Diversity vs Specificity: Immunoglobulins (Bowden) Flashcards
Naive B cells
Mature B cells that have not encountered antigen
Epitope
the parts of antigens that are recognized by antibodies
Lymphocyte Development
1) commitment of progenitor cells
2) Proliferation of progenitors
3) Sequential and ordered rearrangment of antigen receptor genes
4) Selection events (of repertoire)
5) Differentiation of effectors
Immunoglobulins
AKA Antibodies
All Ab’s are made by a clone that has a very specific antigen specificity
Structure of Antibody
Two heavy chains
Two light chains
Immunoglobulin superfamily
IgM
T-cell receptor
HLA (MHC class I and class II)
Ig-alpha/Ig-beta heterodimer (b-cell receptor co)
Immune repertoire
what the body will respond to
Clonal selection
Gene rearrangement events occur in absence of antigen
1) Lymphocyte clones mature in generative lymphoid organs (bone marrow or thymus) in the absence of antigens
2) clones of mature lymphocytes (T or B cells) specific for diverse antigens enter lymphoid tissues
3) Antigen-specific clones are activated (selected) by antigens
4) Antigen-specific immune responses occur
Clone
lymphocyte of 1 specific specificity and it’s progeny
you hope when you are exposed to an antigen that you have made a clone or two that the antigen can select…. for protection
1 gene- 1 polypeptide theory
1 gene for each antibody clone
a large amount of genome would be required to generate this level of diversity
would be the size of a mouse! too big
Primary Immunoglobulin Rearrangement
1) Multiple germ line genes
- combinatorial diversification (V-J or VDJ recombinations)
2) Junctional diversity
- Addition of nucleotides during process of D-J or V to DJ joining
Germ lines
have 2 copies (ma and pa)
Secondary Immunoglobulin rearrangement
Somatic Hypermutation
- point mutations occurring in fully assembled V-J or VDJ regions during an immune response
- provides a significant source of Ab diversity (MAGIC)
IL-3
influences immature progenitors (lymphoid progenitor or myeloid)
made by T cells
multi lineage Cytokine
IL-7
necessary for commitment to lymphoid lineages
Stem Cell to Pro-B
IL-3 and IL-7 directed toward lymphoid lineage so start to proliferate clones at this point
in bone marrow
unrecombined germline DNA
no Ig expression
no response to antigen
Heavy chain
chromosome 14
has diversity region (not on light chains)
Light chain kappa
only 1 constant (C) region b/c there is only one type of kappa light chain
several joining (J) exons b/w C and V genes
have 35 V regions
chromosome 2
light chain lambda
4 C regions
30 V genes
-each V region gene is composed of two eons, one L that codes for leader and the other V that codes for the most variable region
J regions in between each C region
chromosomes 22
C on Ab gene
Constant regions
on downstream end (3’ end)
only 1 on kappa chain
7 on lambda
J
Joining regions
5 on kappa chain
leader sequence for constant region on lambda chain (so in front of constant regions)
D
diversity regions
23 on heavy chain
V
Variable regions
100 on heavy chain
35 on kappa chain
30 on lambda
Allelic exclusion
Have both ma and pa chromosomes
express heavy chain and light chain from only one chromosome
ensures that B cells never contain more than one light and heavy chain
essential for antigen specificity b/c the expression of both alleles would render B cell multispecific
Pro B to Pre-B
proliferating
start upregulating enzyme expression (RAG and TdT)
still germiline, unrecombined
no antigen
still in bone marrow
Making a heavy chain
D joined to J
Add V to DJ region
Gene becomes transcriptionally active (b/c Promoter is close to Enhancer)
Introns removed
Resulting mRNA’s have L, V, D, J, and Cmu or Cdelta exons
mRNAs are translated in the cyotplasm
Combinatorial diversity
diversity of antigen receptors produced by use of different combinations of V,D, and J gene segments in different clones of lymphocytes
primary Ig rearrangement
RSS
recombination signal sequences
Rag-1 and Rag-2
Catalyze recombination events
aka VDJ recombinase
Tdt
catalyzes random polymerization of nucleotides that are not part of germline genes into DNA without the need for a template
adds P and N nucleotides
leads to further diversity in the third hypervariable region (idiotype)
P nucleotide
added by tdt to asymmetrically cleaved hairpins in a templated manner
Junctional diversity
largest contribution to diversity of antigen receptors
changes in nucleotides sequences introduced at the junctions of the recombining V,D, and J gene segments (done using Tdt)
Primary Ig rearrangement
problem with this is that you are adding alot of stop codons and other useless shit (not perfect)… adding frameshifts (since in non-templated manner) so it is not read by machinery and will not make a functional protein
N nucleotides
added by tdt in a non-templated manner
Rearrangment of heavy chain…
must be successful for cell survival
if not then undergoes apoptosis
if successful go from preB to immature B cell
Pre B cell receptor
Complex of IgM chain and surrogate light chains (just imposters)
IgBeta and IgAlpha are the coB cell receptors that are in conjunction now with our Pre-B receptor
now have recombined H chain gene (VDJ), IgM mRNA heavy chain
cytoplasmic mu and pre-B receptor associated mu
still in bone marrow
starting to pick up markers on surface
no antigen
checking for if it works 3-dimensionally
Pre B cell receptor
Complex of IgM (Ig mu heavy chain protein), surrogate light chains (just imposters), and Ig-alpha and Ig-beta
Ig-alpha and Ig-beta are signaling molecules
pre-BCR delivers signals that promote survival and proliferation of B lineage cells that have made functional (proper) rearrangement of H-chain (this is the first check point)
Pre-B to Immature B
Once checked for 3-D working properly then start to proliferate again
Rag expression once again
recombine a light chain!!
Pre-B to Immature B
Once checked for 3-D working properly then start to proliferate again
Rag expression once again
recombine a light chain!!
Making a light chain
Always make kappa first
V joined with J
premRNA is spliced and V/J region joined with C region
now have RNA that is translated to make light chain
Making a light chain
Always make kappa first
V joined with J
premRNA is spliced and V/J region joined with C region
now have RNA that is translated to make light chain
After we make the light chain, what are the bone marrow stromal cells checking?
Checking for recognition of self
checked by being presented bits and pieces of HLA
negative selection vs. positive selection
After we make the light chain, what are the bone marrow stromal cells checking?
Checking for recognition of self
checked by being presented bits and pieces of HLA
negative selection vs. positive selection
Receptor editing
This is something unique to light chains
Nonproductive light chain rearrangements can be rescued by further gene arrangement
If 1st is nonproductive then the V can go back and select another J…. can do this up to 5 times b/c there are 5 J regions
If it grabbed the 5th joining region, and it is not recombined successfully, then move on to lambda light chain
Receptor editing
This is something unique to light chains
Nonproductive light chain rearrangements can be rescued by further gene arrangement
If 1st is nonproductive then the V can go back and select another J…. can do this up to 5 times b/c there are 5 J regions
If it grabbed the 5th joining region, and it is not recombined successfully, then move on to lambda light chain
Immature B cell
Have IgM (light and heavy chain) on surface
going through negative selection and clonal deletion to cells that respond avidly
receptor editing is part of this
Immature B cell
Have IgM (light and heavy chain) on surface
going through negative selection and clonal deletion to cells that respond avidly
receptor editing is part of this
Alternative splicing of heavy chain mRNA
in the first heavy chain rearrangement we removed introns, so the preMRNA was processed in two ways (one to bring VDJ next to Cmu or close to Cdelta)
have both IgM and IgD on membrane surface of B cell b/c of alternative splicing
IgM–> mu heavy chain
IgD–> delta heavy chain
Alternative splicing of heavy chain mRNA
in the first heavy chain rearrangement we removed introns, so the preMRNA was processed in two ways (one to bring VDJ next to Cmu or close to Cdelta)
have both IgM and IgD on membrane surface of B cell b/c of alternative splicing
IgM–> mu heavy chain
IgD–> delta heavy chain
clonal selection
The process of gene rearrangement of the heavy and light chains and the combinatorial association of these chains occurs during B cell development in the bone marrow and is independent of antigen.
BCR
B cell receptor
this is a surface bound IgM plus Ig-alpha and Ig-beta
Negative selection by BM stromal cells
if the immature b cell responds to avidly with self-Antigens they are targeted for apoptosis
Negative selection by BM stromal cells
if the immature b cell responds to avidly with self-Antigens they are targeted for apoptosis
there are a lot of self-antigens located in the bone marrow
also… if it does bind too tightly it can activate VDJ recombinase enzyme, undergo light chain recombination (V grabs a different J) and then once again change the specificity of the antigen receptor
Positive selection by BM stromal cells
if the immature b cells bind with low avidity, clones are selected to be released into the periphery
Positive selection by BM stromal cells
if the immature b cells bind with low avidity, clones are selected to be released into the periphery
Mature B cell
has Cmu and Cdelta mRNA
membrane IgD and IgM
now in the periphery
activated by antigens that select their clone
Mature B cell
has Cmu and Cdelta mRNA
membrane IgD and IgM (due to alternative splicing)
now in the periphery
activated by antigens that select their clone
how are TCReceptors made?
same exact way as BCR
how are TCReceptors made?
same exact way as BCR
same V, D, J, N
bigger potential repertoire
Pre-B cell
now have recombined H chain gene (VDJ),
defined by presence of the Ig mu heavy-chain protein in cytoplasm and some mu on the cell surface in association with surrogate light chains
mu chain and surrogate light chains associate with Igalpha and Igbeta signaling molecules to make pre-BCR
still in bone marrow
no antigen
pre-BCR checking for if it works 3-dimensionally
how many progenitor cells actually make it to the end
12.5 percent
clonal selection
The process of gene rearrangement of the heavy and light chains and the combinatorial association of these chains occurs during B cell development in the bone marrow and is independent of antigen.
secondary rearrangement
Somatic Hypermutation
Tahir Muhtar 6 month old
IgG: low
IgM: low
IgA: mildly low
WBC: Low CD19 low CD3 low CD8 low CD4 low CD56 (NK) high
Kid with SCID
Had homozygous mutations in RAG1 and RAG2 gene
What is the result?
Cannot rearrange his germiline for heavy or light chains for both B’s and T’s
so cannot make immune response
RAG mutations account for approximately 3-4 percent of SCID
SCID
Bubble kids
50 percent of SCID cases are result of mutations in cytokine IL-7
the result of this is that neither lymphocyte pool matures into effector cells
X-linked Agammaglobulinemia (Bruton’s)
Bruton Tyrosine Kinase (Btk) delivers signals from pre-BCR for survival of cell
so basically there is no survival of B or T cells, decreased immune response
The primary development of B cells is …
antigen independent (DOES NOT SEE ANTIGEN)
