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Abbas Chapter 8
Lymphocyte Development and the
Rearrangement and Expression of
Antigen Receptor Genes
Introduction
Lymphocyte development (maturation)
The process by which lymphocyte
progenitors in central lymphoid organs
such as the thymus and bone marrow
differentiate into mature lymphocytes
that populate peripheral lymphoid
tissues
Introduction
Immune repertoire
Collection of antigen receptors and
specificities expressed by B and T
lymphocytes
Overview of Lymphocyte
Development
Central lymphoid organs
Commitment of progenitor cells
Rearrangement of antigen receptor
genes and expression of antigen
receptor proteins
Selection events that preserve cells that
produced correct antigen receptor and
eliminates autoreactive cells
Proliferation of immature cells
Differentiation of B and T cells into
distinct populations
Commitment to B/T cells
Maturation of B cells occurs in the
bone marrow and fetal liver
Fetal liver gives rise to B-1 cells
Bone marrow gives rise to follicular
(circulating) B cells
Maturation of T cells occurs in the
thymus
γδ T cells originate in the fetal liver
αβ T cells originate in the bone marrow
Commitment to T/B cells
Early proliferation is stimulated by IL-7
Produced in the stromal cells in the
bone marrow and thymus
Mutations in the IL-7 receptor leads to
SCIDS (common γ chain)
Receptor Gene Rearrangement
In B cells, the first receptor gene to be
rearranged is the Ig heavy chain
In T cells, the β chain is rearranged first
If rearrangement is successful, next
chain can rearrange
If rearrangement is not successful, cell
dies by apoptosis
Receptor Gene Rearrangement
In B cells, the first receptor gene to be
rearranged is the Ig heavy chain
In T cells, the β chain is rearranged first
If rearrangement is successful, next
chain can rearrange
If rearrangement is not successful, cell
dies by apoptosis
Gene Rearrangment
Gene Rearrangment
Positive selection
Preservation of useful specificities
Negative selection
Process that eliminates (or alters)
developing lymphocytes whose antigen
receptors bind strongly to self
In T cells, this occurs by clonal deletion
In B cells, this is corrected by receptor
editing
Gene Rearrangement
Genes are generated by rearrangement
in individual lymphocytes of
Variable (V) region
Diversity (D) region
Joining (J) region
V(D)J rearrangement
V(D)J Recombination
DNA recombination event
Mediated by the coordinated activities
of several enzymes
Some of which are found only in
developing lymphocyte
Others are ubiquitous DNA double
strand break repair enzyme
V(D)J Recombination
Enzymes recognize DNA sequences called
recombination signal sequences (RSS)
Located 3’ of each V segment, 5’ of each J
segment, and each side of the D segment
Highly conserved heptamer
Usually CACAGTG
Followed by 12-23 nonconserved nucleotides
Followed by AT rich stretch of 9 nucleotides
V(D)J Recombination
Most gene rearrangements occur by
deletion
50% of κ light chain rearrangements
occur by inversion
V(D)J Recombination
Synapsis
Portions of the antigen receptor
chromosome are made accessible to
the recombination machinery, and two
selected coding segments and their
adjacent RSSs are brought together by
a chromosomal looping event and held
in position for subsequent cleavage,
processing, and joining
V(D)J Rearrangement
Cleavage
Double-strand breaks are enzymatically
generated at RSS-coding sequence
junctions using machinery that is
lymphoid specific
V(D)J Rearrangement
Coding and Processing
The broken coding ends (but not the
signal/RSS ends) are modified by the
addition or removal of bases, and thus
greater diversity is generated
V(D)J Rearrangement
Joining
The broken coding ends as well as the
signal ends are brought together and
ligated by a double-strand break repair
process found in all cells that is called
non-homologous end joining
V(D)J Rearrangement
RAG 1/RAG 2
Recognizes DNA sequence at the
junction b/w a heptamar and a coding
segment and cleaves it
Also hold the hairpin ends and the blunt
ends together prior to the modification
of the coding ends
V(D)J Rearrangement
Ku70 and Ku80
DNA end binding proteins that bind ot the breaks
and recruit DNA-PK, a DNA repair enzyme
Artemis
An endonuclease that opens the hairpins
DNA Ligase IV
Ligases broken ends
Terminal deoxynucleotidyl transferase (TdT)
Catalyzes the addition of junctional N nucleotides
Generation of Diversity
Combinatorial diversity
Different combinations of V, D, and J
segments that are possible as a result
of somatic recombination of DNA
Junctional diversity
Largest contribution to the diversity of
antigen receptors
Removal or addition of nucleotides
between segments at the time
Generation of Diversity
Greatest variability is at the junction of
the V and C regions, which is called
CDR3
B Cell Development
Pro-B cell
Earliest bone marrow committed cell
CD19+
First recombination of Ig genes in
heavy chain
Pre-B cell
Has productive Igμ
Has surrogate light chain
B Cell Development
Pre-B cell receptor
μ
Surrogate light chain
Igα
Igβ
Allelic exclusion
An individual B cell can express heavy
chain proteins encoded by only one of
the two inherited alleles
B Cell Development
Bruton’s tyrosine kinase (Btk)
Delivers signals from the pre-BCR that
mediates survival, proliferation, and
maturation beyond pre-B cell stage
B Cell Development
Immature B Cell
Expresses IgM
CD19+
Leave bone marrow
Receptor editing
B cells that recognize self antigens with high
avidity
RAG genes are reactivated
Additional light chain VJ recombination
Expression of a new, different light chain
B Cell Development
Mature B cell
aka follicular B cell
IgM
IgD
Ability to recirculate from one lymphoid
organ to another in B cell follicles
Naïve
B Cell Subsets
B-1
Develop from fetal liver
Express CD5 (Ly-1)
Found in peritoneum and mucosal sites
Limited repertoire
Secrete IgM (natural Ab) that react with
microbial polysaccharides and lipids
May also secrete IgA
Analogous to γδ T cells
B Cell Subsets
Marginal Zone B cells
Marginal sinus in the spleen
Similar to B-1 cells
T cell independent responses
T Cell Maturation
Thymocytes
Developing T cells in the thymus
First found in subcapsular sinus and
outer cortical region
Migrate through cortex where most of
the maturation occurs
Migrate to medulla and exit the thymus
T Cell Maturation
Pro-T cells
Double negative thymocytes
New arrival from bone marrow
No TCR, CD3, ζ chains, CD4, CD8
DJ rearrangements
Pre-T cells
V to DJ rearrangement
TCR β chain
Pre-Tα
T Cell Maturation
Double Positive T Cell
CD4+
CD8+
CCR7 (guides to medulla)
TCRα gene rearrangement
May occur on both chromosomes
Up to 30% of mature T cells have 2
different α chains (but only one β chain)
T Cell Maturation
Double Positive Thymocytes
αβTCR, CD3 and ζ chains are all
required for surface expression
Positive and Negative Selection
Single Positive Thymocytes
Immature
Lose CD4 or CD8 designation
Naïve mature T cells
Leave thymus
T Cell Maturation
Positive selection
Process in which thymocytes whose
TCRs bind with low avidity to self
peptide-self MHC complexes are
stimulated to survive
Negative selection
Process in which thymocytes whose
TCRs bind strongly to self peptide
antigens in association with self MHC
molecules are deleted
T Cell Maturation
Central Tolerance
Tolerance induces in immature
lymphocytes by recognition of self
antigens in the generative (or central)
lymphoid organs
T Cell Maturation
Autoimmune Regulator (AIRE)
Secreted by medullary epithelial cells
Induces expression of a number of
tissue-specific genes in the thymus
makin a host of tissue-specific peptides
available for presentation in the thymus
to double-positive and single positive T
cells during negative selection
Animation
http://www.studentconsult.com/content
/9781416031222/animation/T_cell.swf
Summary
B and T cells arise from a common bone
marrow derived precursor that becomes
committed to the lymphocytes lineage
B cell maturation proceeds in the bone
marrow
T cells progenitors migrate to the thymus
Early maturation is characterized by cell
proliferation which is induced by IL-7
Summary
B and T cell maturation involves the
somatic rearrangement of antigen
receptor gene segments and the initial
expression of Ig heavy chain proteins
in B cell precursors and TCR β
molecules in T cell precursors
Summary
The expression of pre-antigen
receptors and antigen receptors is
essential for survival and maturation of
developing lymphocytes and for
selection processes that lead to a
diverse repertoire of useful antigen
specificities
Summary
The antigen receptors of B and T cells are
encoded by genes formed by the somatic
rearrangement of a limited number of gene
segments that are spatially segregated in the
germline antigen receptor loci
There are separate loci encoding the Ig
heavy chain, Ig κ light chain, Ig λ light chain,
TCR α, β, and γchains