otogeny of B+T cells

where do T cells mature? B cells?
thymus, bone marrow
what are the important common events in development of B and T cells?
1) commitment of progenitor cells to the B or T lineage 2) temporally ordered process of rearrangement of antigen receptor genes and expression of antigen receptor status 3) selection events that preserve cells that have produced correct antigen receptor proteins and elimination events to remove potentially dangerous cells, (failure to do this = autoimmunity). T cells undergo additional selection processes to ensure they recognize MHC. 4) differentiation of B+T cells into functionally/phenotypically distinct populations, (CD4+,CD8+, B1,B2)
where do B cells come from?
they begin as stem cells that go through pro, pre, and immature stages finally emerge mature and naive from the bone marrow
where do T cells come from?
they begin as stem cells that move to the thymus, go through pro, pre, and immature stages finally emerge from there mature and naive
what happens to mature naive cells when they are exposed to anitgen?
they become differentiated to effector lymphocytes, (B cells differeniate to plasma cells which produce antibody. T cells differentiate to CD4+, T helper cells that release cytokines/mediate cell-cell interactions or CD8+ cell that are cytolytic)
what does antigen dependence refer to?
what antigens are driving the receptor rearrangement events that occur at different differentiation states
where are immature lymphocytes found?
immature lymphocytes are found in the primary lymphoid tissue, (bone marrow, thymus)
where are mature lymphocytes found?
in secondary lymphoid organs, (peripheral organ or tissue)
why is there no requirement for antigen up until the immature state of differentiation?
because the rearrangement events that occur at the level of antigen receptor genes are random
when do the selection processes for antigen start? what is the driving force here?
at the immature cell phase. the driving antigens for this process are self-antigens which can render the cell functionally inactive, (a state called anergy), or inducing another round of rearrangement
where do B and T cells derive from originally?
hematopoietic stem cells that mature into a common lymphoid progenitor, (CLP)
where is CLP located?
CLP, (common lymphoid progenitor), is located in fetal liver and adult bone marrow
even though the link between B+T cells and CLP is unknown, what is early B and T cell development characterized by?
a proliferation of progenitors, stimulated by IL-7, particularly for T cells
how does IL-7 stimulate B and esp T cell progenitors?
IL-7 results in marked increase in cell numbers, ensuring a large enough pool of progenitors to generate highly diverse repertoire of antigen-specific lymphocytes
how is IL-7 made?
IL-7 is made by stromal cells in the bone marrow and thymus
what do mutations in the IL-7 receptor cause?
SCID, (x-linked) – severe combined immunodeficiency, boy in the bubble
what precedes antigen receptor rearrangement?
proliferation
what occurs prior to pre-B cell stage differentiation in terms of Igs? what enzyme is active in correlation with this?
heavy chain rearrangement, which RAG, (recombinase activating gene), is highly active in.
what has happened by the time the cell gets to pre-B cell?
by the time the cell gets to pre-B cell, the heavy chain has recombined, and the first heavy chain protein, u is produced and depending on whether it is a large or small pre-B cell stage, the protein will be at the cell surface or can accumulate in cytoplasm
what is a successful recombination at the heavy chain locus and successful expression of the pre-B cell receptor a strong signal for?
it is a strong signal for the cell to start rearranging the light chains
what are the first light chains to be rearranged? what if this fails?
the kappa light chains. if both kappa chains fail, the cell will revert to the lambda chains and try to rearrange them
at the immature state, what does the B cell express? what is this stage marked by?
a heavy and a light chain successfully rearranged. this stage is marked by expression of IgM
what marks a B or T cell as mature?
expresion of IgM and IgD
where do negative selection events occur?
the immature stage
how long does Ig DNA remain unrecombined?
up until the pre-B stage, which is where you see recombined H gene, (VDJ) and u,(mu) mRNA
what does Ig DNA, RNA look like at the immature B stage?
recombined H chain gene, (VDJ), kappa or lambda genes, (VJ), and u or kappa or lamba mRNA
what does Ig DNA, RNA look like at the mature phase?
alternative splicing of VDJ-C RNA to form Cu and C gamma mRNA
how are Ig genes organized and why? can they be expressed in other cells?
Ig genes, (and those for the alpha/beta chains of the T cell receptor), are organized differently from other genes to achieve the very large number of antibody specificities.
can the Ig and the alpha/beta TCR genes be expressed in other cells?
in cells other than B cells, Ig genes are fragmented in a way that cannot be expressed, they are in germline configuration, which means there have been no deletions, and in order to express these genes, a rearrangement must occur.
what loci make up the heavy chain?
multiple V,D,J, and C exons
what loci make up the kappa light chain?
multiple V, J, and one C exon
what loci make up the lambda light chain?
multiple V, and C+J are interspersed between each other
how do sections of the heavy chain protein compare to the genes coding for it?
the V region domain has elements encoded by 1 V region gene segment, 1 D and 1 J region gene segment. the C region is encoded by 1 C exon which is downstream of V regions
how do sections of the light chain protein compare to the genes coding for it?
V region is encoded by a single V region gene segment, and a single J gene segment. the C region is encoded by for by 1 C exon
how does the T cell receptor compare to the B cell light chains in terms of gene coding for proteins?
the beta-chain encoded by V, D and J gene segments (similar to heavy chain), and a chain has just V and J (similar to light chain). both b and a chain are structurally similar to light chain
this is because they are members of the immunoglobulin super family, and therefore they have globular structures
what is the pro B cell/what part of the B cell lineage does it make up? does it produce Ig? does any re-arrangement occur w/it? does it express any B cell lineage-restricted markers?
the pro B cell is the earliest bone marrow cell commiteted to the B cell lineage, it does not produce Ig, but this is where Ig heavy chain rearrangement occurs. it does express other B cell lineage-restricted markers, (CD19 and CD10)
what are the rearrangements in the early pro-B cell stage? what are the rearrangements at the later pro-B cell stage?
at the earliest pro B cell stage, there is D-J rearrangement, (brings D and J close together). at later pro-B cell stage there is V-DJ rearrangement,(brings V close to DJ segments). ALPHABETICAL ORDER
what is driving the entire Ig gene rearrangement process? what is the basic process?
Recombinase Activating Gene (RAG). the general process is DNA loops out, excised, degraded, 2 ends ligased
what do you get w/a RAG deficiency?
no immune system
what happens to the Ig gene after rearrangement?
the gene is shorter because DNA is missing
what does rearrangement of the heavy chain mark?
rearrangement of heavy chain gene marks the cell as B lymphocyte
what is the specific mechanism of recombination of the B cell heavy chains? what is an RSS?
each V,D,J gene segment is flanked by non-coding “recombination signal sequence” RSSs and the protein products of RAG-1 AND RAG-2 genes mediate recombination at these junctions between RSS/coding sequences. through the action of recombinases, exonucleases, and DNA ligases, (prob part of RAG 1/2 complex), the unwanted DNA is looped out, excised, degraded, and the wanted DNA is re-ligated.
what follows the successful production of one heavy chain locus?
this causes production of the u heavy chain protein, which will eventually be IgM when complexes with light chain
what happens if the heavy chain locus rearrangement fails?
the cell has another copy of the locus from the host’s other parent which it can try using, (usually it would be turned off -> allelic exclusion), but if heavy chain recombination fails in both alleles, the cell dies
what is the first stage of pre-B cell development? how does this stage relate to the overall development of B cells?
the large, less mature B cells complex the newly synthesized u heavy chain with a surrogate light chain, (composed of VpreB, ~variable region, and lamba5, ~constant domain), some of these pre-B cell receptors go to the surface, most remain in the ER. this is an important checkpoint in B cell differentiation that confirms the productive rearrangement of the heavy chain gene.
what does presences of a pre-B cell receptor instigate in the cell?
presence of pre B cell receptor halts rearrangement of other heavy chain locus and synthesis of surrogate light chains, the large pre B cell proliferates and yields small pre B cells
what are small pre-B cells? where is the u heavy chain? when can they makde IgM?
more mature than large pre-B cells, these have no pre-B receptors, the u heavy chain is restricted to the cytoplasm and rearrangement of the light chain proceeds, once this accomplished successfully, IgM can be made
how does light chain rearrangement occur?
kappa first then lambda. this happens the same way heavy chain rearrangement happened, but in one step b/c all that has to happen is joining of V+J via recombinases, exonucleases and ligases.
what happens in light chain rearrangement if the kappa gene fails to rearrange successfully?
usually the first or second kappa gene rearrangement is successful, but if they fail then the first and second lamba genes are tried. (this is why only 15% of B cells have lamba b/c one of the kappas is usually successful). however, if the lamba fails, apoptosis. if the lamba succeeds, IgM is expressed with the lambda light chain.
what marks the end of the pre-B cell stage and the beginning of the immature stage of B-cell development?
expression of either a successfully rearranged u:k or u:l IgM
where are B cells at the beginning of the immature phase and what are they expressing?
immature B cells express only IgM and they are still in bone marrow.
what is the biological assumption if an immature B cell interacts with antigen? what happens if this is the case?
that it is self antigen and one of three things will happen. 1)apoptosis, (seems to occur more with surface antigens) 2)anergy, cell inactivated, not dead, can no longer respond to antigenic stimulation, this seems to happen with more soluble antigens. 3) undergoes receptor editing, recombinase enzymes are reactivated and the light chains are preferentially targeted.
once immature B cells are checked for self antigen, (negative selection), what do mature B cells do? are they responsive to antigen?
mature B cells emigrate from the bone marrow, and via alternative splicing co-express IgM and IgD. mature B cells are then fully responsive to antigen.
do IgM and IgD expressed on naive mature cells have the same or different specificity?
the same, the V,D,J regions have already been compiled and don’t change after initial rearrangement
how does co-expression of IgM and IgD occur?
co-expression occurs due to alternate mRNA splicing. on a primary RNA transcript there is a VDJ region, then a C u and C delta. This will get alternatively spliced, and make 2 transcripts, one with C u and one with C delta. remember-> receptor specificity does not change, the only thing that is different is whether or not C region is u or delta
what do mature naive cells have on them from primary transcript?
in mature naive cells, IgM and IgD are the only 2 immunoglobins expressed from primary transcript
what things does the development of T cell have in common with the development of B cells?
stem cells for both arise in the bone marrow, both undergo antigen receptor gene rearrangement, both must undergo negative selection
what things does the development of T cell not have in common with the development of B cells?
T cell precursors must emigrate from the bone marrow and go to the thymus, developing T cells must undergo positive selection to ensure that the receptor can recognize either class I or II MHC, finally T cell receptor genes are NOT Ig genes
how are T cells arranged in the thymus?
immature T cells, (thymocytes), are embedded in a network of thymic epithelial cells called stroma that together form a outer, close-packed cortex and an inner, less dense medulla
where do pre-thymic stem cells come from? do they have markers of mature T cells? where do they enter the thymus? can they recognize antigen?
pre-thymic T stem cells come from the bone marrow, and even though they are committed to the T lineage, they do not have markers of mature T cells. they enter the thymus at the subcapsular region and cannot recognize antigen, (receptor genes are in the germline config, – not rearranged).
what is the first stage of differentiation for T cells? what is going on with the TCR at this point? do they express CD4 or CD8?
pro T cells have RAG1,2 gene expression, meaning the beta chain of the TCR is being rearranged. they express neither CD4 or CD8, (CD 4-/CD8-)
what will most pro T cells become?
CD4+ and CD8+ T cells bearing the alpha/beta TCR
what marks pro T cells becoming pre T cells and how is this expressed? do pre T cells have CD4/8 expression?
successful rearrangement of the TCR beta chain gene, (via RAG1,2), marks pre T cells, and the beta chain is expressed on the surface with an invariant “surrogate” alpha chain protein called a pre-T alpha chain receptor. pre T cells are double negative for CD4/8
what does successful expression of a pre-T cell receptor stimulate?
rearrangement of the alpha chain and expression of CD3 and gamma signaling complexes
what is the beta chain encoded by in T cells?
V, D, J and a transmembrane protein gene, all procesed by the RAG complex
what is the alpha chain encoded by in T cells?
V, J and a transmembrane protein gene, all procesed by the RAG complex
what happens to pre T cells that successfully rearrange alpha and beta genes of the TCR?
they become thymocytes expressing both CD4+ and CD8+, (double positive), and also express a complete TCR, (alpha&beta)w/associated CD3 complex and gamma chains. this 80% of thymic cells, and now they must undergo positive and negative selection.
how does positive selection of double positive alpha/beta TCR thymocytes occur?
this occurs in the cortex and involves interaction of double positive cells with thymic epithelial cells. these cells express class I+II MHC cells. if the thymocyte sees class I it is stimulated to make CD8, if thymocyte sees class II it is stimulated to make CD4. if it sees neither=apoptosis.
what is a mnemonic for which CD recognizes what MHC?
CD (4) x MHC (2) = 8
CD (8) x MHC (1) = 8
what does positive thymocyte selection halt?
alpha chain rearrangement
how does negative selection of thymocytes occur?
thymocytes interact with anigen presenting cells such as TEC, dendritic cells or macrophages. negative selection occurs if the alpha/beta TCR T cell binds to the MHC so tightly that it also binds to the self peptide, this suggests cells are self-reactive and could lead to auto-immunity. cells that are negatively selected are deleted via apoptosis or rendered anergic, (if managed to exit the thymus). if the cell has a moderate interation, (not tight), it goes on to become either CD4 or CD8
what % of thymic cells die due to failure to either positively or negatively select?
90%
what can happen in the proliferation, (first), phase to both B and T cell differentiation that will lead to SCID?
lack of IL-7 receptors that drive proliferation
what do both pre-B and T cells have in common?
expression of a preantigen receptor, for B cells, this is a u with a prelambda5 (surrogate light chain), in the case of T cells this is a rearranged beta chain with a pre-alpha chain (surrogate light chain).
what do immature B and T cells express?
IgM on B cells, fully rearranged alpha and beta chain of T cell TCRs
how does negative selection occur in B and T cells? positive selection in T cells?
in both B and T, if antigen binds receptors tightly it means it recognizes self. in T cells only, positive selection is designed to determine whether or not T-cell receptor sees MHC class I or II, (B cells do not interact with MHC)
do B and T cells have receptor editing if negatively selected?
only B cells are known to be able to do this
what T cells are CD1 markers seen on?
thymocytes and langerhans cells, (terminally differentiated macrophages in the skin)
what T cells are CD3 markers seen on?
any cell with rearranged TCR complex, thymocytes and mature T cells
what T cells are CD4 markers seen on?
these function as co-receptors on T helper cells. they recognize Class II MHC. they are expressed on double positive thymocytes and any CD4 thymocytes and helper T cells
what T cells are CD5 markers seen on?
CD5 is an important marker for co-stimulation on T cells and a subset of B cells
what T cells are CD8 markers seen on?
CD8 is a co-receptor for cytolytic T cells and precursors and interacts with Class I MHC. CD8 is expressed on double positive thymocytes and cells that recognize class I MHC, CD8+ single positive
what B cells are CD10 markers seen on?
also called CALLA (common acute lymphocytic leukemia antigen), CD10 markers are seen on pre-B cells and germinal center B cells (mature)
what B cells are CD19 markers seen on?
pre b cell and mature b cells, not on plasma cells, (expressed before CD20)
what B cells are CD20 markers seen on?
pre b cell and mature b cells, not on plasma cells, (expressed after CD19)
what B cells are CD21 markers seen on?
CD21 is a receptor for Epstein Barr Virus (EBV). it is seen on mature B cells and follicular dendritic cells
what B cells are CD23 markers seen on?
activated mature B cells
what B cells are CD79a markers seen on?
pre B cells and mature B cells
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