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Transcript
T-CELL DEVELOPMENT
CENTRAL TOLERANCE
ARPAD LANYI PhD
The cellular organization of the thymus
The proportion of the thymus that produces
T-cells decreases with age
DIFFERENTIATION OF T-CELLS
IN THE THYMUS
REGULATED T-CELL DIFFERENTIATION
CD4-CD8-
pro T-cell
NO T –CELL
RECEPTOR
CD4+CD8+
preTCR
cell
preEpithelial
T-cell
SIGNALING RECEPTOR
CD4+/CD8+
TCR
naive T-cell
ANTIGEN RECOGNIZING
immature
T-cell
RECEPTOR
T-CELL DIFFERENTIATION
T-cell precursors that enter the thymus express the hematopoietic
stem-cell marker CD34 and the adhesion molecule CD44, but none of
the characteristic markers of the T-cell lineage (CD2, CD3, CD5).
Upon interaction with thymic stromal cells, the progenitor
signaled to divide and differentiate. After around a week,
have lost stem-cell markers and have become thymocytes
committed to the T-cell lineage (pro T-cell), as seen
expression of the T-cell specific adhesion molecule CD2.
cells are
the cells
that are
by their
Commitment to the T-cell lineage is driven by the receptor Notch 1.
Notch 1 on the thymocyte binds to its ligand on thymic epithelium.
This interaction induces a protease to cleave the intracellular domain
from the plasma membrane. The soluble intracellular domain is
translocated to the nucleus, where it turns on the expression of genes
essential for T-cell development by removing repressive transcription
factors and recruiting co-activating transcription factors.
T-CELL DIFFERENTIATION
Commitment to the T-cell lineage changes receptor expression
Cells committed to the T-cell lineage express CD2,
CD5, CD1a and IL7-receptor, but do not express T-cell
receptor, CD4 or CD8 (double negative thymocytes).
Lack of IL7 signaling (IL7 or IL7R) stalls early
T-cell development.
SCIDs.
Cells are beginning to rearrange
the TCR genes.
TCR β-, α-, δ- and γ-chain loci
Each germline TCR locus includes variable (V), joining (J)
and constant (C) gene segments.
TCR β and TCR δ loci also have D segments,
like the Ig heavy chain locus.
The basic rules of TCR rearrangement are identical to
that of the BCR.
δ gene segments are embedded within the a-chain locus.
α-chain gene rearrangement results in the deletion of
the δ-chain locus.
V(D)J
RECOMBINATION
Recombination signal sequences:
conserved hepta- and nonamer sequences
CACAGTG; ACAAAAACC
spacer regions: 12/23bp
V(D)J recombinases
Recognize RSSs and bring together two coding
segments.
RAG1 makes a nick:
generates free 3’-OH and 5’-P
Endonuclease.
3’-OH
attacks
phosphodiester
bond on
the
Opens
up thea hairpins
at the coding
ends.
other strand
forming
Mutation
of Artemis:
T- aB-hairpin.
NK+ SCID
The blunt signal ends are ligated together
and discarded.
Double-stranded DNA repair enzyme.
Activates Artemis.
Mutation of DNA-PK: T- B- NK+ SCID
Adds bases to broken DNA ends.
Lymphoid-specific.
V(D)J
RECOMBINATION
Combinatorial diversity
V(D)J rearrangement brings together multiple germline gene segments that
may combine randomly, and different combinations produce different antigen receptors.
Junctional diversity
The largest contribution to the diversity of antigen receptors is made by the
removal or addition of nucleotides at the junctions of the V and D, D and J, or V and J segments.
P nucleotides: nucleotides added to the asymmetrically cleaved hairpin ends
N nucleotides: random added (up to 20) non–template-encoded nucleotides by TdT
The y- and δ-chain loci contain fewer V gene segments,
BUT during δ-gene rearrangement two D segments can
be incorporated into the final gene sequence.
increase in junctional diversity
(extra N nucleotides between the two D segments)
(+more combinations)
T-CELL DIFFERENTIATION
Rearrangement of the δ-, γ- and β-chain genes leads to early commitment of
some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before
β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further
rearrangement. γ:δ T-cells mature, leave the thymus and travel to other
tissues via the blood.
variable region (V)
constant region (C)
transmembrane region
cytoplasmic tail
γδ T-cells
•MHC-independent, CD1c and CD1d dependent.
•Double megative.
•Comprise about 1-5% of the T-cells found in the
circulation, but can be the dominant (up to 50%) T-cell
population in epithelial tissue.
•A population that is expanded in intra- (Mycobacterium
tuberculosis and Listeria monocytogenes) and extracellular
infections (Borrelia burgdorferi) and certain disease states
such as celiac disease.
T-CELL DIFFERENTIATION
Rearrangement of the δ-, γ- and β-chain genes leads to early commitment of
some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before
β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further
rearrangement. γ:δ T-cells mature, leave the thymus and travel to other
tissues via the blood.
The more frequent outcome of the competition between the β-, y- and δchain genes is for a productive β-chain gene rearrangement to be made
before both productive y- and δ-chain rearrangements occur.
TCR β-CHAIN GENE
REARRANGEMENT
1ST CHECKPOINT
Rearrangement of a Vβ, a Dβ and a Jβ gene segment
After translocation
tofunctional
the endoplasmic
reticulum
creates the
V-domain
exon. β-chain is
tested for its capacity to bind to an invariant polypeptide
called pTα,
Unused V and
J genes
between
the rearranged
which
acts as
a surrogate
α-chain. V and J
genes are deleted.
TCR β-CHAIN GENE
REARRANGEMENT
This possibility is not available to the immunoglobulin heavychain genes, because a nonproductive rearrangement
deletes all the non-rearranged D segments.
A nonproductively rearranged β-chain gene
can also be rescued by a second
rearrangement at the same locus.
If a rearrangement at one β-chain locus is
nonproductive, a thymocyte can attempt a
rearrangement at the β-chain locus on the
homologous chromosome.
Thymocytes can make four
attempts to rearrange the
β-chain gene
The potential for up to four β-chain gene rearrangements
means that 80% of thymocytes make a productive
rearrangement of the β-chain gene, compared with a 55%
success rate for heavy-chain gene rearrangement by
developing B cells.
T-CELL DIFFERENTIATION
Rearrangement of the δ-, γ- and β-chain genes leads to early commitment of
some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before
β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further
rearrangement. γ:δ T-cells mature, leave the thymus and travel to other
tissues via the blood.
The more frequent outcome of the competition between the β- y- and δchain genes is for a productive β-chain gene rearrangement to be made
before both productive y- and δ-chain rearrangements occur.
If the β-chain binds to pTa, this heterodimer assembles with the CD3
complex and ζ-chain to form the pre-T-cell receptor. Pre-TCR is sufficient
for signaling and there is no requirement for binding a ligand. Pre-TCR
induce pre T-cell to stop gene rearrangement (suppressing RAG1/2),
proliferate and express CD4 and CD8 co-receptors (double-positive
thymocytes).
At this stage the recombination machinery is reactivated and targeted to
the α, γ, and δ loci, but not to the β-chain locus. A minority of the doublepositive thymocytes give rise to additional γ:δ T-cells.
Upon rearrangement of the α-chain locus, the δ-chain locus it contains is
eliminated as part of an extrachromosomal circle.
TCR α-CHAIN GENE
SUCCESSIVE
REARRANGEMENT
2ND CHECKPOINT
After translocation to the
endoplasmic reticulum
α-chain is tested for its capacity
to bind the β-chain and assemble a
T-cell receptor.
A Vα gene segment rearranges to a
Jα gene segment to create a
functional exon encoding the V
domain.
It continues until either a productive
rearrangement occurs or the supply of
V and J gene segments is exhausted,
whereupon the cell dies
(like Ig light chain).
T-CELL DIFFERENTIATION
Rearrangement of the δ-, γ- and β-chain genes leads to early commitment of
some cells to the γ:δ T-cell lineage. δ- and γ-chain genes rearrange before
β-chain and γ:δ receptor assembles. Signals through γ:δ TCR stop further
rearrangement. γ:δ T-cells mature, leave the thymus and travel to other
tissues via the blood.
The more frequent outcome of the competition between the β- y- and δchain genes is for a productive β-chain gene rearrangement to be made
before both productive y- and δ-chain rearrangements occur.
If the β-chain binds to pTa, this heterodimer assembles with the CD3
complex and ζ-chain to form the pre-T -cell receptor. Pre-TCR is sufficient
for signaling and there is no requirement for binding a ligand. Pre-TCR
induce pre T-cell to stop gene rearrangement (suppressing RAG1/2). The
cells proliferate and express CD4 and CD8 co-receptors (double-positive
thymocytes).
At this stage the recombination machinery is reactivated and targeted to
the α, γ, and δ loci, but not to the β-chain locus. A minority of the doublepositive thymocytes give rise to additional γ:δ T-cells.
Upon rearrangement of the α-chain locus, the δ-chain locus it contains is
eliminated as part of an extrachromosomal circle.
Productive α-chain gene rearrangements produce double-positive CD4 CD8
α:β T-cells. This ends the early stage of T-cell development.
GENE EXPRESSION THROUGH THE
STAGES OF
α:β T-CELL DEVELOPMENT
Signals from the pre-T-cell receptor depend on
the expression of the co-receptors CD4 and
CD8, the signaling complex CD3, the tyrosine
kinase ZAP-70, which is involved in relaying
signals from the receptor, and the tyrosine
kinase Lck, which is involved in signaling from
the co-receptors.
Ikaros regulates Notch target gene expression.
Th-POK is required for the development of
single-positive CD4 T-cells from double-positive
thymocytes.
Uncommitted progenitors: survival
Committed thymocytes: optimal β selection
CD4 T-cells: survival
Effector CD4 T-cells: Th2 polarization
Only a small fraction of T-cells mature into functional T-cells
POSITIVE SELECTION
-Occurs in the cortex, requires thymic epithelial cells.
-αβ double-positive thymocytes must recognize selfMHC. T-cells expressing TCRs that can bind to the self
MHC/self-peptide complex on the surface of cortical
epithelial cells will survive, but the others will die due
to the lack of survival signals (death by neglect).
-Selection continues until a successful rearrangement
on the TCRα locus occurs (3-4 days).
-Ca. 2% of thymocytes survive!!
Positive selection --- results in clones that are
reactive to SELF MHC.
BASIS OF MHC RESTRICTION!!!
THE KEYWORD: RECOGNITION
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
SIDE BY SIDE VIEW OF
T-AND B-CELL DEVELOPMENT
Nemazee Nature Reviews Immunology 6, 728–740 (October 2006) | doi:10.1038/nri1939
The response of the immune system
to the stimuli of the outer and inner environment
Environment
Immune system
Tolerance
Self
Non-self
Dangerous
Pathogenic
Immune
response
IMMUNOLOGICAL
TOLERANCE
Immunological tolerance
Definition:
Unresponsiveness to a given antigen induced by the interaction of that
antigen with the lymphocytes.
ANTIGEN SPECIFIC!!!
Unlike immunosuppresion.
Why is this important?
-All individuals are tolerant to their own antigens (self tolerance).
-Failure of self tolerance results in autoimmunity.
-Terapeutic potential:
Treat autoimmune diseases, allergic reaction or even tissue rejection.
T-cells with high affinity TCR towards the self MHC/self peptide
complex are eliminated, but clones with intermediate affinity survive.
NEGATIVE SELECTION
Elimination of potentially
CENTRAL
TOLERANCE
KEYWORD: AFFINITY
of T-cells in THE
the thymus
autoreactive clones
A percentage of self-reactive T-cells – that have high affinity
TCRs, bordering negative selection – will survive the negative
selection process and differentiate into regulatory T-cells.
Central and peripheral tolerance to self antigens
Central tolerance:
Elimination of self-reactive clones.
BUT!!! Some T-cell clones escape.
Peripheral tolerance:
Elimination of „fugitive” or altered
clones is an important role for
regulatory T-cells.
SUMMARY
CENTRAL T-CELL TOLERANCE IS SURPRISINGLY
EFFECTIVE. MEDIATED MAINLY BY NEGATIVE
SELECTION.
THE QUESTION:
HOW CAN TISSUE-RESTRICTED ANTIGENS BE
EXPRESSED IN MEDULLARY THYMIC EPITHELIAL CELLS?
THE ANSWER:
AUTOIMMUN REGULATOR (AIRE)
A transcription factor expressed in the medulla of the thymus and
induces expression of many tissue-specific genes
AIRE DEFICIENCY
DEFICIENCY IN ESTABLISHING CENTRAL T-CELL TOLERANCE
Lack of proper negative selection allows too many self reactive
T-cell clones to leave the thymus
AUTOIMMUNE POLYENDOCRINOPATHY- CANDIDIASISECTODERMAL DYSTROPHY (APECED)
Rare disease, but more frequently seen in inbred populations Finnish, Iranian
Jews and in the island of Sardine
SYMPTOMS OF APECED
• Anti-Th17 specific
antibodies!!!!!
• Role of Th17 discovered by
studying a rare
immunodeficiency.
• https:///jimneydandme.wordp
ress.com/james-story
THE END
V(D)J RECOMBINATION
1. Synapsis:
 V(D)J recombinases recognizes recombination signal sequences (conserved hepta- and nonamer sequences
flanking the V,D,J segments; spacer regions: 12/23bp).
 Recombination-activating gene 1-2 (Rag-1 and Rag-2):
 lymphoid specific
 expressed mainly in the G0 and G1 stages
 inactivated in proliferating cells
 Rag-1 is enzymatically active only when complexed with Rag-2.
 mutation of RAG enzymes – Omen syndrome, T- B- NK+ SCID
 Two selected coding segments and their adjacent RSSs are brought together by a chromosomal looping event.
2. Cleavage:
 Rag-1 makes a nick (on one strand) between the coding end and the heptamer.
 The released 3′ OH of the coding end attacks a phosphodiester bond on the other strand, forming a covalent hairpin.
 The signal end (including the heptamer and the rest of the RSS) does not form a hairpin and is generated as a blunt
double-stranded DNA terminus that undergoes no further processing.
3. Hairpin opening and end-processing:
 The broken coding ends are modified by the addition or removal of bases, and thus greater diversity is generated.
• Artemis:
 endonuclease, opens up the hairpins at the coding ends
 mutation of Artemis: T- B-NK+ SCID
• Terminal deoxynucleotidyl transferase (TdT)
 lymphoid-specific
 adds bases to broken DNA ends
4. Joining:
 The broken coding ends as well as the signal ends are brought together and ligated.
 Double-stranded break repair process: nonhomologous end joining.
• DNA-dependent protein kinase (DNA-PK)
 double-stranded DNA repair enzyme
 activates Artemis
 mutation of DNA-PK: T- B-NK+ SCID
 Ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4.
TCR β-CHAIN GENE
REARRANGEMENT
Rearrangement of a Vβ, a Dβ and a Jβ gene segment
creates the functional V-domain exon.
Unused V and J genes between the rearranged V and J
genes are deleted.
1ST CHECKPOINT
After translocation to the endoplasmic reticulum β-chain is
tested for its capacity to bind to an invariant polypeptide
called pTα,
which acts as a surrogate α-chain.
SUCCESSIVE
TCR α-CHAIN GENE
REARRANGEMENT
2ND CHECKPOINT
After translocation to the
endoplasmic reticulum
α-chain is tested for its capacity
to bind the β-chain and assemble a
T-cell receptor.
A Vα gene segment rearranges to a
Jα gene segment to create a
functional exon encoding the V
domain.
It continues until either a productive
rearrangement occurs or the supply of
V and J gene segments is exhausted,
whereupon the cell dies
(like Ig light chain).
NEGATIVE SELECTION
of T-cells in the thymus
CENTRAL TOLERANCE
Elimination of potentially
autoreactive clones
T-cells with high affinity TCR towards the self MHC/self peptide
complex are eliminated, but clones with intermediate affinity survive.
A percentage of self-reactive T-cells – that have high affinity
TCRs, bordering at negative selection – will survive the negative
selection process and differentiate into regulatory T-cells.
THE KEYWORD: AFFINITY