t-cell development central tolerance
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T-CELL DEVELOPMENT CENTRAL TOLERANCE
ARPAD LANYI PhD The cellular organization of the thymus
The figure shows the detailed structure of a lobus.The left panel
shows a microscopic image of a stained lobus section, and the right
panel shows the same structures schematically.Large number of
immature thymocytes (blue) is found in the darkly staining cortex,
together with the network of cortical epithelial cells (orange).The
medulla contains mature thymocytes, medullar epithelial cells (dark
orange), dendritic cells and macrophages (yellow).It also contains
the Hassal's corpuscules. The proportion of the thymus that
produces T-cells decreases with age
Starting at birth, the T-cell producing tissue of the thymus is
gradually replaced by fatty tissue. This process is called the
involution of the thymus. The graph shows the percentage of thymic
tissue that is still producing T-cells at different ages. The
micrograph in panel a shows a section through the thymus of a
3-day-old infant; the micrograph in panel b shows a section through
the thymus from a 70-year-old person for comparison. Tissue is
stained with hematoxylin and eosin (red and blue). Magnification x
20. Micrographs courtesy of Yasodha Natkunam. DIFFERENTIATION OF
T-CELLS
IN THE THYMUS REGULATED T-CELL DIFFERENTIATION ANTIGEN RECOGNIZING
RECEPTOR
CD4+CD8+preTCR CD4+/CD8+ TCR CD4-CD8- naive T-cell pre T-cell
Epithelial cell ANTIGEN RECOGNIZING RECEPTOR pro T-cell immature
T-cell SIGNALING RECEPTOR NO 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 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 T-cell specific adhesion molecule CD2. 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 -, -, - and -chain loci
Each germline TCR locus includes variable (V), joining (J) and
constant (C) gene segments. TCR and TCR loci also have D segments,
like the Ig heavy chain locus. The basic rules of TCR rearrangement
are identical to that of the BCR. gene segments are embedded within
the a-chain locus. -chain gene rearrangement results in the
deletion of the -chain locus. In all cells with the exception of
matured lymphocytes TCR (and immunoglobulin) genes are in germline
configuration. Transcription of these genes is possible only after
the gene rearrangement. V(D)J recombination occurs only during
lymphocyte development. V(D)J RECOMBINATION spacer regions:
12/23bp
Recombination signal sequences: conserved hepta- and nonamer
sequences CACAGTG; ACAAAAACC V(D)J recombinases Recognize RSSs and
bring together two coding segments. RAG1 makes a nick: generates
free 3-OH and 5-P Endonuclease. Opens up the hairpins at the coding
ends. Mutation of Artemis: T- B- NK+SCID Double-stranded DNA repair
enzyme. Activates Artemis. Mutation of DNA-PK: T- B- NK+SCID
Lymphoid specific . Expressed mainly in the G0 and G1 stages.
Inactivated in proliferating cells. Rag-1is enzymatically active
only when complexed with Rag-2. Mutation of RAG enzymes Omen
syndrome, T- B- NK+ SCID 3-OH attacks a phosphodiester bond on the
other strand forming a hairpin. The blunt signal ends are ligated
together and discarded. Adds bases to broken DNA ends.
Lymphoid-specific. Combinatorial diversity
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)
nontemplate-encoded nucleotides by TdT The y- and -chain loci
contain fewer V gene segments, BUT during -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 -, - and -chain genes leads to early
commitment of some cells to the : T-cell lineage. - and -chain
genes rearrange before -chain and : receptor assembles. Signals
through: TCR stop further rearrangement. : T-cells mature, leave
the thymus and travel to other tissues via the blood. variable
region (V) constant region (C) transmembrane region cytoplasmic
tail 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 -, - and -chain genes leads to early
commitment of some cells to the : T-cell lineage. - and -chain
genes rearrange before -chain and : receptor assembles. Signals
through: TCR stop further rearrangement. : T-cells mature, leave
the thymus and travel to other tissues via the blood. The more
frequent outcome of the competition between the -, y- and -chain
genes is for a productive -chain gene rearrangement to be made
before both productive y- and -chain rearrangements occur. TCR
-CHAIN GENE REARRANGEMENT
1ST CHECKPOINT Rearrangement of a V,a D and a J gene segmentcreates
the functional V-domain exon. After translocation to the
endoplasmic reticulum -chain is tested for its capacity to bind to
an invariant polypeptide called pT, which acts as a surrogate
-chain. Unused V and J genes between the rearranged V and Jgenes
are deleted. Thymocytes can make four attempts to rearrange the
-chain gene
TCR -CHAIN GENE REARRANGEMENT This possibility is not available to
the immunoglobulin heavy-chain genes, because a nonproductive
rearrangement deletes all the non-rearranged D segments. A
nonproductively rearranged-chain gene can also be rescued by a
second rearrangement at the same locus. If a rearrangement at one
-chain locus is nonproductive, a thymocyte can attempt a
rearrangement at the -chain locus on the homologous chromosome.
Thymocytes can make four attempts to rearrange the -chain gene The
potential for up to four -chain gene rearrangements means that 80%
of thymocytes make a productive rearrangement of the -chain gene,
compared with a 55% success rate for heavy-chain gene rearrangement
by developing B cells. T-CELL DIFFERENTIATION
Rearrangement of the -, - and -chain genes leads to early
commitment of some cells to the : T-cell lineage. - and -chain
genes rearrange before -chain and : receptor assembles. Signals
through: TCR stop further rearrangement. : T-cells mature, leave
the thymus and travel to other tissues via the blood. The more
frequent outcome of the competition between the - y- and -chain
genes is for a productive -chain gene rearrangement to be made
before both productive y- and -chain rearrangements occur. If the
-chain binds to pTa, this heterodimer assembles with the CD3
complex and -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 , , and
loci, but not to the -chain locus. A minority of the
double-positive thymocytes give rise to additional : T-cells. Upon
rearrangement of the -chain locus, the -chain locus it contains is
eliminated as part of an extrachromosomal circle. After
translocation to the endoplasmic reticulum
TCR -CHAIN GENE REARRANGEMENT SUCCESSIVE 2ND CHECKPOINT After
translocation to the endoplasmic reticulum -chain is tested for its
capacity to bind the -chain and assemble a T-cell receptor. A V
gene segment rearranges to a J 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 -, - and -chain genes leads to early
commitment of some cells to the : T-cell lineage. - and -chain
genes rearrange before -chain and : receptor assembles. Signals
through: TCR stop further rearrangement. : T-cells mature, leave
the thymus and travel to other tissues via the blood. The more
frequent outcome of the competition between the - y- and -chain
genes is for a productive -chain gene rearrangement to be made
before both productive y- and -chain rearrangements occur. If the
-chain binds to pTa, this heterodimer assembles with the CD3
complex and -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 , , and
loci, but not to the -chain locus. A minority of the
double-positive thymocytes give rise to additional : T-cells. Upon
rearrangement of the -chain locus, the -chain locus it contains is
eliminated as part of an extrachromosomal circle. Productive -chain
gene rearrangements produce double-positive CD4 CD8 : T-cells. This
ends the early stage of T-cell development. GENE EXPRESSION THROUGH
THE STAGES OF : 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
selection CD4 T-cells: survival Effector CD4 T-cells: Th2
polarization POSITIVE SELECTION THE KEYWORD: RECOGNITION
Only a small fraction of T-cells mature into functional T-cells
POSITIVE SELECTION -Occurs in the cortex, requires thymic
epithelial cells. double-positive thymocytes must recognize
self-MHC. 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 TCR 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 T-AND B-CELL DEVELOPMENT
SIDE BY SIDE VIEW OF T-AND B-CELL DEVELOPMENT Common lymphoid
progenitors (CLPs) give rise to both B-cell and T-cell lineages,
but the development of B-cells and T-cells occurs separately, with
B-cells developing in the bone marrow (bottom) and T-cells in the
thymus (top). In progenitor (pro)-B-cells and progenitor
(pro)-T-cells, the first antigen-receptor chain locus undergoes
V(D)J recombination, generating the immunoglobulin heavy (IgH)
chain in B-cells and the -chain of the T-cell receptor (TCR) in
T-cells. (In addition, the genes that encode TCR and TCR recombine
in pro-T-cells, sometimes giving rise to T-cells.) The first
receptor chain then associates with the surrogate second receptor
chain 5VpreB in precursor (pre)-B-cells, and the precursor
(pre)-TCR -chain (pT) in pre-T-cells yielding a pre-B-cell receptor
(pre-BCR) or pre-TCR complex, signaling through which mediates
proliferation and developmental progression. Next, lymphocytes stop
dividing and recombine the genes that encode the second receptor
chain, generating immunoglobulin light (IgL) chain in B-cells and
TCR in T-cells. In the main pathway of T-cell development (top),
continued rearrangement at the TCRA locus often occurs because of
autoreactivity or lack of positive selection, creating either a
non-functional rearrangement (denoted by the reverse arrow) or an
edited receptor with a new TCR. Positive selection ultimately stops
gene rearrangements and promotes the loss of either CD4 or CD8. In
B-cell development (bottom), successful recombination of the locus
that encodes the IgL chain leads to cell-surface BCR expression on
immature B cells. If the BCR is bound by autoantigen, these cells
continue V(D)J recombination, resulting in receptor editing,
particularly at loci that encode the IgL chain. Innocuous
(non-self-reactive) receptors promote positive selection, and these
B cells are subsequently released into the periphery. Ig:IgH chain
with constant region. Nemazee Nature Reviews Immunology 6, 728740
(October 2006) | doi: /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 The
response of the immune system to the stimuli of the outer and inner
environment. Antigens that are not harmful for the body will be
tolerated by the immune system: examples are the molecules of our
food or the body's own proteins. Pathogens that pose a danger will
generate an immune response.The "black box" represents the immune
system processing the stimuli from the environment. Immune response
IMMUNOLOGICAL TOLERANCE Immunological tolerance Unlike
immunosuppresion.
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. NEGATIVE SELECTION of T-cells in the thymus
Elimination of potentially
T-cells with high affinity TCR towards the self MHC/self peptide
complex are eliminated, but clones with intermediate affinity
survive. NEGATIVE SELECTION of T-cells in the thymus Elimination of
potentially autoreactive clones THE KEYWORD: AFFINITY CENTRAL
TOLERANCE Development of central T-cell tolerance is an integral
part of T-cell differentiation. T-cells incapable of interacting
with self-MHC/self-antigen complexes are eliminated during positive
selection, whereas clones with intermediate or high affinity
towards the same complexes will survive and become single-positive
T-cells. On the other hand, autoreactive clones are eliminated
during negative selection by dendritic cells and other cells at the
corticomedullary boundary of the thymus. T-cells with high affinity
TCR towards the self MHC/self peptide complex are destroyed
(negative selection, deletion), but clones with intermediate
affinity survive. A percentage of self-reactive T-cells will
survive the negative selection process, and differentiate into
regulatory T-cells, an interesting cell type that have high
affinity TCRs, bordering at negative selection, and have important
roles in maintaning peripheral tolerance (discussed later).
Immunological Tolerance and Autoimmunity : Self-Nonself
Discrimination in the Immune System and Its Failure Abbas, Abul K.,
MBBS, Basic Immunology: Functions and Disorders of the Immune
System, Chapter 9, 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. Central and peripheral tolerance to self
antigens. Central tolerance: Immature lymphocytes specific for self
antigens may encounter these antigens in the generative (central)
lymphoid organs and are deleted; B lymphocytes change their
specificity (receptor editing); and some T lymphocytes develop into
regulatory T-cells. Some self-reactive lymphocytes may complete
their maturation and enter peripheral tissues. Peripheral
tolerance: Mature self-reactive lymphocytes may be inactivated or
deleted by encounter with self antigens in peripheral tissues, or
suppressed by regulatory T-cells. Immunological Tolerance and
Autoimmunity : Self-Nonself Discrimination in the Immune System and
Its Failure Abbas, Abul K., MBBS, Basic Immunology: Functions and
Disorders of the Immune System, Chapter 9, Peripheral tolerance:
Elimination of fugitive or altered clones is an important role for
regulatory T-cells. SUMMARY THE QUESTION: THE ANSWER:
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 DEFICIENCY IN
ESTABLISHING CENTRAL T-CELL TOLERANCE
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-
CANDIDIASIS-ECTODERMAL 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 bystudying a rareimmunodeficiency.
https:///jimneydandme.wordp ress.com/james-story THE END V(D)J
RECOMBINATION 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-1is
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 -CHAIN GENE REARRANGEMENT
Rearrangement of a V,a D and a J gene segmentcreates the functional
V-domain exon. Unused V and J genes between the rearranged V and
Jgenes are deleted. 1ST CHECKPOINT After translocation to the
endoplasmic reticulum -chain is tested for its capacity to bind to
an invariant polypeptide called pT, which acts as a surrogate
-chain. After translocation to the endoplasmic reticulum
SUCCESSIVE TCR -CHAIN GENE REARRANGEMENT 2ND CHECKPOINT After
translocation to the endoplasmic reticulum -chain is tested for its
capacity to bind the -chain and assemble a T-cell receptor. A V
gene segment rearranges to a J 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 Elimination of potentially
autoreactive clones CENTRAL TOLERANCE 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
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