Transcript Lecture 18-Chap18

Chapter 18
Somatic
Recombination and
Hypermutation in the
Immune System
18.1 The Immune System: Innate and
Adaptive Immunity
• antigen – A molecule that can bind specifically
to an antigen receptor, such as an antibody.
• B cell – A lymphocyte that produces antibodies.
Development occurs primarily in bone marrow.
– B cells emerging from the marrow undergo further
differentiation in the bloodstream and peripheral
lymphoid organs.
18.1 The Immune System: Innate and
Adaptive Immunity
• T cells – Lymphocytes of the T (thymic) lineage.
T cells differentiate in the thymus from stem cells
of bone marrow origin.
– They are grouped in several functional types
(subsets) according to their phenotype, mainly
expression of surface proteins CD4 and CD8.
– Different T cell subsets are involved in different cellmediated immune responses.
18.1 The Immune System: Innate and
Adaptive Immunity
• adaptive (acquired) immunity – The response
mediated by lymphocytes that are activated by
their specific interaction with antigen.
– The response develops over several days as
lymphocytes with antigen-specific receptors are
stimulated to proliferate and become effector cells.
– It is responsible for immunological memory.
18.1 The Immune System: Innate and
Adaptive Immunity
• B cell receptor (BCR) – The receptor for
antigen expressed on the surface of B
lymphocytes.
– The BCR has the same structure and specificity of the
antibody that will be produced by the same B cell
after its activation by antigen.
18.1 The Immune System: Innate and
Adaptive Immunity
• T cell receptor (TCR) – The antigen receptor on
T lymphocytes.
– It is clonally expressed and binds to a complex of
MHC class I or class II protein and antigen-derived
peptide.
• Antibody response – An immune response that
is mediated primarily by antibodies. It is defined
as immunity that can be transferred from one
organism to another by serum antibody.
18.1 The Immune System: Innate and
Adaptive Immunity
• innate immunity – A response triggered by
receptors whose specificity is predefined for
certain common motifs found in bacteria and
other infective agents.
– The receptor that triggers the pathway is typically a
member of the Toll-like receptor (TLR) family, and the
pathway resembles the pathway triggered by Toll
receptors during embryonic development.
– The pathway culminates in activation of transcription
factors that cause genes to be expressed whose
products inactivate the infective agent, typically by
permeabilizing its membrane.
18.2 The Innate Response Utilizes
Conserved Recognition Molecules and
Signaling Pathways
• Innate immunity is triggered by pattern recognition
receptors (PRRs) that recognize highly conserved
microbe-associated molecular patterns (MAMPs) found
in bacteria, viruses, and other infectious agents.
• PAMPs – Pathogen-associated molecular patterns, now
referred to as MAMPs, are broadly conserved microbial
components, including bacterial flagellin and
lipopolysaccharides, that are recognized by PRRs, which
critically initiate innate immune responses.
18.2 The Innate Response Utilizes
Conserved Recognition Molecules and
Signaling Pathways
Figure 18.01: Innate immunity; a summary of MAMPs and PRRs.
18.2 The Innate Response Utilizes
Conserved Recognition Molecules and
Signaling Pathways
• Toll-like receptors (TLRs) are important PRRs that
directly activate innate immune responses and direct the
initial stages of adaptive responses. TLRs are expressed
in dendritic cells (DCs), macrophages, neutrophils, B
lymphocytes, and some T lymphocytes.
• TLR signaling pathways are highly conserved from
invertebrates to vertebrates and an analogous pathway
is found in plants.
18.2 The Innate Response Utilizes
Conserved Recognition Molecules and
Signaling Pathways
• Toll/interleukin 1/resistance (TIR) domain –
The key signaling domain unique to the TLR
system.
– TIR is located in the cytosolic face of each TLR, and
also in TLR adaptors.
– Similar to the TLRs, the adaptors are conserved
across many species.
– These adaptors are MyD88, MyD88-adaptor-like
(MAL, also known as TIRAP), TIR-domain-containing
adaptor protein inducing IFN (TRIF; also known as
TICAM1), TRIF-related adaptor molecule (TRAM; also
known as TICAM2), and sterile armadillo-motifcontaining protein (SARM).
18.3 Adaptive Immunity
• Helper T (Th) cells produce signals required by B cells
to enable them to differentiate into antibody-producing
cells.
• complement – A set of ~20 proteins that function
through a cascade of proteolytic actions to lyse infected
target cells, or to attract macrophages.
• cell-mediated response – The immune response that is
mediated primarily by T lymphocytes, and defined based
on immunity that cannot be transferred from one
organism to another by serum antibody.
18.3 Adaptive Immunity
Figure 18.04: Free antibodies bind to antigens to form antigen-antibody complexes.
18.3 Adaptive Immunity
• Cytotoxic T cells (CTLs) or
killer T cells are responsible for
the cell-mediated response in
which fragments of foreign
antigens are displayed on the
surface of a cell.
– These fragments are recognized
by the TCR expressed on the
surface of T cells.
Figure 18.05: Cell-mediated immunity.
18.3 Adaptive Immunity
• In TCR recognition, the antigen must be presented in
conjunction with a major histocompatibility complex
(MHC) molecule.
• autoimmune disease – A pathological condition in
which the immune response is directed to self antigen.
18.4 Clonal Selection Amplifies Lymphocytes
That Respond to a Given Antigen
• clonal selection – The theory proposed that
each lymphocyte expresses a single antigen
receptor specificity and that only those
lymphocytes that bind to a given antigen are
stimulated to proliferate and to function in
eliminating that antigen.
– Thus, the antigen "selects" the lymphocytes to be
activated.
– Clonal selection was originally a theory, but it is now
an established principle in immunology.
18.4 Clonal Selection Amplifies Lymphocytes
That Respond to a Given Antigen
Figure 18.06: The B cell and T cell repertoires include BCRs and TCRs with a variety of
specificities.
18.4 Clonal Selection Amplifies Lymphocytes
That Respond to a Given Antigen
• Each B cell expresses a unique BCR and each T cell
expresses a unique TCR.
• A broad repertoire of BCRs/antibodies and TCRs exists
at any time in an organism.
• Antigen binding to a BCR or TCR triggers the clonal
proliferation of that receptor-bearing B or T cell.
18.4 Clonal Selection Amplifies
Lymphocytes That Respond to a Given
Antigen
• hapten – A small molecule that can elicit an
immune response only when conjugated with a
carrier, such as a large protein.
– Once antibodies have been induced by the carrierconjugated hapten, the hapten will in general bind
those antibodies.
• epitope – The portion of an antigen that is
recognized by the antigen receptor on
lymphocytes.
– It is also called the antigenic determinant.
GENE RECOMBINATION VIDEO
http://www.youtube.com/watch?v=AxIMmN
ByqtM
18.5 Ig Genes are Assembled from Discrete
DNA Segments in B Lymphocytes
Figure 18.07: An antibody
(immunoglobulin, or Ig) molecule is a
heterodimer consisting of two identical
heavy chains and two identical light chains.
• An antibody
(immunoglobulin) consists of
a tetramer of two identical
light (L) chains and two
identical heavy (H) chains.
There are two families of L
chains (Igλ and Igκ) and a
single family of IgH chains.
• Each chain has an Nterminal variable (V) region
and a C-terminal constant
(C) region. The V region
recognizes the antigen and
the C region mediates the
effector response.
18.5 Ig Genes are Assembled from Discrete
DNA Segments in B Lymphocytes
• V and C regions are separately encoded by V(D)J gene
segments and C gene segments.
• A gene coding for a whole Ig chain is generated by
somatic recombination of V(D)J genes (variable,
diversity, and joining genes in the H chain; variable and
joining genes in the L chain) giving rise to V domains, to
be expressed together with a given C gene (C domain).
18.6 L Chains Are Assembled by a Single
Recombination Event
• A λ chain is assembled through a single recombination
event involving a Vλ gene segment and a Jλ-Cλ gene
segment.
• The Vλ gene segment has a leader exon, an intron, and
a Vλ-coding region. The Jλ-Cλ gene segment has a short
Jλ-coding exon, an intron, and a Cλ-coding region.
18.6 L Chains Are Assembled by a Single
Recombination Event
Figure 18.08: The Cl gene segment is preceded by a J segment, so that Vl-Jl recombination
generates a productive Vl-JlCl.
18.6 L Chains Are Assembled by a Single
Recombination Event
• A κ chain is
assembled by a
single
recombination
event involving a
Vκ gene segment
and one of five Jκ
segments
preceding the Cκ
gene.
Figure 18.09: The Ck gene segment is preceded by multiple J
segments in the germ line.
18.7 H Chains Are Assembled by Two
Sequential Recombination Events
• The units for H chain recombination are a VH gene, a D
segment, and a JH-CH gene segment.
• The first recombination joins D to JH-CH. The second
recombination joins VH to DJH-CH to yield VH-DJH-CH.
• The CH segment consists of four exons.
18.7 H Chains Are Assembled by Two
Sequential Recombination Events
Figure 18:10: Heavy genes are assembled by sequential recombination events.
18.8 Recombination Generates Extensive
Diversity
• The human IgH locus can generate in excess of 104
VHDJH sequences.
• Imprecision of joining and insertion of unencoded
nucleotides further increases VHDJH diversity to 106
sequences.
• Recombined VHDJH-CH can be paired with in excess of
104 recombined VκJκ-Cκ or VλJλ-Cλ chains.
Figure 18.11: The lambda family
consists of Vl gene segments and a
small number of Jl-Cl gene segments.
Figure 18.12: The human and mouse Igk
families consist of Vk gene segments and
five Jk segments linked to a single Ck gene
segment.
18.8 Recombination Generates
Extensive Diversity
Figure 18.13: A single gene cluster in humans contains all the information for the IgH chain.
18.9 V(D)J DNA Recombination Uses RSS
and Occurs by Deletion or Inversion
• The V(D)J recombination machinery uses consensus
sequences consisting of a heptamer separated by either
12 or 23 base pairs from a nonamer (recombination
signal sequence, RSS).
Figure 18.14: RSS sequences are present in inverted orientation at each pair of
recombining sites.
18.9 V(D)J DNA
Recombination Uses RSS
and Occurs by Deletion or
Inversion
• Recombination occurs by doublestrand DNA breaks (DSBs) at the
heptamers of two RSSs with different
spacers: “12/23 rule.”
Figure 18.15: Breakage and recombination at RSSs generate
VJC sequences.
Adapted from D. B. Roth, Nat. Rev. Immunol. 3 (2003): 656-666.
18.9 V(D)J DNA Recombination Uses RSS
and Occurs by Deletion or Inversion
• The signal ends of the DNA excised between two DSBs
are joined to generate a DNA circle or signal circle. The
coding ends are ligated to join VL to JL-CL (L chain), or
D to JH-CH and VH to DJH-CH (H chain). If the
recombining genes lie in an inverted rather than direct
orientation, the intervening DNA is inverted and retained,
instead of being excised as a circle.
18.10 Allelic Exclusion Is Triggered by
Productive Rearrangements
• V(D)J gene rearrangement is productive if it leads to
expression of a protein.
• A productive V(D)J gene rearrangement prevents any
further rearrangement of the same kind from occurring,
whereas a nonproductive rearrangement does not.
• Allelic exclusion applies separately to L chains (only
one VκJκ or VλJλ may be productively rearranged) and to
VHDJH chains (one H chain is productively rearranged).
18.10 Allelic Exclusion Is Triggered by
Productive Rearrangements
Figure 18.16: A successful rearrangement to produce an active light or heavy chain
suppresses further rearrangements, resulting in allelic exclusion.
18.11 RAG1/RAG2 Catalyze Breakage and
Religation of V(D)J Gene Segments
• The RAG proteins are necessary and sufficient
for the cleavage reaction.
– RAG1 recognizes the nonamer consensus sequences
for recombination.
– RAG2 binds to RAG1 and cleaves DNA at the
heptamer.
– The reaction resembles the topoisomerase-like
resolution reaction that occurs in transposition.
18.11 RAG1/RAG2 Catalyze Breakage and
Religation of V(D)J Gene Segments
Figure 18.17: Processing of coding ends
introduces variability at VkJk, VlJl, or
VH-D-JH junctions. Depicted is a Vk-Jk
junction.
18.11 RAG1/RAG2 Catalyze Breakage and
Religation of V(D)J Gene Segments
• The reaction proceeds through a hairpin intermediate at
the coding end; opening of the hairpin is responsible for
insertion of extra bases (P nucleotides) in the
recombined gene.
• Terminal deoxynucleotidyl transferase (TdT) inserts
additional unencoded N nucleotides at the V(D)J
junctions.
• The DSBs at the coding joints are repaired by the same
mechanism that has generated the whole V(D)J
sequence.
18.11 RAG1/RAG2 Catalyze Breakage and
Religation of V(D)J Gene Segments
• severe combined immunodeficiency (SCID) –
A syndrome that stems from mutations in
different genes that result in B and/or T cell
deficiency.
– X-linked SCID is due to IL-2R γ chain gene mutations;
autosomal recessive SCID can be due to
RAG1/RAG2 mutations, Artemis gene mutations,
Jak3 gene mutations, ADA gene mutations, IL-7R αchain mutations, CD3 δ or ε mutations, or CD45 gene
mutations.
18.12 B Cell Differentiation: Early IgH Chain
Expression Is Modulated by RNA
Processing
• All B lymphocytes newly emerging from the bone
marrow express the membrane-bound
monomeric form of IgM (Igμm).
• As the B cell matures after exiting the bone
marrow, it expresses surface IgD at a high
levels. Such IgD consists of Igδm containing the
same VHDJH sequence paired with the same
recombined κ or λ chain as the IgM expressed
by the same cell.
18.12 B Cell
Differentiation: Early
IgH Chain Expression
Is Modulated by RNA
Processing
Figure 18.19: The 3 end of each CH gene
cluster controls the use of splicing junctions
so that alternative forms of the heavy gene
are expressed.
• A change in RNA splicing
causes μm to be replaced
by the secreted form
(Igμs) after a mature B cell
is activated and begins
differentiation to antibodyproducing cells in the
periphery.
18.13 Class Switching Is Effected by DNA
Recombination (Class Switch DNA
Recombination, CSR)
• Igs comprise five classes according to the type of CH
chain.
Figure 18.20: Immunoglobulin type and functions are determined by the H chain.
18.13 Class Switching Is Effected by DNA
Recombination (Class Switch DNA
Recombination, CSR)
• Class switching is
effected by a
recombination between S
regions that deletes the
DNA between the
upstream CH region gene
cluster and the
downstream CH region
gene cluster that is the
target of recombination.
Figure 18.21: Class switching of CH genes.
18.13 Class Switching Is Effected by DNA
Recombination (Class Switch DNA
Recombination, CSR)
• CSR relies on a molecular machinery that is different
from that of V(D)J recombination and is acting later in B
cell differentiation.
18.14 CSR Involves AID and Elements of
the NHEJ Pathway
• CSR requires activation of intervening promoters (IH
promoters) that lie upstream of each of the two S regions
involved in the recombination event and germline IH-CH
transcription through the respective S regions.
• S regions contain highly repetitive 5′-AGCT-3′ motifs. 5′AGCT-3′ repeats are the main targets of the CSR
machinery and DSBs.
18.14 CSR Involves AID and Elements of
the NHEJ Pathway
Figure 18.22: Class switching passes occurs through sequential and discrete stages.
18.14 CSR Involves AID and Elements of
the NHEJ Pathway
• activation-induced (cytidine) deaminase
(AID) – An enzyme that removes the amino
group from the cytidine base in DNA.
– AID mediates the first step (deoxycytidine
deamination) in the series of events that lead
to insertion of DSBs within S regions; the
DSBs’ free ends are then religated through
an NHEJ-like reaction or A-EJ pathway.
18.15 Somatic Hypermutation (SHM)
Generates Additional Diversity
• Somatic hypermutation (SHM) introduces somatic
mutations in the antigen-binding V(D)J sequence.
• Such mutations occur mostly as substitutions of
individual bases.
Figure 18.24: Somatic mutation
occurs in the region surrounding
the V segment and extends over
the recombined V(D)J segment.
18.15 Somatic Hypermutation (SHM)
Generates Additional Diversity
• In the IgH chain locus, SHM depends on the iEμ
and 3′Eκ that enhance VHDJH-CH transcription.
• In the Igκ chain locus, SHM depends on iEκ and
3′Eκ that enhance VκJκ-Cκ transcription.
– The λ locus transcription depends on the weaker λ2-4
and λ3-1 enhancers.
18.16 SHM Is Mediated by AID, Ung, Elements of
the Mismatch DNA Repair (MMR) Machinery, and
Translesion DNA Synthesis (TLS) Polymerases
• SHM uses some of the same critical elements of CSR.
– Like CSR, SHM requires AID.
• Ung intervention influences the pattern of somatic
mutations.
• Elements of the MMR pathway and of TLS polymerases
are involved in SHM and CSR.
18.16 SHM Is Mediated by AID, Ung, Elements of
the Mismatch DNA Repair (MMR) Machinery, and
Translesion DNA Synthesis (TLS) Polymerases
Figure 18.25: Deamination of C by AID gives rise to a U:G mispair.
18.17 Chromatin Modification in V(D)J
Recombination, CSR, and SHM
• Chromatin modification in V(D)J recombination,
CSR, and SHM are induced by the same stimuli
that drive these processes.
• Transcription factors and transcription target
histone posttranslation modifications.
• Histone modifications are read and transduced
by chromatin-interacting factors.
18.18 Expressed Igs in Avians Are
Assembled from Pseudogenes
• An Ig gene in chickens is generated by copying a
sequence from one of 25 pseudogenes into the
recombined (acceptor) V gene: gene conversion.
Figure 18.26: The chicken l light
chain locus has 25 V pseudogenes
upstream of the single functional VlJl-C region.
18.18 Expressed Igs in Avians Are
Assembled from Pseudogenes
• The enzymatic machinery of gene conversion depends
on AID and enzymes involved in homologous
recombination.
• Ablation of certain DNA homologous recombination
genes transforms gene conversion into SHM.
18.19 B Cell Differentiation in the Bone
Marrow and the Periphery: Generation of
Memory B Cells Enables a Prompt and
Strong Secondary Response
• Mature B cells that emerge from the bone marrow and
are recruited in the primary response express a BCR
with only a moderate affinity for antigen.
• Toward the end of the primary response, B cells
expressing BCRs with a higher affinity for antigen are
selected and later revert back to a resting state to
become memory B cells.
• Re-exposure to the same antigen triggers a secondary
response through rapid activation and clonal expansion
of memory B cells.
18.19 B Cell
Differentiation in the
Bone Marrow and the
Periphery: Generation of
Memory B Cells Enables
a Prompt and Strong
Secondary Response
Figure 18.29: B cell differentiation is
responsible for acquired immunity.
18.20 The T Cell Receptor for
Antigen (TCR) Is Related to the BCR
• T cells use a mechanism of V(D)J recombination similar
to that of B cells to express either of two types of TCR.
• TCRαβ is found on more than 95% and TCRγδ on less
than 5% of T lymphocytes in the adult.
Figure 18.31: The human TCR locus
contains interspersed and segments.
Figure 18.32: The TcR locus contains many V
gene segments spread over ~500 kb that lie
~280 kb upstream of the two D-J-C clusters.
18.20 The T Cell Receptor for
Antigen (TCR) Is Related to the BCR
• The organization of the TCRα locus resembles that of
the Igκ locus; the TCRβ resembles the IgH locus, the
TCRγ, the Igλ locus.
18.21 The TCR Functions in
Conjunction with the MHC
• The TCR recognizes a short
peptide set in the groove of an MHC
molecule on the surface of an
antigen-presenting cell (APC).
Figure 18.34: T cell development
proceeds through sequential
stages.
Figure 18.35: The two chains of the T cell receptor
associate with the polypeptides of the CD3 complex.
18.21 The TCR Functions in Conjunction with
the MHC
• The recombination process to generate functional TCR
chains is intrinsic to the development of T cells.
• The TCR is associated with the CD3 complex that is
involved in transducing TCR signals from the cell surface
to the nucleus.
18.22 The Major Histocompatibility Complex
(MHC) Locus Comprises a Cohort of Genes
Involved in Immune Recognition
• The MHC locus codes for Class I, Class II, and Class III
molecules.
Figure 18.37: The MHC region extends for >2 Mb.
18.22 The Major Histocompatibility Complex
(MHC) Locus Comprises a Cohort of Genes
Involved in Immune Recognition
• Class I proteins are the
transplantation antigens
distinguishing “self” from
“nonself.”
• Class II proteins are involved
in interactions of T cells with
APCs.
Figure 18.36: Class I and class II MHC have a
related structure.
18.22 The Major Histocompatibility Locus
Comprises a Cohort of Genes Involved in
Immune Recognition
• MHC class I molecules are heterodimers consisting of a
variant α chain and the invariant β2 microglobulin.
• MHC class II molecules are heterodimers consisting of
an α chain and a β chain.
Figure 18.38: Each class of MHC genes
has a characteristic organization, in
which exons represent individual
protein domains.