What are the 3 phases of B-cell development in the bone marrow?
Development of B-cells in the bone marrow occurs in 3 phases and is associated with Ig gene expression.
1. Generation of diverse and clonally expressed B-cell antigen receptors (i.e., immunoglobulin or Ig)
2. Alteration of B-cell antigen receptors (Ig) or elimination of B-cells that express B-cell antigen receptors (Ig) that binds to self-antigens.
3. Promotion of immature B-cells who antigen receptor will recognize foreign antigen and become mature B-cells in secondary lymphoid tissues.
What are the 6 stages of B-cell development? What do these stages coincide with? (hint: has to do with Ig genes)
Phase 1: Development of B-cells surface Ig occurs in 6 stages:
Early pro-B cell
Late pro-B cell
Large pre-B cell
Small pre-B cell
Immature B cell
B-cell development stages coincide with Ig gene rearrangement.
Explain the status of H-chain gene rearrangement, L-chain gene rearrangment, and Ig status in each of the 6 stages of B cell development.
attached is slide 6 of PP
What is allelic exclusion? What is the purpose of allelic exclusion? What is the excpetion ot this rule and when in B-cell development does it occur?
Allelic exclusion. To prevent the formation of Ig with two different heavy chains and two different light chains (and decreasing avidity of the antibody) the Ig genes rearrange on a single chromosome. The other chromosome is silent. The exception of the rule is D to J gene rearrangement. In this instance both chromosomes are active at the same time. D to J gene segment joining occurs on both chromosomes in early pro-B cells.
Explain the selection process that occurs in the early pro-
B cell and late pro-B cell development.
What is the approximate percentage of B-cells that continue past these stages to become large pre-B cells?
What is the surrogate light chain? What does it interact with? When is it expressed?
The V to DJ gene segment joining only occurs on one chromosome in late pro-B cells. If it is a nonproductive rearrangement occurs on one chromosome, then the next V gene segments joining with DJ gene on the other chromosome will occur.
- If both rearrangement attempts are nonproductive, the late pro B-cell will die by apoptosis since no receptor can be formed.
- Once heavy chain variable genes are rearranged the mu (μ) heavy chain is made. Heavy chain μ proteins join together with surrogate light chain inside large pre-B-cell. The surrogate light chain signals the cell to continue development.
- 50% of cells die by apoptosis and the other 50% continue on in B cell development.
After the pre B-cell receptor (mu heavy chain) is made it will be expressed in the cytoplasm of the large-pre B-cell to wait for an Ig light chain to form.
The cell advances to the next stage of development called the small pre-B cell stage.
Now that the cell is in the small pre-B cell development stage, it can begin light chain gene rearrangment. Explain this process. State which light chain gene begins rearrangment first.
What happens to cells that are unable to proceed past the small pre-B cell stage? Why does this occur?
- The Kappa light chain V-J gene segments will rearrange on one chromosome. Productive rearrangement will lead to the formation of IgM.
- If it is a nonproductive rearrangement of genes occurs then the kappa light chain V-J genes on the other chromosome rearrange.
- If this rearrangement fails then the rearrangement of V-J gene segments of the lambda light chain occurs.
- When rearrangements of V-Js on the lambda light chain on one chromosome is non productive then the V-J gene segments on other chromosome rearrange
- If rearrangement of the last lambda light chain gene leads to a non-productive light chain gene rearrangement then the pre-B cell will die. This ensures that B-cells will only leave the bone marrow with an intact IgM molecule expressed on the B-cell surface.
Before B-cells can be released to the periphery, what process must occur? (Just state the name of this process)
Explain the process of negative selection of B-cells. Explain what occurs if a B-cell originally "fails" at the first trial of negative selection.
What happens to B-cells that successfully complete the negative selection process?
Negative selection of B-cells responding to self antigens: Before exiting the bone marrow B-cells expressing surface IgM will be selected for their ability to recognize self-components (i.e., self-antigens). If the B-cell recognizes self-antigens it will be induced to rearrange its light chain genes. The IgM made by the B-cell will have a different specificity. If the surface IgM continues to bind to self antigens and no other rearrangement of light chain genes are possible the B-cell undergoes apoptosis. (the lymphocyte arranges all available light chain genes until has productive rearrangment but if not, then undergoes apoptosis).
Once auto-reactive B cells are eliminated from the bone marrow IgD production occurs and the B-cell so-expresses both IgM and IgD.
The IgM and IgD co-expressing B cells will travel to secondary lymphoid tissues where it will remain until they encounter antigen.
What happens to B-cells that do not recognize non-self?
They undergo apoptosis.
List the order in which thymocytes express their receptors in the thymus.
State what parts of the thymus the thymocytes are in when they develop each receceptor. State what cells they come in contact with in these regions.
Where do antigen specific naive T cells exit the thymus? Where do they go?
Developing thymyocytes express receptors in this order in different sites of the thymus:
1.beta TCR chain
2.both CD4 and CD8,
3. alpha TCR chain
4. Finally the thymocytes develops into CD4 only or CD8 only mature T-cells.
- Thymocytes originally mature in the subcapsular region where they generate the beta chain the TCR. Then they migrate to the cortical region where they express CD4 and CD8 and the alpha chain of the TCR.
- Next they come into contact with cortical epithelial cells. From here they migrate to the medullary region where they come into contact with medullary epithelial cells, dendritic cells and macrophages.
- The surviving thymoyctes lose either CD4 or CD8 and become antigen specific naïve T-cells. The antigen specific naïve T-cells exit the thymus through venules that line the medullary region and travel to secondary lymphoid organs to encounter dendritic cells expressing their specific peptide antigen in the context of MHC molecules.
We know that the beta chain of the TCR rearranges first. Explain what occurs in the process of this rearrangment.
How many attemps are made to rearrange the segments for the beta chains?
What happens to the beta chain if productive rearrangment occurs? What is the purpose of this?
What happens if non-productive rearrangment occurs?
The first stage of development occurs when the committed T-cell progenitor begins to rearrange their V, D and J chains to form the beta TCR chain (first D-J, then V-DJ. Two attempts are made to rearrange the VDJ chains for the beta TCR chain (two chromosomes). Unlike Ig formation allelic exclusion is not required since the alpha and beta chains that form the TCR do not have to be identical.
If a productive rearrangement occurs the beta-chain of the TCR expressed on thymocyte cell surface with pseudo-alpha chain (forming a superdimer, see slide 24 of PP). This complex has the ability to interact with one another and form a dimer. The dimer triggers proliferation of the thymocyte.
If non-productive rearrangement occurs, the thymocyte undergoes apoptosis and are ingested by thymic macrophages.
Note that rearranged and surface expression of a TCR beta chain is the first check-point in process to generate an antigen specific TCR (see attached pic on next card).
What occurs after productive rearrangment of the beta chain? (What does the T-cell begin to express? What processes must it undergo to do this?)
At this point the thymocyte expressing a beta TCR chain will begin to express both the CD4 and CD8 molecules. Next V and J alpha chain of the alpha TCR gene segments rearrange. The chain locus can sustain many attempts at a functional rearrangment. If a productive TCR alpha chain is formed the alpha chain TCR protein will displace the pseudo-alpha chain of the TCR in order to form the TCR complex with the beta TCR.
Once both chains of a TCR are expressed by the thymocyte, what process in development occurs next? (just name the process)
What is positive selection? Explain what occurs in this process.
Explain the fate of thymocytes that pass and those that fail positive selection.
What thymic cells participate in this process?
Once both chains of a TCR are expressed the thymocyte begins to select for their ability to recognize MHC molecules. This process is know as positive selection. Positive selection is a way in which a thymocyte matures further but only if they are capable of recognizing self peptides presented by MHC molecules on cortical thymic epithelial cells. If they are unable to engage MHC then they will die. If they recognize self peptides presented by MHC molecules then they advance to the next selection process.
Thymoctyes that have weak or no binding with self peptides presented by MHC molecules undergo apoptosis. Thymocytes that have moderate to strong binding move on to the next stage.
After positive selection of thymocytes, what process occurs next? What thymic cells are involved? How do thymocytes move past this selection process? What occurs if they do not?
The next stage of thymocyte development is called negative selection. Thymocytes that have undergone positive selection will come into contact with dendritic cells and macrophages. If they moderately bind self-peptides, they go on to the next stage in development. If they strongly bind self-peptides, they undero apoptosis.
If thymocytes successfully complete negative selection, what is the next stage in development? What determines the fate of this process?
What is the thymocyte called after this process? Where does it go?
If they do not respond they survive and they begin to be selected for expression of either CD4 or CD8. The loss of either CD4 or CD8 expression on thymocytes is random but typically the ratio of CD4 to CD8 T-cells in the blood is 2:1. At this stage of development the thymocyte is an antigen specific naïve T-cell that will travel to the secondary lymphoid tissue to encounter specific peptide antigens presented in the context of MHC molecules by dendritic cells or macrophages.
What happens to the thymus with age? When does this begin to occur? Why does it occur?
What is the response of the thymus to novel antigens after this time period?
Naïve T-cell development in the thymus diminishes with age: As one gets older, and most often after puberty, T-cells production in the thymus diminishes. This occurs because the repertoires of T-cells in the periphery that are available to deal with any antigenic challenge are made by the time we reach puberty. Typically the thymus will contract over time after puberty. However, during infections or exposure the to novel antigens the thymus can fill up again after puberty. As one ages further (>50 years old) the thymus begins to fill with adipocytes.