and T-cell diversity_Development of lymphocytes_LAx

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Transcript and T-cell diversity_Development of lymphocytes_LAx

GENERATION OF B-AND T-CELL
DIVERSITY
DEVELOPMENT OF LYMPHOCYTES
LÁNYI ÁRPÁD PhD
[email protected]
GENERATION OF B-CELL RECEPTOR
DIVERSITY
In the absence of (foreign) antigens
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.
????
SOMATIC RECOMBINATION
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
CDR1 és CDR2 CDR3
Rearrangement of gene segments encoding the V domain is random
RANDOM REARRANGEMENT OF GENE SEGMENTS
ENCODING THE VARIABLE DOMAIN OF KAPPA CHAIN
Vκ
B-cell 2
Vκ
Vκ
Jκ
5 Jκ
35 Vκ
Vκ
Vκ
Germline
Jκ Jκ Jκ Jκ
During B-cell development
B-cell 1
Vκ
Vκ
Vκ
Jk Jκ Jκ Jκ
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
HEAVY CHAIN V REGION IS ASSEMBLED FROM
THREE GENE SEGMENTS
CDR1 és 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!!
In practice, some H + L combinations do not occur as they are unstable
Certain V and J genes are also used more frequently than others
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
MOLECULAR EXPLANATION OF THE 12-23 RULE
12-mer
One turn
23-mer
Two turns
V7
9
9
V4
7D J
Intervening DNA
of any length
V2
9
• Heptamers and nonamers align back-to-back
9
23-mer
12-mer
7
V1
7
• An appropriate shape can not be formed if two
23-mer (or 12-mer) flanked elements attempted
to join
V3
DJ
V5
Loop of
intervening
DNA is
excised
V9
V6
V8
V7
e
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.
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)
Combinatorial diversity: random selection of gene segments: ~2x106 possibilities
Junctional diversity increases overall diversity by a factor of up to 3×107
GENERATION OF TCR DIVERSITY
TCR β-, α-, δ- and γ-chain loci
The basic rules of TCR rearrangement are identical to that of the BCR
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
δ gene segments are embedded within the a-chain locus
α-chain gene rearrangement results in the deletion of the δchain locus
POTENTIAL DIVERSITY OF B/TCR
Constant segments (C)
9
1(κ) 4(λ)
2
1
DEVELOPMENT OF LYMPHOCYTES
EARLY PHASE
Generation of antigen recognition receptors
SELECTION
Deletion
Anergy
BONE MARROW
CD19
CD127
CD
34
CLP
pro
B
pre
B
IgM
pre pre pre
preB
B
B
Heavy chain
Light chain
B
rearrangement pre pre pre rearrangement Imm
B
B B B
Commitment
CD2
CD127
THYMUS
pro
T
CENTRAL
TOLERANCE
Allelic exclusion
Receptor
editing
1st checkpoint
2nd checkpoint
β-chain
rearrangement
γ-, δ-chain
rearrangement
γδ
T
Negative
selection
α-chain
rearrangement
pre pre
T pre
T
γ-, δ-chain
pre pre T rearrangement
pre
T
T
pre pre T
γδ
T
T
T
DP
T
Negative
selection
Positive
selection
SECONDARY LYMPHOID
TISSUES
IgM>IgD
IgM<IgD
Imm
B
Mat
B
Treg
CD4
CD8
MHC
restriction
Death by neglect
Deletion
DEVELOPMENT OF B LYMPHOCYTES
PRO-B CELLS DEVELOP FROM
THE PLURIPOTENT HEMATOPOIETIC STEM CELL
IL-7Rα
B-CELL DEVELOPMENT IS STIMULATED BY BONE MARROW
STROMAL CELLS
STAGES OF B-CELL DEVELOPMENT DEFINED BY
THE SEQUENTIAL
REARRANGEMENT OF THE IMMUNOGLOBULIN GENES
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
Non-productive rearrangement: incorrect reading
frame, no translation
Productive rearrangement: correct reading frame,
translation
1ST CHECKPOINT
Monitoring the quality of immunoglobulin heavy chain
Large pre-B-cells: preBCR assembles: μ heavy
chain,surrogate light chain - VpreB, λ5 – CD79
A/B (Igα, Igβ) (µ, λ5, lgα, lgß deficiency: non Bruton
agammaglobulinemia)
• Signaling receptor
• ALLELIC EXCLUSION
• Suppression of RAG transcription, RAG protein
degradation, chromatin reorganization: inhibition of
gene rearrangement on heavy chain locus
ALLELIC EXCLUSION
Successful somatic recombination on one chromosome
inhibits gene rearrangement on the other
Allelic exclusion at the immunoglobulin loci gives
rise to B-cells having antigen receptors of a single specificity
Needed for efficient clonal selection
Hybrid antibodies
Autoreactive antibodies
Deletion, anergy: holes in the repertoire
1ST CHECKPOINT
Monitoring the quality of immunoglobulin heavy chain
Large pre-B-cells: preBCR assembles: μ heavy
chain,surrogate light chain - VpreB, λ5 – CD79
A/B (Igα, Igβ) (µ, λ5, lgα, lgß deficiency: non Bruton
agammaglobulinemia)
• Signaling receptor
• ALLELIC EXCLUSION
• Suppression of RAG transcription, RAG protein
degradation, chromatin reorganization: inhibition of
gene rearrangement on heavy chain locus
• Proliferation
• Small pre-B-cells
• Shut off of surrogate light chain transcription
• RAG induction, stimulation of κ light chain recombination
NONPRODUCTIVE LIGHT CHAIN GENE REARRANGEMENTS
CAN BE SUPERSEDED BY FURTHER GENE REARRANGEMENT
Max. 5 attempts can be made at each of the four light chain loci
SUCCESSFUL REARRANGEMENT OF THE IMMUNOGLOBULIN
LIGHT-CHAIN GENES IN PRE-B CELLS LEADS TO THE
EXPRESSION OF CELL-SURFACE IGM
Rearrangement of light chain loci
continues until either a productive
rearrangement occurs or the supply
of V and J gene segments is
exhausted, whereupon the cell dies
2ND CHECKPOINT
Monitoring the quality of
light chain
BCR assembles
FATE DETERMINING CHECKPOINTS
NEGATIVE SELECTION I.
Immature B-cells with specificity for multivalent self antigens are
retained in the bone marrow
RECEPTOR EDITING
By changing their antigen specificities receptor
editing rescues many self-reactive B-cells
CLONAL
DELETION
NEGATIVE SELECTION II.
Immature B-cells specific for soluble monovalent self antigens
develop a state of anergy
Anergic B-cells have a half life of 45 days (10% that of regular B-cells)
COEXPRESSION OF IgD AND IgM IS REGULATED
BY RNA PROCESSING
MATURATION AND SURVIVAL OF IMMATURE B-CELLS
REQUIRES ACCESS TO LYMPHOID FOLLICLES
Immature B-cell: IgM>IgD
Mature B-cell: IgM<IgD
SUMMARY
DEVELOPMENT OF T LYMPHOCYTES
T-CELLS DEVELOP IN THE THYMUS
T-CELL DIFFERENTIATION
T-cell precursors that enter the thymus express the hematopoietic
stem-cell marker CD34 and adhesion molecule CD44, but none of the
characteristic markers of T-cell lineage (CD2, CD3, CD5).
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. On
interaction with thymic stromal cells, the progenitor cells are signaled
to divide and differentiate. After around a week, the cells have lost
stem-cell markers and have become thymocytes that are committed to
the T-cell lineage (pro T-cell), as seen by their expression of the Tcell specific adhesion molecule CD2.
Lack of IL7 signaling (IL7 or IL7R) stalls early Tcell development
SCIDs
Cells are beginning to rearrange the
TCR genes
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)
•MHC-independent, CD1c and CD1d dependent.
constant region (C)
•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
transmembrane region
tissue.
•A population that is expanded in intra- (Mycobacterium tuberculosis
citoplasmic tail
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
δ-chain 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
This possibility is not available to the immunoglobulin heavychain genes, because the V-DJ recombination excises all the
non-rearranged D segments.
A nonproductively rearranged β-chain gene
can 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 βchain gene
80% of thymocytes make a productive rearrangement of the
β-chain gene, compared with a 55% success rate for heavychain 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
δ-chain genes is for a productive β-chain gene rearrangement to be
made before both productive y- and δ-chain rearrangements occur.
After translocation to the endoplasmic reticulum β-chain is tested for
its capacity to bind to an invariant polypeptide called pTα.
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 coreceptors (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 double-positive 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.
SUCCESSIVE GENE REARRANGEMENTS
ON TCRα LOCI
Multiplicity of V (~70) and J gene ( 61)
segments allows many attempts
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
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
δ-chain genes is for a productive β-chain gene rearrangement to be
made before both productive y- and δ-chain rearrangements occur.
After translocation to the endoplasmic reticulum β-chain is tested for
its capacity to bind to an invariant polypeptide called pTα.
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 coreceptors (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 double-positive 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 concludes the early stage of T-cell
development.
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/auto-antigen complex on the surface of cortical
epithelial cells will survive, but others will die due to
the lack of survival signals (death by neglect).
-Continuing α-chain gene rearrangement increases the
chance for positive selection
-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
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.
SUMMARY

THANK YOU
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 – T- B- NK+ SCID; Omenn syndrome
 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.
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)
•MHC-independent, CD1c and CD1d dependent.
constant region (C)
•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
transmembrane region
tissue.
•A population that is expanded in intra- (Mycobacterium tuberculosis
citoplasmic tail
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
δ-chain genes is for a productive β-chain gene rearrangement to be
made before both productive y- and δ-chain rearrangements occur.
After translocation to the endoplasmic reticulum β-chain is tested for
its capacity to bind to an invariant polypeptide called pTα,
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 coreceptors (double-positive thymocytes).
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
δ-chain genes is for a productive β-chain gene rearrangement to be
made before both productive y- and δ-chain rearrangements occur.
After translocation to the endoplasmic reticulum β-chain is tested for
its capacity to bind to an invariant polypeptide called pTα,
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 coreceptors (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 double-positive 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 concludes the early stage of T-cell
development.
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