Lymphocyte Development and Ag receptor Gene Rearrangement Part II Flashcards
Before birth, B lymphocytes develop from committed precursor in what organ?
The fetal liver
After birth, where are B lymphocytes generated?
In the bone marrow
Arising from adult bone marrow, what do progenitors initially not have?
Do not express Ig
When do immature B cells leave the bone marrow to further develop?
When immature B cells express membrane-bound IgM
After leaving the bone marrow, immature B cells primarily go to mature further where?
In the spleen
How long does it take for the development of a mature B cell from a lymphoid progenitor?
Estimated to take 2 to 3 days
What is the earliest bone marrow cell committed to the B cell lineage?
Pro-B cell
Pro-B cells do not produce ___
Ig
What are first expressed on pro-B cell?
Rag-1 and Rag-2 proteins
In pro-B cells, the first recombination of Ig genes occurs at what locus ?
At the heavy chain locus
What is the function of the TdT enzyme?
Catalyzes the non-templated addition of junctional N nucleotides
When is the TdT enzyme expressed most abundantly?
During the pro-B stage when VDJ recombination occurs at the lg H locus
When do levels of TdT decrease?
Before L chain gene V-J recombination is complete
Junctional diversity attributed to the addition of N nucleotides is more prominent in the rearrangement of what genes?
More in H chain genes than in light chain genes since TdT is most abundantly expressed during VDJ recombination at the lg H locus
How does the H chain C region exons remain separated from the VDJ complex?
By DNA containing the distal J segments and the J-C intron
The rearranged Ig H chain gene is transcribed into what?
A primary transcript that includes the rearranged VDJ complex and the C μ exons
What does the C μ nuclear RNA undergo?
Undergoes splicing in which the introns are removed and exons joined together
For a rearrangement to be productive (in the correct reading frame) what must happen?
Bases must be added or removed at junctions in multiples of three
How many of all pro-B cells make productive rearrangements at the lg H locus?
about 50%
What cells survive and differentiate further?
Only cells that make productive rearrangements
When are pre-BCRs expressed?
During the pre-B cell stage of maturation
What is the pre-BCR composed of?
μ IgH chains and an invariant surrogate IgL chain
What is the surrogate IgL chain composed of?
The V pre-B protein ( a homolog of a variable domain of IgL)
And λ5 protein (a homolog of a constant domain of IgL)
How is the λ5 protein attached to the μ IgH chain?
Covalently attached by a disulfide bond
What is the pre-BCR associated with?
The Igα and Igβ signaling molecules that are part of the BCR complex in mature B cells
What forms the pre-BCR?
Complexes of μ IgH, surrogate IgL chains and Igα and Igβ
What do invariant λ5 and V pre-B proteins composed? What are they structurally homologous to?
They compose the surrogate IgL chain
Invariant λ5 and V pre-B proteins are structurally homologous to k and λ light chains
Igα and Igβ also form part of what in mature B cells?
B cell receptor
What are responsible for the largest proliferation expansion of B lineage cells in the bone marrow?
Signals from the pre-BCR
What Ag is recognized by the pre-BCR?
It is not known
It is speculated that pre-BCR is activated by what?
By the process of assembly in a ligand-independent manner
How is the importance of pre-BCRs illustrated?
Illustrated by markedly reduced numbers of mature B cells in KO mice deficient in μ Igh or surrogate IgL chains
Rearrangement of IgH locus is initiated at what stage?
pro-B-cell stage
If rearrangement of IgH locus is successful, what will it give rise to?
The Ig μ chain that is expressed on the cell surface in the form of the pre-B cell receptor at the large pre-B cell stage
What does signaling from the pre-BCR induce?
Clonal proliferation and recombination of (IgL) genes
What results in the expression of a complete BCR on immature B cells?
In-frame IgL gene rearrangements in small pre-B cells
What is the first checkpoint in B cell maturation?
The expression of the pre-BCR
A number of signaling molecules linked to pre-BCR are required for what?
For cells to successfully negotiate the pre-BCR-mediated checkpoint at the pro-B to pre-B cell transition
What kinase is activated downstream of the pre-BCR?
Bruton’s tyrosine kinase (Btk)
What is Btk required for?
For the delivery of signals from this receptor that mediate survival, proliferation, and maturation at and beyond the pre-B cell stage
What does mutations in the Btk gene result in?
A disease called x-linked agammaglobulinemia (XLA), which is characterized by a failure of B cell maturation
If a μ IgH is produced from one chromosome and forms a pre-BCR, what does this receptor signal?
Signals to irreversibly inhibit rearrangement of the IgH chain locus on the other chromosome
What does allelic exclusion involve?
involves changes in chromatin structure in the IgH chain locus that limit accessibility to the V(D)J recombinase
An individual B cell can express an IgH chain protein encoded by what?
ONLY ONE of the two inherited alleles
What happens to the other IgH chain allele not expressed by B cells?
It is retained in the germline configuration
What occurs if both alleles undergo nonproductive IgH gene rearrangements?
A pre-BCR dependent survival signal is not generated and the cell dies by apoptosis
Following the pre-B cell stage, each developing B cell initially rearranges what?
a k IgL gene
If the rearranged k IgL gene is productive, what will it produce?
Produce a k IgL protein, which associates with the previously synthesized μ IgH to produce a complete IgM protein
What does the production of k IgL protein prevent?
Prevents λ rearrangement
How many of the two types (k and λ) of IgL can be expressed?
Only ONE, The phenomenon is called light chain isotype exclusion
If the rearrangement of k locus is nonproductive, what happens?
The cell can rearrange the λ locus to produce a complete IgM molecule
What happens if both k and λ chains are nonfunctional?
The developing B cell don’t receive survival signals that are normally generated by the BCR and dies
The assembled IgM (BCR) is associated with what in the BCR complex?
Igα and Igβ (signaling subunits)
What does the assembled BCR provided?
Ag-independent tonic signals that keep the B cell alive and also mediate the shut off of RAG gene expression that prevents further Ig gene rearrangement
In response to Ags, what do immature B cells do?
They DO NOT proliferate and differentiate
Instead, if BCRs recognize Ags in the bone marrow with high avidity, the B cells may undergo receptor editing or apoptosis
Receptor editing or apoptosis are important for what?
Negative selection of strongly self-reactive B cells
What happens to B cells that are not strongly self-reactive?
They leave the bone marrow and complete their maturation in the spleen before migrating to other peripheral lymphoid organs
Immature B cells that recognize self Ags with high avidity may be induced to do what?
Change their specificities by a process called receptor editing
Ag recognition by immature B cells induce reactivation of RAG genes and what?
the rearrangement and production of a new Ig light chain, allowing the cell to express a different (edited) B cell receptor that is not self-reactive
What happens to the original VJk exon encoding the IgVL chain gene that was self reactive?
It is typically deleted and replaced by a new rearrangement involving an upstream Vk and a downstream Jk gene segment
If the editing process fails to generate an in-frame productive k IgL, what happens?
The activated immature B cell may then go on to rearrange the λ light chain locus
Almost all B cells bearing λ light chains are cells that were once what?
Self-reactive and have undergone receptor editing
If receptor editing fails, what happens?
The immature B cells that express high-affinity receptors for self Ags die by apoptosis
What is the process called that follows receptor editing failure?
Negative selection
The Ags mediating negative selection are usually what?
Abundant or polyvalent self Ags such as nucleic acids, membrane bound lipids and membrane proteins
What are responsible for maintaining B cell tolerance to self Ags that are present in the bone marrow?
Receptor editing and negative selection
What do most B cells that differentiate into B-1 lineage develop from?
Fetal liver-derived stem cells
B lymphocytes that give rise to the B-2 lineage arise from what?
Bone marrow precursors after birth
The affinity of the BCRs for self Ags may contribute to differentiation into:
Follicular B cells and Marginal zone B cells
What are recirculating lymphocytes?
Follicular B cells
What cells are abundant in the spleen and also are found in LNs?
Marginal zone B cells
B1 and B2 B cell lineages seem to be ________ regulated and undergo tightly controlled developmental processes
Independently
What do B1 B cells develop from?
HSCs in the bone marrow or fetal liver
What B cells are self-renewing and produce natural Abs involved in self-defense?
B1 B cells
Where do B2 B cells develop from?
From HSCs in the bone marrow
When do immature B2 B cells relocate to the spleen?
Following rearrangement of their BCR chain genes and removal of autoreactive cells via central tolerance
Immature B2 B cells that escape the processes of peripheral tolerance differentiate into:
MZ B cells or mature follicular B2 cells
What B cells develop into long-lived plasma cells or memory B cells upon T-cell-dependent activation?
Only mature follicular B2 cells
Where are marginal zone (MZ) B cells localized?
To the splenic marginal zone
What do MZ B cells respond to?
Bloodborne Ags
Responses are independent of T cell help
What do follicular B cell respond to?
Protein Ags in a T cell-dependent manner
Which B cells progressively undergo immunoglobulin isotype switching and affinity maturation?
Follicular B cells
Which B cells comprise a much smaller population?
B-1 B cells
What B cells predominates in the pleural and peritoneal cavities and contributes most of the serum IgM during the early phases of infection?
B1 B cells
What B cells are predominantly self-renewing?
MZ and B-1 B cells
Which B cell needs constant replenishment from bone marrow?
Follicular B cells
What B cells develop from fetal liver-derived HSCs?
B-1 cells
What B cells expresses limited BCR diversity and why?
B-1 cells because TdT is not expressed in the fetal liver
A large number of B-1 cells are found as what?
Self-renewing populations in the peritoneum and mucosal sites
B-1 cells develop when?
Earlier during ontogeny than FBCs and MZ B cells do
What do B-1 cells spontaneously secrete?
Secrete IgM Abs that often react with microbial polysaccharides and lipids as well as oxidized lipids
IgM secreted by B-1 cells are sometimes called what?
Natural antibodies because they are present in individuals without overt immunization
What do most mature B cells belong to? What do they produce?
The FBC subset and produce both IgM and IgD with the same Ag specificity
What do FBC coexpress?
μ and δ IgH using the same VDJ exons and same k or λ IgL to produce two membrane receptors
How is simultaneous expression of IgM and IgD in a single B cell achieved?
By alternative RNA splicing
A long primary RNA transcript (pre mRNA) containing what?
The rearranged VDJ unit as well as both Cμ and Cδ genes
What allows a B cell to simultaneously produce mature mRNAs and proteins of two different heavy chain isotopes?
Selective polyadenylation and alternative splicing
Where are MZ B cells located?
Primarily in the vicinity of the marginal sinus of the spleen
Similar to B-1 cells, MZ B cells have BCRs of what?
limited diversity which respond to polysaccharide Ags and to generate natural Abs
What two places can MZ B cells be found?
Spleen and lymph nodes
What do MZ B cells respond very rapidly to?
Blood-borne microbes
What do MZ B cells differentiate into after Ag activation?
Short-lived IgM-secreting plasma cells
Although MZ B cells generally mediate T cell-independent humoral immune responses to circulating pathogens, they also appear capable of what?
Mediating some T cell-dependent immune responses
The development of mature T lymphocytes from committed progenitors involves:
The sequential rearrangement and expression of TCR genes
Cell proliferation
Antigen-induced selection
Commitment to phenotypically and functionally distinct subsets
Is T cell maturation similar to B cell maturation.
Yes, in many ways
What does the thymic environment provide?
Provides stimulii that are required for the proliferation and maturation of thymocytes
What is the unique feature of T cell maturation?
It is the selection of mature T cells with specificity for self MHC-associated peptide Ags
What is the major site of maturation of T cells?
The thymus
The thymus as a major site for T cell maturation was first suspected why?
Because of immunologic deficiencies associated with the lack of a thymus (DiGeorge syndrome)
If the thymus is removed from a neonatal mouse, what happens?
This animal fails to develop mature T cells
The thymus involutes with age and is virtually undetectable in post-pubertal humans resulting in what?
In a somewhat reduced output of mature T cells, however, maturation of T cells continues throughout adult life
Why is there a reduced output of mature T cells post-puberty?
Because memory T cells have a long lifespan (perhaps longer than 20 years) and accumulate with age
The need to generate new T cells decreases as individuals age
What do T lymphocytes originate from?
From precursors that arise in the fetal liver and adult bone marrow and seed the thymus
Describe the precursors for T lymphocytes
Multipotent progenitors that enter the thymus from the blood stream
What are developing T cells in the thymus called?
Thymocytes
Where does maturation of thymocytes mostly occur?
Occurs in the cortex region of the thymus
What do αβ T cells mature into?
Either CD4 class II MHC-restricted or CD8 Class I MHC-restricted T cells as they leave the cortex, enter the medulla and exit the thymus through the circulation
Where do many stimuli required for the proliferation and maturation of thymocytes come from?
Other thymic cells
What do thymic cortical epithelial cells form?
A meshwork of long cytoplasmic processes which allows physical interactions with thymocytes necessary for their maturation
What do epithelial cells present in the medulla serve a unique role as?
As APCs for the negative selection of developing T cells
Where are bone marrow-derived DCs present?
At the cortico-medullary junction and within the medulla
Where are macrophages present primarily?
Within the medulla
What do epithelial cells, DCs, and Mo in the thymus express? How are they important?
Class I and class II MHC molecules They are important for the selection of the mature T cell repertoire
What is the movement of cells into and through the thymus driven by?
Chemokines
What chemokine recognized on precursors control the entry into the thymus?
CCL25,
It is recognized by the CCR9 on precursors
What are recognized by thymocytes that mediate the guided movement of developing T cells from the cortex to the medulla?
CCL21 and CCL19 are recognized by thymocytes via CCR7
What do generated T lymphocytes express to exit the thymic medulla? How does this work?
Express sphinosine-1 phosphate receptor and exit the thymic medulla
They follow a gradient of sphingosine-1 phosphate into the blood stream
What induces the proliferation of thymocytes?
IL-7 produced by epithelial and other stromal cells
The rates of cell proliferation and apoptotic death are extremely (high/low) in cortical thymocytes
HIGH
95% of thymocytes die by apoptosis due to:
- failure to rearrange the TCR β chain gene
- failure to be positively selected by self MHC molecules
- self-Ag-induced negative selection
Precursors of T cells travel from the _______ through the _____ to the ______
Bone marrow
Blood
Thymus
In the thymic cortex, progenitors of αβ T cells express what?
TCRs and CD4 and CD8 coreceptors
What does negative selection eliminate?
Twice self-reactive T cells in the cortex at the double-positive (DP) stage and also single-positive (SP) thymocytes in the medulla
What do TCRs bind to that promotes survival of thymocytes?
TCRs bind to self MHC molecules with low affinity to promote survival
Where does functional and phenotypic differentiation into CD4+CD8- or CD8+CD4- T cell occur?
Occurs in the medulla, and mature T cells are released into the circulation
What do some double-positive cells differentiate into?
regulatory T cells
What do cortical thymocytes contain?
What are these cells called?
TCR genes in their germline configuration and do NOT express TCR, CD3, ζ chains, CD4, or CD8
These cells are called double-negative thymocytes and are considered to be pro-T cells
What are first expressed in the double-negative stage of T cell development?
Rag-1 and Rag-2
What are Rag-1 and Rag-2 required for?
The rearrangement of TCR genes
What rearrangement of the β chain locus occurs first?
The DJ
When do V(DJ) rearrangement occur?
At the pro-T to pre-T stage transition during αβ T cell development
90% of the double-negative thymocytes that survive thymic selection will ultimately give rise to what?
To αβ TCR CD4+ and CD8+ T cells
What will the other 10% of double-negative thymocytes that survive thymic selection give rise to?
γδ T celsl
When are pre-T cell receptors expressed?
During the pre-T cell stages of maturation
What is the pre-T cell receptor composed of?
The TCR β chain and the invariant pre-T α (pTα) chain
What does the pre-T cell receptor associate with that are part of the TCR complex in mature T cells?
With the CD3 and ζ proteins
What does the pre-TCR mediate?
The development and selection of pre-T cells
What do signals from the pre-TCR mediate?
The survival of pre-T cells and contribute to the largest proliferation during T cell development
Also β chain allelic exclusion
What do pre-TCR signals initiate and drive?
TCR α chain locus recombination (a second wave of RAG expression)
Drives the transition for double-negative to the double-positive stage of thymocyte development
How is pre-TCR signaling initiated?
in a ligand-independent manner by assembly of the pre-TCR complex
How has the essential function of the pre-TCR complex in T cell maturation been demonstrated?
By numerous studies showing that lack of the TCR β chain, pre-Tα, CD3 or ζ chains results in a block in the maturation of T cells at the double-negative stage
In contrast to the TCR β chain locus, there is ____ or __ allelic exclusion in the α chain locus
little or no
Because there is no allelic exclusion in the α chain locus, productive TCR α rearrangements may occur where?
What can this result in?
On both chromosomes, and if this happens, the T cell will express two α chains
How many mature peripheral T cells do express two different TCRs, with different α chains but the same β chain?
up to 30%
It is possible that only one of the two different TCRs participates in what?
self MHC-driven positive selection
What does unsuccessful rearrangements of the TCR α gene on both chromosomes leads to what?
A failure of positive selection and apoptosis
Double-positive thymocytes are produced without what?
What do they express?
Ag stimulation
They express αβ TCRs with randomly generated specificities
What are the only APCs that mediate the positive selection by displaying a variety of self peptides bound to class I and class II MHC molecues
Cortical epithelial cells
Weak recognition of self peptide-self MHC complexes promotes what for T cells?
The survival of the T cells
Explain death by neglect
Thymocytes whose receptors do not recognize self MHC molecules die by apoptosis
What is positive selection involving T cells?
Is the process in which thymocytes whose TCRs bind with low avidity to self peptide-self MHC complexes are stimulated to survive
What does positive selection ensure?
That T cells are self MHC-restricted
During the transition from double-positive to single-positive cells, what do thymocytes with class I MHC-restricted TCRs become?
CD8+CD4-
During the transition from double-positive to single-positive cells, what do thymocytes with class II MHC-restricted TCRs become?
CD4+CD8-
Double-positive T cells express TCRs that may recognize what?
Either self class I or self class II MHC
The commitment of immature T cells towards either lineage may depend on what?
The random probability of a double-positive cell differentiating into a CD4 or a CD8 T cell
A cell that recognizes self class I MHC may randomly differentiate into a CD8 T cell and do what?
A CD8 T cell (with the appropriate coreceptor) can survive
The same cell that recognizes self class I MHC may randomly differentiate into a CD4 T cell and do what?
A CD4 T cell has the wrong coreceptor and may fail to receive survival signals
What is a more widely accepted view of how T cells actively induce expression of the correct coreceptor and shut off expression of the other coreceptor?
That class I MHC-and class II MHC-restricted TCRs deliver different signals that induce the correct coreceptor
It is known that double-positive cells go through a stage at which they express what?
High CD4 and low CD8
For a class I MHC-restricted cell, when it sees class I MHC, what will happen?
It will receive a weak signal because levels of the CD8 coreceptor are low
What do weak signals to a class I MHC-restrictied cell that sees a class I MHC activate?
Activates Runx3 that maintains the CD8 T cell phenotype by regulating the expression of CD8
For a class II MHC-restricted, when it sees class II MHC, what signals will it receive? What will these signals activate?
It will receive a stronger signal because of high CD4 levels and these strong signals activate GATA3, which commits cells toward a CD4 fate
The high-avidity recognition of self Ags triggers what?
apoptosis and death resulting in negative selection of the T cell repertoire
Both DCs and Mo in the medulla and medullary thymic epithelial cells are what?
What does this process control?
APCs that mediate negative selection
Controls the central tolerance to self Ags
However, thymocytes high-avidity recognition of self Ags may also differentiate into what?
Regulatory T cells that function to prevent autoimmune reactions
What does the survival of naive lymphocytes require?
Survival signals before they encounter the foreign Ag
The same self peptides involved in the selection of double-positive thymocytes in the thymus may be involved in what?
The survival in the periphery
What may positive selection allow many different T cell clones to do?
survive and differentiate
Many of the T cells that have positive selection recognize self peptides with (high/low) affinity
low
Matured T cells strongly recognize foreign Ags and become what?
Activated and generate an immune response
Recombination of TCR γ and δ loci proceeds in a fashion similar to that of TCR β and α, although the order of rearrangement appears to be what?
less rigid
In a developing double-negative T cell, rearrangement of TCR β,γ, or δ loci is initiated _______.
Simultaneously
If a cell first succeeds in productively rearranging its TCR γ as well as its TCR δ before it makes a productive TCR β rearrangement, it is selected into what?
the γδ T cell lineage
How often do developing double-negative T cells become γδ T cells?
10%
How often do developing double-negative T cells become αβ T cells?
90%
Why does the limited diversity of expressed γδ TCRs occur?
Because only a few of the available V, D, and J segments are used in mature γδ T cells, for unknown reasons
