ICL 2.0: TCR & Ig Diversity Flashcards
what does RAG stand for?
recombination activating gene
assembly of immunoglobin genes is tightly controlled and requires the recombination activating gene (RAG) gene products
what does AID stand for?
activation-induced cytidine deaminase enzyme
what does TdT stand for?
terminal deoxynucleotidyl transferase enzyme
how is the adaptive immune system able to have such a specific response to an antigen?
the highly specialized immune response is produced because each B lymphocyte and each T lymphocyte has a different receptor able to recognize a distinct unique structure (called epitope) on a pathogen (antigen)
consequently, during an adaptive immune response only B and T lymphocytes bearing receptors that recognize the infecting pathogen are selected for the adaptive immune response
these selected B and T lymphocytes proliferate and differentiate into effector lymphocytes through the process called clonal selection and clonal expansion
what is clonal selection?
the processes that select lymphocytes for proliferation and differentiation into effector lymphocytes
this is needed because during an adaptive immune response only lymphocytes bearing receptors that recognize the infecting pathogen are selected for the response
When clonal selection gets out of control, this is how you get lymphomas!
how are lymphocytes formed?
a common progenitor called pluripotent hematopietic stem cells derive multidifferential stem cells, which derive lymphoid stem cells and subsequently lymphocytes
multidifferential stem cells derive also myloid progenitor cells which next give rise to red blood cells (erythrocytes), and platelets (megakarocytes)
the same myloid progenitor cells derive also neutrophil, eosinophil, and basophil lines
how do B and T cells get receptor diversity?
it stems from the fact that the diversity of receptors for B and T cells are produced by mixing of gene fragments encode receptors
the assembly of these fragments may encode endless possibilities for the extensive diversity of immunoglobin (Ig) or B cell receptor (BCR) and T cell receptor (TCR)
what is allelic exclusion?
allelic exclusion control ensures that each B cell expresses only one type of binding site because an antibody binding site is determined by the combination of a particular heavy and light chain
it protects a single receptor specific for each B cell clone
there’s two copies of each Ig gene cluster but clonal selection requires just one H and one L chain to be produces so that there’s only one receptor type per B cell
if there’s no allelic exclusion then there would be 4 light chains and 2 heavy
what is the numbers paradox?
we think there’s about 10ˆ9 different antibodies, each with their own specific receptors
but the number of genes in the human genome is no more than 2x10ˆ4 genes
the calculation of required specificities and available number of genes suggests that there’s 50,000 more Ab proteins than genes in the cell….
so how is it possible to achieve such high diversity numbers with minimal number of available genes???
this can be accomplished through the process of gene fragments rearrangements in a fashion producing practically an infinite numbers of possible specificities
what’s the structure of an antibody?
Y shaped - consists of two heavy chains and two light chains
the AA sequence of each H and L chain is known as a variable region or V region and a constant C region
the variability portion in V region is the reason for the great diversity of antigen-binding specificities because paired V regions of H and L chains form the antigen binding site
remaining parts of H and L chains have much more limited variations in amino acid sequence and are called the constant region
slide 11
what forms the antigen binding site?
the variability portion in V region is the reason for the great diversity of antigen-binding specificities because paired V regions of H and L chains form the antigen binding site
Each B cell and each T cell recognizes only one epitope!
what is the combinatorial joining of Ig gene segments theory?
one gene even when assembled from different fragments is producing one protein
extensive diversification of germline sequences and combinatorial joining in the evolution of immunoglobulin heavy chain and light chain produces multiple specificities
there are 3 fragments in H chain = V, D, and J and there are 2 fragments in L chain = V and J for rearrangement of the variable region
after rearrangement, mRNA is produced, spliced and transcribed into a protein
if there are 5 fragments of 2 regions and one fragment from each region would make a variable fragment; then you do the math and there should be 25 possibilities of recombination rearrangements
when one of these possibilities is selected B cell does not express other possibilities
what is the combinatorial joining of Ig gene segments in light chains of Ig?
there are 2 fragments in the V region of L chain = V and J for rearrangement of the variable region
there are 35 Vϰ fragments with 5 J fragments = 175 possibilities
there are 30Vλ fragments with 4 J fragments = 120 possibilities
175+120 possibilities = 300 possibilities for the L chain
at the moment that one of these 300 possibilities is successfully selected, other options are not used
what is the combinatorial joining of Ig gene segments in heavy chains of Ig?
there are 3 fragments in the variable region of H chain = V, D, and J
there are 40 V fragments, 23 D fragments, and 6 J fragments = 5520 possibilities
again, when one of these 5520 possibilities is successfully selected, other options are not used
how does a B cell decide which recombination group to pick to make up its epitope?
a single B cell selects one of 5520 combinations of the H chain and one of 300 combinations for the L chain to encode these two fragments as its antigen (epitope) recognition site
what does RSS stand for?
recombination signal sequences
what does the RSS do?
RSS = recombination signal sequences
each V, D, or J gene segment in H chain and each V and J gene segment in L chain are flanked by the RSS
what are the types of RSS?
- a nanomer (9-bp) and heptamer (7-bp) separated by a 12 bp spacer
- a nanomer (9-bp) and heptamer (7-bp) separated by a 23-bp spacer
the heptamer (7-bp) is located at the nicking site of the gene fragment and nanomer (9-bp) as a positioning site for enzyme involved in cutting
which RSS type flanks H chain fragments?
V and J fragments are flanked by RSS with a 23-bp spacer
D fragments are flanked by RSS with a 12-bp spacer
which RSS type flanks L chain fragments?
V fragments are flanked by RSS with a 23-bp spacer
J fragments are flanked by RSS with a 12-bp spacer
what is the 12/23 rule?
only gene segments flanked by an RSS of different length can rearrange efficiently
the signaling system with 12-bp and 23-bp spacers create very effective mechanism for proper rearrangements as only a 23-bp spacer will connect to a 12-bp spacer. These two RSS signals create a 12/23 rule
only a pair of dissimilar spacer RSS signals will be efficiently recombined = this mechanism guarantees efficient rearrangements of L and H chains
this insures that there’s not V-V joining, for example
what do RAG1 and RAG2 do?
the process of rearrangements must be controlled and executed by enzymes which are able to separate, shuffle and rejoin the V, D, and J fragments for H chain as well as V and J fragments for L chain –> RAG1 and RAG2 are at the ends of VDJ genes that separate, shuffle, and rejoin the VDJ genes
RAG1 and RAG2 encode enzymes that play an important role in the rearrangement and recombination of gene fragments of immunoglobulin and T cell receptor molecules during the process of recombination
the diversity of antibodies (or BCRs) and TCRs are achieved by VDJ recombination
they work as multi-subunit complexes to cleave a single double stranded DNA between the antigen receptor coding segment and RSS signal
when are RAG1 and RAG2 active?
both RAG1 and RAG2 enzymes are active exclusively during the development process of B and T cells
what happens when there’s a RAG1 or RAG2 mutation?
RAG mutations lead to a Severe combined ImmunoDeficiency = SCID
there is partial V(D)J recombinase activity in an inherited disorder called Omenn syndrome
this is an absence of circulating B cells and infiltration of skin with activated oligoclonal T cells
what is Omenn syndrome?
an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by erythroderma, desquamation, alopecia, chronic diarrhea, failure to thrive, lymphadenopathy, eosinophilia, hepatosplenomegaly
caused by amino acid substitution mutations in the RAG genes that reduce their activity to 1–25% of normal
give an example of how RAG works
RAG enzymes flank a 23-bp spacer and a 12-bp spacer
one RAG complex binds to the 23-bp spacer and second RAG complex to the 12-bp spacer to attach RSS signal site containing a 12-bp spacer to RSS signal site containing a 23-bp spacer
this fulfills the 12/23 rule
the sequences are broken at the heptamer sequences
the fragment between joined ends is excised as a circle
what is the combinatorial joining of heavy and light chains theory?
to calculate the possibilities for V regions rearrangements: 3 L variable chains with 4 H variable chains would produce 12 antibodies, each with a unique antigen specificity
but don’t forget there’s 5520 possible H-chain combination and 300 possible L chain combinations
so that gives you 1.7x10^6 possible Ab molecules!
also, there can be imprecise joining of V and J fragments in L chains as well as V,D, and J fragments for H chains which would produce additional diversity! aka changing a couple nucleotides at the joining site could give you extra combinations because AAs would change
how does addition of untemplated nucleotides increase Ab diversity?
another way to increase diversity is addition of N-nucleotides or non-templated nucleotides producing an additional N-region diversification
this process is under the control of TdT
TdT adds N-nucleotides to the V, D and J exons during antibody gene recombination enabling the phenomenon of additional functional diversity
this process occurs more commonly in H-chains; and it occurs at amino (N) end of V, D or J segments during joining
N-nucleotides or non-templated nucleotides are believed to exist only to create diversity at VJ and VDJ recombination
what does TdT do?
it’s an enzyme that’s a specialized DNA polymerase
it’s expressed in immature, pre-B and pre-T cells
where is the process of non-templated nucleotides additions more likely to occur?
H chains
it occurs at amino (N) end of D or J segments during joining
what are the three ways to increase Ab diversity?
- combinatorial joining of H and L chains
- imprecise VJ or VDJ joining = junctional diversification
- N-region diversification
- somatic hypermutation (B cells only**)
ALL THE SAME WAYS TO INCREASE DIVERSITY IS PRESENT IN B AND T CELLS DURING THEIR MATURATION!
what is somatic hypermutation?
a process that allows B cells to mutate the genes that they use to produce antibodies specific for certain pathogens; it involves a programmed process of mutation affecting the variable regions in immunoglobulin genes
somatic hypermutation improves specificity of antibody = affinity
it is associated with affinity maturation of BCR and Ig produced by this B cell
somatic hypermutation requires the activity of AID**
unlike germline mutation somatic hypermutation affects only individual B cell and such mutations are NOT transmitted to the offspring!!!
SOMATIC HYPERMUTATION IS EXCLUSIVELY FOR B CELLS! NEVER FOR T CELLS!
what does AID do in somatic hypermutation?
somatic hypermutation requires the activity of activation induced cytidine deaminase (AID)
AID is an enzyme that creates deliberate mutations in DNA by resining the amino group from a cytidine base which turns it into a uridine; which is recognized as a thymidine
aka it changes a CG base pair into a UG base pair and since the cell’s DNA repair machinery recognizes it as a TG base pair, it finally converts up as a TA base pair
so this causes mutations that produce antibody diversity but that same mutation can lead to B-cell lymphoma!!
what does AID deficiency cause?
autosomal recessive form of the hyper-IgM syndrome (HIGM2)
characterized by normal or elevated serum IgM levels with absence of IgG, IgA, and IgE, resulting in profound susceptibility to bacterial infections
AID deficiency causes hyper IgM syndrome because you can’t delete anything and our bodies are just gonna produce the first coding region they come across so you can’t do class switching without AID
what are CDRs?
CDR = complementarity determining regions
the CDRs within antibody or T cell receptor proteins are complementary to the antigen’s shape (they’re in the variable region of Abs)
CDR regions determine the protein’s affinity and specificity for a unique antigen
CDR regions are the most variable part of the antibody and TCR and they contribute to the diversity of these molecules. Such adjustments during the immune response allow the antibody to mount very specific response
which genes does a B cell have?
each B cell expresses only one ϰ OR λ light chain gene
it also expresses only one heavy chain gene
ϰ = chromosome 2
λ = chromosome 22
H = chromosome 14
what is class switching?
class switching (CSR) = isotope switching
switching puts the same antigen specificity
onto different constant regions with differing functions
it’s a biological mechanism that changes a B cell’s production of immunoglobulin from one type to another, such as from the isotype IgM to the isotype IgG
on chromosome 14 where the heavy chain is, there are 5 different regions that determine which class the antibody will be (IgG, IgM, IgD, IgA, or IgE)
we have different classes of antibodies because they function differently in order to provide us with the best protection against invading pathogens so we need to be able to make all of these types of antibodies
AID enzyme helps to delete the portion of DNA in between the switch sequences so that different classes of antibody can be made
IgM is always the first to be made because it’s coding region is first in the linear organization of DNA so in order for transcription factors to reach the regions that code for other types of antibodies, we need to be able to skip over the IgM portion or any antibody region that comes before whichever antibody we’re trying to make
AID deletes the preceding gene regions so we’re able to produce different kinds of antibodies
AID deficiency causes hyper IgM syndrome because you can’t delete anything and our bodies are just gonna produce the first coding region they come across so you can’t do class switching without AID
what are the 5 Ig classes?
IgM
IgD
IgG
IgE
IgA
what is the function of AID?
it’s a B cell-specific enzyme that induces DNA damage by cytidine deamination and accounts for the B cell predisposition to cancer-associated chromosome translocations
AID is the master regulator of secondary antibody diversification – involved in somatic hypermutation, class switch recombination and gene conversion
how are the constant regions of the H chain diversified?
germ line arrangement of H chain constant region exons
there are secreted and membrane-bound forms
how are T cell receptors different than B cell receptors?
TCRs are:
– monovalent
– have no secreted version
– have no equivalent of class switching or hypermutation
how are T cell receptors the same as B cell receptors?
Like B cell receptors, TCRs:
– undergo V(D)J joining via RAG
– are subject to N-region diversification
– use variable regions on two chains to define specificity
– rearrange one chain first () and express with surrogate
what is the structure of the TCR antigen recognition site?
TCR antigen recognition site is composed of TCRα and TCRβ chains
which chromosome are the TCRα and TCRβ chain genes located?
TCRα = chromosome 14
TCRβ = chromosome 7
what is the TCRα chain structure?
it’s similar to the L chain of antibody
it has two fragments: V and J
70-80 V fragments
61 J fragments =
4880 rearrangement possibilities
what is the TCRβ chain structure?
it’s similar to the H chain of antibody
3 fragments = V, J, D
how is the TCRαβ receptor produced?
rearrangement of TCRα and TCRβ chains
the TCRα- and TCRb-chain genes are composed of segments that are joined by somatic recombination during development of the T cell
for TCRα, a V gene segment rearranges to a J gene segment to create a functional V-region exon. Next, the transcription and splicing of the VJ exon to C exon generates the mRNA that is translated to yield the TCRα chain protein
for TCRβ, there are three fragments V, D, and J which are rearranged to generate a functional VDJ exon that is transcribed and spliced to join to a C fragment
the rearranged TCRalpha and TCRbeta mRNAs are translated to produce a single TCRalpha/beta receptor for a T cell
