Lymphocyte Development and
Antigen Receptor Gene Rearrangement
浙江大学医学院免疫所
汪洌 wanglie@zju.edu.cn
OVERVIEW OF LYMPHOCYTE DEVELOPMENT
1. The commitment of progenitor cells to the B cell or T cell
lineage.
2.Proliferation of progenitors and immature committed cells at
specific early stages of development, providing a large pool of
cells that can generate useful lymphocytes.
3.The sequential and ordered rearrangement of antigen receptor
genes and the expression of antigen receptor proteins.
OVERVIEW OF LYMPHOCYTE DEVELOPMENT
4.Selection events that preserve cells that have produced correct
antigen receptor proteins (Positive selection) and eliminate
potentially dangerous cells that strongly recognize self antigens
(Negative selection)
5.Differentiation of B and T cells into functionally and
phenotypically distinct subpopulations.
Stages of lymphocyte maturation
Pluripotent stem cells give rise to distinct B and T lineages.
Early B and T cell development is characterized by the proliferation of
committed progenitors induced by cytokine-derived signals (IL7)
In 1993, it was reported that the γ chain was defective in patients
with X-linked severe combined immunodeficiency (XSCID; the
disease is formally designated as SCIDX1)
1.Absent or profoundly diminished numbers of T cells and mitogen
responses.
2. Absence of NK cells
3. Normal numbers of B cells, but defective B-cell responses.
4. IgM can be normal, but greatly diminished immunoglobulins of
other classes.
SCID Patient with severe Candida in mouth.
Checkpoints in lymphocyte maturation
Central tolerance
is the mechanism by
which newly developing
T cells and B cells are
rendered non-reactive
to self during their
development in thymus
and bone marrow.
Positive and negative selection during lymphocyte maturation
Thymocytes are positive selected by MHC/self
peptides
Positive selection:
During positive selection Double-Positive T cells
that can recognize self MHC‘s are selected for
proliferation, and those T cells that do not
recognize self MHC die via Apoptosis.
Positive selection also assures TCR to recognize
peptide/MHC complex and also go with the appropriate
CD4 or CD8. For example, TCR‘s specific for MHC II need to
retain CD4, and lose CD8. If the reverse occurs, they will
die via apoptosis. The same is true for the T cells that are
specific for MHC I, which need to retain CD8, and lose
CD4.
Central Tolerance is achieved by negative
selection
Negative selection:
T cells that are strongly activated by self
MHC plus self peptides need to be
eliminated in the thymus. complex.
If they escape this elimination, they may
subsequently react against self antigens, and
cause Autoimmune disease.
Development and migration of B cells (An overview)
variable region and constant region
variable region(V region)
Light Chain - VL (110 aa)
Heavy Chain - VH (110 aa)
•HVR: hypervariable regions
•CDR: the complementarity
determining regions.
•CDR are found in both the
H and the L chains.
Germline organization of human Ig loci
Domains of Ig proteins
Antigen receptor gene rearrangements
Diversity of antigen receptor genes
V(D)J recombination
Critical lymphocyte-specific factors that mediate V(D)J
recombination recognize certain DNA sequences called
recombination signal sequences (RSSs), located 3′ of each
V gene segment, 5′ of each J segment, and flanking each
D segment on both sides.
V(D)J recombination
V(D)J recombination
Recombination occurs between two segments only if one
of the segments is flanked by a 12-nucleotide spacer and
the other is flanked by a 23-nucleotide spacer; this is
called the 12/23 rule.
Transcriptional regulation of Ig genes
Portions of the chromosome on which
the antigen receptor gene is located are
made accessible to the recombination
machinery. 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.
Double-stranded breaks are
enzymatically generated at RSS-coding
sequence junctions by machinery that is
lymphoid specific. Two proteins
encoded by lymphoid-specific genes,
called recombination-activating gene 1
and recombinationactivating
gene 2 (Rag-1 and Rag-2), form a
tetrameric complex that plays an
essential role in V(D)J recombination.
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. After the formation of doublestranded breaks,
hairpins must be resolved (opened up) at the coding junctions, and bases may be added to or
removed from the coding ends to ensure even greater diversification. Artemis is an
endonuclease that opens up the hairpins at the coding ends.A lymphoid-specific enzyme,
called terminal deoxynucleotidyl transferase (TdT), adds bases to broken DNA ends and will be
discussed later in this chapter in the context of junctional diversity.
The broken coding ends as well as the signal ends are brought together and ligated by
a doublestranded break repair process found in all cells that is called nonhomologous
end joining. A number of ubiquitous factors participate in nonhomologous end joining.
Ku70 and Ku80 are DNA end-binding proteins that bind to the breaks and recruit the
catalytic subunit of DNA-dependent protein kinase (DNA-PK) a double-stranded DNA
repair enzyme, Ligation of the processed broken ends is mediated by DNA ligase IV
and XRCC4, the latter being a noncatalytic but essential subunit of this ligase.
Combinatorial diversity
Junctional diversity
Stages of B cell maturation
Ig heavy and light chain gene recombination
Ig heavy and light chain gene expression
Pre-B cell receptor
X-linked agammaglobulinemia
Mutation in the btk gene
Bruton's agammaglobulinemia
tyrosine kinase (btk).
Failure in B cell development
Males are more affected
Figure 9-8
Ligation of the pre-B cell receptor
1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B
Unconfirmed ligand of
pre-B cell receptor
1. Ensures only one specificty of
Ab expressed per cell
Stromal cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the allele
on the second chromosome
Evidence for allelic exclusion
ALLOTYPE- polymorphism in the C region of Ig – one allotype
inherited from each parent
Allotypes can be identified by staining B cell surface Ig with antibodies
B
a
AND b
b
B
B
Y
Suppression of H chain rearrangement by pre-B
cell receptor prevents expression of two
specificities of antibody per cell
B
Y
b
Y
B
Y
a
a/b
b/b
Y
Y
a/a
a
Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell
Y
Y Y
S. aureus
Y
Y
Y
Y
Anti
S. aureus
Antibodies
B
S. aureus
Anti
brain
Abs
Y
Y
Y
Y
Self antigen
expressed by
e.g. brain cells
Y Y
B
Anti
S. aureus
Antibodies
Suppression of H chain gene rearrangement
ensures only one specificity of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens
Allelic exclusion is needed for efficient clonal selection
Antibody
S. typhi
S. typhi
All daughter cells must express the same Ig specificity
otherwise the efficiency of the response would be compromised
Suppression of H chain gene rearrangement helps prevent the emergence of
new daughter specificities during proliferation after clonal selection
Allelic exclusion is needed to prevent holes in the repertoire
One specificity of Ag
receptor per cell
IF there were two specificities
of Ag receptor per cell
Anti-brain Ig
Anti-brain Ig
AND
anti-S. aureus Ig
B
B
Exclusion of anti-brain B cells i.e.
self tolerance
B
Deletion
OR
B
BUT anti S.aureus B cells will
be excluded leaving a
“hole in the repertoire”
Anergy
B
S. aureus
Ligation of the pre-B cell receptor
1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B
Unconfirmed ligand of
pre-B cell receptor
1. Ensures only one specificity of
Ab expressed per cell
Stromal cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
Acquisition of antigen specificity creates a need
to check for recognition of self antigens
B
B
Y
Y
Y
Y
Small pre-B cell
Immature B cell
No antigen receptor at cell surface
Unable to sense Ag environment
!!May be self-reactive!!
Cell surface Ig expressed
Able to sense Ag environment
1.
2.
3.
Can now be checked for self-reactivity
Physical removal from the repertoire
Paralysis of function
Alteration of specificity
DELETION
ANERGY
RECEPTOR EDITING
B cell self tolerance: clonal deletion
Small
pre-B
B
B
Immature
B
B
YY
Small pre-B cell
assembles Ig
Immature
B cell recognises
MULTIVALENT
self Ag
Clonal deletion by
apoptosis
B cell self tolerance: anergy
IgD normal IgM low
Small
pre-B
B
YY
Immature
B
B
IgM
IgD
B
IgD
IgD
B
Small pre-B cell
assembles Ig
Immature
B cell recognises
soluble self Ag
No cross-linking
Anergic B cell
Receptor editing
A rearrangement encoding a self specific receptor can be replaced
V
V
V
V
D J
C
!!Receptor
recognises
self antigen!!
B
Arrest development
And reactivate
RAG-1 and RAG-2
V
B
V
V
B
D J
Apoptosis
or anergy
C
Edited receptor now recognises
a different antigen and can be
rechecked for specificity, self-reactive κ
light chain genes
B lymphocyte subsets
Coexpression of IgM and IgD
Two B cell lineages
B cell precursor
Mature B cell
B
B
Plasma cell
PC
B2 B cells
CD5
?
B
Distinct B cell
precursor
B
Y
Y
Y
Y
Y
YY
Y
Y
Y
?
IgG
B1 B cells
‘Primitive’ B cells found in
pleura and peritoneum
IgM - no other isotypes
B-1 B Cells
IgM uses a distinctive & restricted range of V regions
CD5
B
Y
Y
Few non-template encoded (N) regions in the IgM
Y
Y
IgM
Recognises repeating epitope Ag such as phospholipid
phosphotidyl choline & polysaccharides
NATURAL ANTIBODY
NOT part of adaptive immune response:
No memory induced
Not more efficient on 2nd challenge
Present from birth
Can make Ig without T cell help
Comparison of Different B cell subtypes
T lymphocyte:
a key regulator of the immune system
Absence or hyperactivation of T cells leads to immunodeficiency,
respectively chronic inflammation or autoimmune disorders.
Geography of the thymus:
Thymic epithelial cells control the maturation of thymocytes
The thymus, which lies in the midline of the body, above the heart, is made of several lobules, each of which contains discrete cortical
(outer) and medullary (central) regions. The cortex consists of immature thymocytes (dark blue in the cartoon) that are closely associated
to branched cortical epithelial cells (pale blue), and scattered macrophages (yellow) that clear apoptotic thymocytes. The medulla, in
contrary consists of mature thymocytes (dark blue), medullary epithelial cells (orange) along with macrophages (yellow) and dendritic
cells (branched, yellow). Thymocytes in the outer cortical layer are proliferating immature cells, while cells of the deeper cortical layer
(cortico-medullary junction) undergo thymic selection.
The cortical epithelium of the thymus is
critical for T cell development
The nude mouse strain is athymic due to a mutation in the Foxn1nu gene
that encodes FOXN1, a transcription factor of the forkhead box family.
FOXN1 is preferentially expressed in the skin and thymus. Alteration of its
expression in the thymus underlies the manifestation of severe
immunodeficiency resulting from total absence of T cell development.
DiGeorge综合征
医学免疫学
上海第二医科大学免疫教研室
Stages of T cell maturation
Germline organization of human TCR loci
Domains of TCR proteins
Maturation of T cells in the thymus
TCR α and β chain gene recombination
TCR α and β chain gene expression
Pre-T cell receptor
CD4 and CD8 expression on thymocytes and positive
selection of T cells in the thymus
Downloaded from: StudentConsult (on 1 June 2006 01:58 PM)
© 2005 Elsevier
Positive Selection
Experiment
•Thymocytes are dependent on the
presence of MHC’s in the thymus for
survival.
•Class I MHC knockout  CD4 is
selected for, but CD8 does not survive.
•Class II MHC knockout  CD8 is
selected for, but CD4 does not survive.
Negative Selection
Experiment
•H-Y peptide expressed form Y
Chromosome (only in males)
•T-cells against the H-Y peptide are
found only in females!!!
•Why?
•Male cell recognizes H-Y peptide as a
self-pathogen  T-cells against H-Y
peptide are negatively selected and
eliminated.
Figure 13-9 part 1 of 2
Slide from Janeway book
Figure 13-9 part 2 of 2
Slide from Janeway book
gd T cells also develop in the thymus
THYMUS
CD4
~4%
CD8
Developing T cells with no b chain: including gd
Natural killer T cells(NKT )
αGalcer
（α-半乳糖神经鞘胺醇）
NKT cells are traditionally defined as cells that co-express the T cell receptor (TCR) and certain natural killer cell
surface markers (e.g. NK1.1 in some mouse strains). Type I NKT cells, also known as ‘classical’ or ‘invariant’ NKT
cells express an invariant TCR that is composed of a common α-chain(Vα14-Jα18) in combination with a limited
number of β-chains (Vβ8.2, Vβ7 or Vβ2) (or V α 24-J α 18/Vβ11 in human).
The NKT cell system as a functional bridge between
innate and acquired immunity
------------------(Toshinori Nakayama,Nature,2003)
IFN-γ produced by NKT cells activate
NK cells and macrophages in the innate
immune system, thus facilitating inflammatory
responses toward pathogens.
IFN-γ produced by NKT cells
also activates pathogen-specific CD4 T helper
type 1 responses as well as CD8 T cell–mediated
cytotoxic responses against infected targets