Transcript Presentation

Lecture on lymphocyte development
• Generation of antibodies and T cell receptors
by V(D)J recombination
• Lymphocyte development: generation of cells
with functional and useful antigen receptors
• Selection of B cells and T cells based on their
specificity to decrease self-reactivity
• Diseases resulting from defects or errors in
lymphocyte development: immunodeficiencies
and cancers
Generation of antibody & TCR diversity
• How do we make >109 different antibodies or TCRs?
Generation of antibody & TCR diversity
• How do we make >109 different antibodies or TCRs?
• Answer: Genes for antibodies and TCRs are present
in pieces that can be combined in many different
ways in different lymphocytes (combinatorial
diversity) and diversity is further increased by
adding or deleting nucleotides at the junctions
(junctional diversity)
VDJ
recombination
•Ordered rearrangement of
gene segments
•Lymphocyte DNA has 1
functional (in-frame) gene
of each type (usually); each
cell is unique until clonal
expansion and selection
•Expression of these
functional genes follows
normal rules of molecular
biology
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100
Cm is always
first heavy
chain type
Generation of Antibody Diversity
- Combinatorial diversity:


-
H chains: 100 VH x 27 DH x 6JH = 16,200
k light chains: 40 Vk x 5 Jk = 175
l light chains: 30 Vl x 4 Jl = 120
295 L chains x 16,200 H chains = 4.8 x 106
- Junctional diversity (addition or deletion
of nucleotides at recombination sites,
especially of H chain), estimated to add up
to 3x107 fold to overall diversity
(J and D form last loop in Ig domain structure; Ab can tolerate
different amino acid lengths resulting from junctional diversity)
Machinery of VDJ recombination
• Rag1, Rag2 (“recombination activating genes”)
– Only expressed in developing lymphocytes
– Recognize “recombination signal sequences”
adjacent to V, D and J segments in DNA
– Which Ig or TCR locus is rearranged is also
regulated
• Artemis and Non-homologous end-joining
recombination machinery (DNA repair system;
expressed in all cells)
• Uncommon forms of SCID resulting from
mutations in Rag1, Rag2 or Artemis
(autosomal; total 5-10% of SCID)
(SCID=severe combined immunodeficiency)
Lymphoid malignancies resulting from errors
in VDJ recombination
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VDJ Recombination reactions contributes to translocation
leading to over-expression of a cellular growth or survival
promoting gene (“oncogene”)
There is an ILM/Problem set on VDJ
recombination and lymphocyte
development on iROCKET
(nothing to hand in)
(answers will be posted this Friday)
Lymphocyte Development
Lymphocyte development is designed
to generate functional lymphocytes
with useful antigen receptors
that are not self-reactive
Much of what happens during lymphocyte
development is designed to improve the
efficiency of adaptive immunity
B cell development
IgH
rearrangement
k or l
rearrangement
•B cell development requires successful VDJ recombination
•Heavy chain protein is required for developing cell to
progress to pre-B cell stage and start rearrangements at Ig
Light chain genes
Pre-BCR and B cell development
•m heavy chain combines with
surrogate light chains to form preBCR (analogous to BCR)
•pre-BCR signals to cell to move to
next stage of development
•Pre-BCR and BCR signaling are
defective in X-linked
agammaglobulinemia due to loss-offunction mutations in Btk: strong
block in B cell development and no
antibodies.
Multiple protein tyrosine
kinases including Btk
Btk: Bruton’s tyrosine kinase
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B cell development
IgH
rearrangement
k or l
rearrangement
•B cell development requires successful VDJ recombination
•Heavy chain protein is required to progress to pre-B cell
stage and start rearrangements at Ig Light chain genes
•Light chain protein is required to progress to B cell stage
and leave the bone marrow
One B cell makes only one heavy chain
and one light chain
• This property of expressing only one of two
Ig alleles is called “allelic exclusion”
One B cell makes only one heavy chain
and one light chain
• This property of expressing only one of two Ig alleles
is called “allelic exclusion”
MECHANISM OF ALLELIC EXCLUSION
• Once a functional heavy chain is made, the pre-BCR
can assemble and send signals, this redirects the
Rag1 and Rag2 proteins away from the IgH locus and
toward the IgL loci
• Once a functional light chain is made, Rag 1 and Rag 2
expression is turned off
One B cell makes only one heavy chain
and one light chain
• This property of expressing only one of two Ig alleles
is called “allelic exclusion”
MECHANISM OF ALLELIC EXCLUSION
• Once a functional heavy chain is made, the pre-BCR can
assemble and send signals, this redirects the Rag1 and
Rag2 proteins away from the IgH locus and toward the
IgL loci
• Once a functional light chain is made, Rag 1 and Rag 2
expression is turned off
• Allelic exclusion enhances the efficiency of antibodies,
since one B cell and its clonal progeny will make
homogenous antibodies with two identical binding sites.
Central Tolerance of B cells
to Self-antigens
CONCEPT: Self-antigen is always present but foreign antigens
are generally not present at sites of development (due to timing
and/or routes of antigen trafficking); therefore developing
lymphocytes that see antigen are typically seeing self-antigen
Central Tolerance of B cells
to Self-antigens
CONCEPT: Self-antigen is always present but foreign antigens
are generally not present at sites of development (due to timing
and/or routes of antigen trafficking); therefore developing
lymphocytes that see antigen are typically seeing self-antigen
Immature B cell + self-antigen:
•B cells can continue to rearrange IgL genes, try to change L
chain and lose self-reactivity (“receptor editing”)
•B cells can die (“clonal deletion” or “negative selection”)
•B cells can become refractory to activation (“clonal
anergy”)
(multiple mechanisms probably reflect the demands of
efficiency and the importance of tolerance to self-antigens)
Development and central tolerance for T cells are
nearly the same as for B cells
The thymus is a specialized organ
for T cell development
The thymus has several specialized
cell types that contribute to T cell
development, primarily by
presentation of MHC + peptides for
selective events
In DiGeorge syndrome, the
thymus gland fails to develop and
T cell development is impaired
(other organs are also affected)
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Early steps in T cell
development
•Stages in development of T cells are defined
by expression of the co-receptors CD4 and CD8
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Early
Lymphoid
progenitor
Early steps in T cell
development
•Stages in development of T cells are defined
by expression of the co-receptors CD4 and CD8
•TCR b is rearranged first; this forms a preTCR together with “pre-TCRa”
•pre-TCR signals to promote T cell development
to the CD4+CD8+ stage, where TCR a
rearranges
Early
Lymphoid
progenitor
Early steps in T cell
development
•Stages in development of T cells are defined
by expression of the co-receptors CD4 and CD8
•TCR b is rearranged first; this forms a preTCR together with “pre-Ta”
•pre-TCR signals to promote T cell development
to the CD4+CD8+ stage, where TCR a
rearranges
•Survival is dependent on cytokines from thymic
environment, especially IL-7
(X-linked form of SCID is caused by mutations
in the gc cytokine receptor chain, which is
required for IL-7 response and response to
some other cytokines)
Early
Lymphoid
progenitor
Positive Selection links the specificity
of a T cell to its development
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Positive Selection of Thymocytes
Receipt of a weak signal through the TCR results in:
• Cessation of VDJ recombination at the TCR a locus
• Loss of expression of the “wrong” co-receptor (e.g.: positive
selection with MHC II leads to retained expression of CD4
and turned off expression of CD8)
• Choice of the corresponding functional lineage (helper vs.
cytotoxic T cell)
• Maturation and eventual export from the thymus to the
recirculating T cell pool
Positive Selection of Thymocytes
Receipt of a weak signal through the TCR results in:
•
•
•
•
Cessation of V(D)J recombination at the TCR a locus
Loss of expression of the “wrong” co-receptor (e.g.: positive
selection with MHC II leads to retained expression of CD4 and
turned off expression of CD8)
Choice of the corresponding functional lineage (helper vs. cytotoxic
T cell)
Maturation and eventual export from the thymus to the
recirculating T cell pool
KEY POINT: Positive selection is the process that links the
specificity of that cell’s TCR for MHC I vs. MHC II to
expression of CD4 or CD8 AND to the functional potential
of that T cell (helper vs. cytotoxic), therefore specificity
and function are matched to give a useful T cell.
Positive and Negative Selection in T cell Development
Positive vs. Negative Selection
NEGATIVE SELECTION: results from strong interaction of a
self peptide/MHC complex with the TCR of a thymocyte. (Foreign
antigens are not brought to the thymus by APC, so typically
antigen in thymus is self)
POSITIVE SELECTION: results from weak interaction of a self
peptide/MHC complex with the TCR of a thymocyte. (Links the
MHC specificity of the TCR to the functional potential of each T
cell)
Resulting T cells are more likely to be useful (again: efficiency)
T cell tolerance
T cell tolerance is created by a combination of
mechanisms, of which negative selection is only one.
In the thymus, some self-reactive T cells become
“regulatory T cells” rather than dying. Regulatory T
cells can suppress T cell immune responses in the
periphery. These cells and other mechanisms of
peripheral tolerance will be discussed next week by
Dr. Abbas.
Inherited Immunodeficiencies
Inherited Immunodeficiencies
•Individual presents with abnormally severe and
frequent infections, often early in life
•Types of infections are often an indication of what
type of immunodeficiency
•Some of the more common immunodeficiencies are Xlinked, so this is potentially important information to
share with parents
•Genetic causes include immune-specific genes and
also genes involved in purine metabolism
•Therapies include replacement therapy (B cell
immunodeficiencies; ADA deficiency) and bone
marrow transplantation
Characteristics of Inherited
Immunodeficiencies
TB also
Figure 12-1 Abbas and Lichtman
“Severe combined immunodeficiency (SCID)”
Defects in Lymphocyte development
leading to Immunodeficiency
Figure 12-2
Abbas and
Lichtman
Immunodeficiencies resulting from
defective lymphocyte activation
Figure 12-4
Abbas and
Lichtman
Note: lymphocyte activation is covered in later lectures