteristics of the TCell receptor for antigen recognition.

The Thymus: What is it?

2-lobed organ, divided into lobules, in thechest, just above the heart

Approximately 2-3 hundred millionthymocytes (in the mouse)

Consists of stromal components andthymocytes at various stages of maturation

T cell development occurs in the thymus

Lack of thymus in mice and human resulted to nude mice and digeorge syndrome in human.

66

Progression through development correlates with rearrangement

67

TCR locus has two D-J clustersAllows a 2nd rearrangement if 1st is nonproductive

Diversity in the TCR gene locus

69

Pre-T cell receptor

How do you test for successful TCRbeta chain rearrangements if you have not rearranged TCRalpha?

Pre-Talpha

The TCR and BCR gene is most variable in the CDR3 region

CDR3

Allelic exclusion occurred in B-TCR but not in a-TCR, So 30% of Ln T has TWO types TCR and co-expressed both allele of a-TCR.

72

73

Thymocytes at different developmental stages are found in distinct parts of the thymus

Maturation

Development of T cells in a mouse.

RAG rearrangement

Tolerance to self antigens encountered in the thymus is achieved by eliminating T cells that are reactive to these antigens.

Major Thymocyte Subsets: (cont.)

CD4-CD8- (Double Negative, DN) cells: 3-5% of total thymocytes Contain least mature cells, considerable cell division 2/3rds are triple negative (TN) based on TCR expression Can be further divided based on CD44 and CD25 (discussed later) TCR and rearrangements occur at this stage 1/3rd are TCR +

CD4+CD8+ (Double Positive, DP) cells: 80-85% of total thymocytes TCR rearrangement occurs at this stageMost have rearranged TCR genes and express low levels (1/10th mature level) of TCR Small subset has high levels of TCR (most mature, positively selected cells) Small subset is actively dividing (earliest DPs) Most apoptosis occurs here, very sensitive to apoptosis inducing agents, especially sensitive to glucocorticoids

CD4+CD8- and CD4-CD8+ (Single positive, SP) cells: 10-15% of total thymocytes Most are mature cells with high levels of CD3 and TCR CD4:CD8 approx 2:1 ratio Most SP cells are functionally mature and are destined to leave the thymus Small subset of SP are immature (ISP) (CD8 in mouse, CD4 in human) and have low CD3 and no TCR - transitional cells that are on the way from DN -> DP

How do DP Cells Become SP?

• Instructive Model– Fitting

• Stochastic Model– Random

Mechanism of Selection

• Avidity– Differences in signal strength is dictated by

TcR:MHC avidity– “Quantitative” Model

• Differential Signaling– Selection outcomes are dictated by different

signals– Qualitative

82

TCR locus has 50 J segments

T Cell Receptors• The TCR protein has 2 subunits

and one antigen binding site.• The alpha subunit has V and J

segments (similar to Ig light chains)

• The beta subunit has V, D and J regions, like the Ig heavy chain.

• Both segments undergo DNA splicing rearrangements like the Ig genes. The joining is not precise and short additions or deletions of bases can occur, as in the Ig genes. However, affinity maturation and somatic hypermutation do not occur.

• The TCR protein is membrane bound. It is only found on T cells.

Sequentially making and expressinga pre-TCR and TCR

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

Summary of Thymic Development

Lineage Commitment Models

DeFranco, Locksley, & Robertson, Immunity, NSP, 2007)

Newer Lineage Commitment Models

DeFranco, Locksley, & Robertson, Immunity, NSP, 2007)

Positive Selection

• Results in MHC restriction

• Mechanism:– Immature thymocytes cluster with MHC

molecules on the cortical cells of the thymus• If TCR interacts with MHC protective signal

results that prevents apoptosis.• If TCR does not interact with MHC no protective

signal and apoptosis occurs.

• Result? Only reactive thymocytes survive.

Negative Selection

• Ensures self-tolerance• Weeds out High affinity thymocytes• Mechanism:

– APC’s bearing MHC’s interact with thymocytes• If avidity is too strong thymocyte undergoes

apoptosis.• Details unknown…

• Result? Only self-tolerant thymocytes survive.

Generation of diversity in the TcR

COMBINATORIAL DIVERSITYMultiple germline segments

In the human TcR

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

JUNCTIONAL DIVERSITYAddition of non-template encoded (N) and palindromic (P) nucleotides atimprecise joints made between V-D-J elements

SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR

ElementImmunoglobulin TcR

Variable segments

Diversity segments

D segments inall 3 frames

Joining segments

Joints with N & Pnucleotides

No. of V gene pairs

Junctional diversity

Total diversity

H

40

27

Yes

6

22360 3640

~1013 ~1013

~1016** ~1016

59

0

-

9

(1)*

52 ~70

2 0

Yes -

13 61

2 1

* Only half of human chains have N & P regions**No of distinct receptors increased further by somatic hypermutation

Estimate of the number of human TcR and IgExcluding somatic hypermutation

Summary: Ig vs TCR

Summary: mechanisms that generate diversity in lymphocyte

receptors

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