Transcript PowerPoint bemutató

GENERATION OF B AND T CELL DIVERSITY
LYMPHOCYTE DEVELOPMENT
GENERATION OF RECEPTOR DIVERSITY
IN B CELLS
1 GENE = 1 PROTEIN??
The total number of antibody specificities available to an individual is known as the
antibody repertoire, that in humans is at least 1013, perhaps many more.
BUT
There are an estimated 20,000-25,000 human protein-coding genes.
????
THEORIES FOR IG-DIVERSITY
1 GENE
Somatic diversification theory
High rate of somatic mutations in the V-region
V
C
Germline theory
Separate genes for each different Ab
V1 C1
V2 C2
Vn
Cn
AMINO ACID SEQUENCE OF IMMUNOGLOBULINS
Multiple myeloma (MM)
Plasma cell tumors – tumor cells reside in the bone marrow
Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/ml
Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification
Gel electrophoresis
L
H
Reduction
50 kDa
Heavy chain
25 kDa
Light chain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Variable
Constant
THE DREYER - BENNETT HYPOTHESIS
The Dreyer - Bennett hypothesis
V
V
V
V
region genes
V
V
V
V
V
V
A single C region gene is
Aencoded
single C in
region
is and
the gene
germline
encoded
in the
germline
and
separated
from
the multiple
V
separated from
the genes
multiple V
region
V
V C
V
V
C
A mechanism
to rearrange
and C genes
A mechanism
toVrearrange
V and C genes
in the genome
exists
so thatexists
they can
in the
genome
sofuse
that they can
to form a complete
Immunoglobulin
fuse to form
a completegene
Immunoglobulin
gene
The germline organization of the human immunoglobulin light-chain loci
L2
Vκ2 Jκ5
Cκ
SOMATIC RECOMBINATION
During B-cell development a Vκ
gene segment rearranges to a Jκ
gene segment to create a functional
exon encoding the V domain
Selection of gene segments encoding the V domain is random
EXPRESSION OF KAPPA CHAIN
Vκ
P
Vκ
Jκ
Jκ
E
Cκ
Jκ
E
Cκ
pA
Vκ-Jκ
Leader
Vκ
Jκ
Primery RNS transcript
Vκ
Jκ Cκ
AAAA
mRNS
Translation
Vκ
Jκ
Cκ
Protein
In developing B cells, the immunoglobulin genes undergo
structural rearrangements that permit their expression. (As
a result of gene rearrangement the promoter of the gene is
placed under the control of the enhancer region.)
The V domains of immunoglobulin light chains are encoded by
two (V and J) distant gene segments, that are brought into
juxtaposition by a SINGLE recombination event.
THE GERMLINE ORGANIZATION OF THE HUMAN
IMMUNOGLOBULIN HEAVY CHAIN LOCUS
HEAVY CHAIN V REGION IS ASSEMBLED FROM
THREE GENE SEGMENTS
L VH
DH
JH
Cμ
Cμ
Cμ
Cμ
Two recombination events are required to form the exon encoding the V domain of the heavy chain:
DH to JH and VH to DHJH
L
VL
JL
CL
A single recombination event is required to form the exon encoding the V domain of the light chain:
VL to JL
V-REGION SEQUENCES ARE ASSEMBLED FROM GENE SEGMENTS
CDR1 and CDR2
CDR3
CDR1 and CDR2
CDR3
In developing B cells, the immunoglobulin genes undergo
structural rearrangements that permit their expression. (As
a result of gene rearrangement the promoter of the gene is
placed under the control of the enhancer region.)
The V domains of immunoglobulin light chains are encoded by
two (V and J) distant gene segments, that are brought into
juxtaposition by a SINGLE recombination event.
The V domain of immunoglobulin heavy chain is encoded by
three (V, D, and J) distant gene segments, that are
brought into juxtaposition by TWO recombination events.
DIVERSITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES
1
2
B-cells
3
4
V-Domains
C-Domains
VH
D
1
VH-D-JH
2
VH-D-JH
JH
VL
JL
VL-JL
VL-JL
ESTIMATED COMBINATORIAL DIVERSITY
46 VH x 23 D x 6JH = 6 348 different heavy chains
38 Vκ x 5 Jκ = 190 combinations
33 Vλ x 5 Jλ = 165 combinations
= 355 different light chains
With random combination of H and L chains:
6 348 x 355 = 2 253 540 different antibodies
Due to the COMBINATORIAL diversity only!!
MECHANISM OF
SOMATIC RECOMBINATION
How is the appropriate order of the
gene segments ensured?
For example VH-JH joining is not
allowed...
RECOMBINATION SIGNAL SEQUENCES (RSS)
Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23,
9 and 12 nucleotides in an arrangement that depended upon the locus
HEPTAMER – Always adjacent to the coding
sequence
Vl
7
Vk
7
23
12
7
23
9
9
12
9
7
12
9
9
9
VH
NONAMER – Separated from the heptamer by a 12or 23-bp spacer
D
23
7
12
9
7
Jl
7
Jk
9
23
7
JH
12-23 RULE – A gene segment flanking by a 23-mer RSS can only be linked
to a segment flanking by a 12-mer RSS
THE MECHANISM OF VDJ RECOMBINATION
JUNCTIONAL DIVERSITY
Junctional diversity: The largest contribution to
antigen receptor diversity is made by removal or
addition of nucleotides at the junctions of
rearranged gene segments
P ( palindromic) nucleotides: nucleotides added
to the asymmetrically cleaved hairpin ends
N nucleotides: random, non–template-encoded
nucleotides added by the polymerase TdT (up to
20)
B CELL DEVELOPMENT
PHASES OF B CELL DEVELOPMENT AND DIFFERENTIATION
Bone marrow
Secondary lymphoid tissues
EARLY STAGES OF B CELL DEVELOPMENT
DEVELOPING B CELLS CAN MAKE TWO ATTEMPTS TO
REARRANGE THE HEAVY CHAIN GENE
A single rearrangement can be made on both
chromosomes: during the first rearrangement all the
non-rearranged D segments are excised
1ST CHECKPOINT
1ST CHECKPOINT
Monitoring the quality of immunoglobulin heavy chain
Signaling receptor:
• Survival signals
• Allelic exclusion
• Proliferation
• Inhibition of the expression of surrogate light chain
• Induction of the rearragment of light chains
NONPRODUCTIVE LIGHT CHAIN GENE REARRANGEMENTS
CAN BE SUPERSEDED BY FURTHER GENE REARRANGEMENT
SUCCESSFUL REARRANGEMENT OF THE IMMUNOGLOBULIN
LIGHT CHAIN GENES IN PRE-B CELLS LEADS TO THE
EXPRESSION OF CELL SURFACE IgM
2ND CHECKPOINT
Monitoring the quality of
light chain
FATE DETERMINING CHECKPOINTS
MATURATION AND SURVIVAL OF IMMATURE B CELLS
REQUIRES ACCESS TO LYMPHOID FOLLICLES
Co-expression of IgD and IgM-type BCR on the surface of naive
B cells is mediated by alternative splicing and polyadenylation
SUMMARY
T CELL DEVELOPMENT
CELLULAR ORGANIZATION OF THE THYMUS
ORGANIZATION AND REARRANGEMENT OF THE T CELL
RECEPTOR GENES
T CELL DEVELOPMENT AND MIGRATION IN THE THYMUS
Only a small fraction of T cells mature into functional T cells
POSITIVE SELECTION
-αβ double-positive thymocytes must recognize selfMHC.
-Continuing α-chain gene rearrangement increases the
chance for positive selection
-Ca. 10-12% of thymocytes survive!
Positive selection --- results in clones that are
reactive to SELF MHC.
BASIS OF MHC RESTRICTION!!!
Positive selection of double
positive (dp) T cells also directs
CD4 and CD8 single positive (sp)
T-cell commitment
THYMIC EPITHELIAL CELLS
ARE MHCI/MHCII POSITIVE!
BARE LYMPHOCYTE SYNDROME (BLS)
Lack of MHC class I – no CD8+ T-cells
Lack of MHC class II – no CD4+ T-cells
CLINICAL SIGNIFICANCE
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