Question

1)You inject a mouse with a protein antigen X. This is the first time the mouse has been exposed to X. In the course of clearing X from their body the mouse will launch an immune response to antigen X. Over the course of the immune response antigen X will encounter naïve, effector and memory lymphocytes (B- and T-cells).  There are distinct differences between the function and role of each type (naïve, effector and memory).Differentiate between naïve, effector and memory lymphocytes.  Focus on their response to antigen (time to respond, magnitude of response, quality/type of response) and their overall function in the immune response.

Note: This question is only worth 5 marks!

• This isn’t a molecular question. It’s a ‘what is the point’, ‘why do I need so many different things’ type of question.
• Think big picture (not lots of molecules) and focus on the underlined words above.
• It is possible to answer the question in a way that is accurate for both T- and B-cells at the same time (i.e., one answer covers both) but you could also describe both independently. Do whichever you feel more comfortable with.
• The mouse, and antigen X, was just an example. You don’t have to mention them in your answer

2)During B-cell activation some activated B-cells undergo differentiation into antibody secreting plasma cells outside of the germinal center and therefore never go through the germinal center responses. Other activated B-cells will return to the follicle forming a germinal center and undergo processes that alter the antibody that will be produced.  

Describe the germinal center responses. Why are these responses important (hint: what is different about the antibody produced)? Why are T cells important in germinal center responses?

3)List the B-cell developmental stages from pre-pro to immature B cells describing B-cell receptor (BCR or Ig) rearrangements occurring and/or key events occurring at each stage. ( Do not Draw it out. Thanks)

4)What is the role of Artemis in V(D)J recombination and why is it important?

Answer 1

1) From this data you will get differenciation points. It is mixed matter.

All lymphocytes that leaves the pivotal(central) lymphoid organs are naive. memory T cell subsets are demonstrably capable of differentiating into both memory and effector subsets whereas effector cells are incapable of differentiating into memory cells in vitroThe supposition that naïve and memory T cells can be distinguished phenotypically is based on the notion that memory T cells retain a permanent imprint of having responded to antigen. Especially prominent are differences in the cell surface expression of adhesion molecules between naïve and memory T cells. Thus, compared to naïve T cells, memory T cells have been reported to express higher levels of β1 (CD29, CD49d and CD49e) and β2 (CD11a, CD11b and CD18) integrins, CD2, CD44, CD54 and CD58. Increased expression of adhesion molecules on recently activated T cells reflects the requirements for effector T cells to enter peripheral tissues at sites of inflammation and interact with target cells, and may similarly affect the function of some memory T cells.

The expression of other molecules involved in lymphocyte migration also differs between naïve and memory T cells. Of particular interest are differences in the expression of two key molecules required for the entry of T cells into lymph nodes through high endothelial venules (HEVs): CD62L and CCR7. CD62L binds to vascular addressins expressed on HEVs and is responsible for the initial stage of adherence of blood-borne T cells to HEVs, while the CCR7 chemokine receptor controls responsiveness to chemokines expressed in HEVs at sites of lymphocyte entry. Whereas naïve T cells are uniform in expressing high levels of both molecules, some memory cells lose expression of CD62L and/or CCR7. However, memory T cells may express receptors for chemokines that direct them to inflammatory sites and for molecules involved in homing to peripheral tissues, such as the cutaneous lymphocyte antigen.Although memory T cells are enriched amongst cells expressing the surface markers, it is also clear that memory cells exhibit substantial phenotypic heterogeneity.Memory T cells are a subset of infection- and cancer-fighting T cells (also known as a T lymphocyte) that have previously encountered and responded to their cognate antigen; thus, the term antigen-experienced T cell is often applied. In comparison to naive T cells, which are T cells have not been exposed to antigens yet, memory T cells can reproduce to mount a faster and stronger immune response.

2) The process is,Germinal centers develop in the B cell follicles of secondary lymphoid tissues during T cell-dependent (TD) antibody responses. The B cells that give rise to germinal centers initially have to be activated outside follicles, in the T cell-rich zones in association with interdigitating cells and T cell help. After immunization with a single dose of protein-based antigen, the germinal centers formed are oligoclonal; on average three B blasts colonize each follicle. These blasts undergo massive clonal expansion and activate a site-directed hypermutation mechanism that acts on their immunoglobulin-variable (Ig-v)-region genes. Mature germinal centers are divided into dark and light zones. The proliferating blasts, centroblasts, occupy the dark zone and give rise to centrocytes that are not in cell cycle and fill the light zone. The light zone contains a rich network of follicular dendritic cells (FDC) that have the capacity to take up antigen and hold this on their surface for periods of more than a year. The antigen is held as an immune complex in a native unprocessed form; but the antigen may be taken up from FDC by B cells, which can process this and present it to T cells. Centrocytes appear to be selected by their ability to interact with antigen held on FDC. There is a high death rate among centrocytes in vivo, and when these cells are isolated in vitro, they undergo apoptosis within hours on culture. The onset of apoptosis can be delayed by cross-linking centrocytes' surface Ig, and long-term survival is achieved by signalling through their surface CD40. After activation through CD40 the centrocytes increase their surface Ig and acquire characteristics of memory and processing of antigen held on FDC and its presentation to T cells that can be induced to express CD40 ligand at the point of cognate interaction. Other signals that induce a proportion of germinal center cells to become plasma cells have also been described. Germinal centers persist for about 3 weeks following immunization, but after this, memory B blasts continue to proliferate in follicles throughout the months of T cell-dependent antibody responses. These cells are probably the source of plasma cells and memory cells required to maintain long-term antibody production and memory after the first 3 weeks of T cell-dependent antibody responses.

T cells are important,because GC formation begins with acquisition of antigen by resting B cells , followed by their migration to the follicle:T-zone (T:B) border, where they receive co-stimulatory signals from CD4+ T cells. This interaction triggers a period of intense proliferation in which responding B cells are located preferentially in the outer B cell follicle.

3)generation Of B cell - B cell development begins in the fetal liver and continues in the bone marrow throughout our lives.

Once a B cell can express both m and L chains on its membrane, it is officially a B cell. However, it is still immature and can be easily killed by contact with self antigen until it also expressed membrane IgD. The mature B cell that moves into the periphery can be activated by antigen and become an antibody-secreting plasma cell or a memory B cell which will respond more quickly to a second exposure to antigen. B cells which fail to successfully complete B cell development undergo apoptosis (programmed cell death).

Lymphoid progenitor cells receive signals from bone marrow stromal cells to begin B cell development. Cytokines induce TdT and recombinase (RAG-1 and RAG-2) synthesis in CD34+ lymphoid progenitors. The cells undergo D-J joining on the H chain chromosome to become early pro-B cells and also begin expressing CD45 (B220) and Class II MHC. Joining of a V segment to the D-JH completes the late pro-B cell stage.

Pro-B cells become pre-B cells when they express membrane m chains with surrogate light chains in the pre-B receptor. Surrogate L chains resemble actual L chains but are the same on every pre-B cell. Signal transduction molecules IgaIgb are also part of the pre-B receptor complex. The cytoplasmic tails of Ig heavy chains are too short to enter the cytoplasm and transmit an antigen-binding signal; Ig a Ig b signal transduction molecules have ITAMs (Immunoreceptor Tyrosine Activation Motifs) which become phosphorylated in response to antigen-BCR binding. The phosphorylation initiates a cytoplasmic signaling cascade. The cell halts recombination of H chain and proliferates into a clone of B cells all producing the same m chain. Since dividing cells are larger than resting cells, this stage is called the large pre-B cell.Following proliferation, small pre-B cells (no longer dividing) undergo V-J joining on one L chain chromosome. Once L chain has been successfully synthesized, it is expressed with m chain on the cell membrane and the cell is called an immature B cell. Immature B cells are very sensitive to antigen binding, so if they bind self antigen in the bone marrow they die. B cells that do not bind self antigen express d chain and membrane IgD with their IgM about the time they leave the marrow and become mature naive (resting) B cells.

B cell regulation- Progenitor cells receive signals from bone marrow stromal cells via cell-cell contacts and secreted signals. This bone marrow microenvironment is responsible for B cell development.Somatic recombination in the developing B cell can be productive (result in synthesis of a functional H or L chain) or nonproductive due to introduction of a stop codon because of frame shift mutations Failure to make productive rearrangements and express Ig at the appropriate times during development results in the death of the developing cell. B cells have two opportunities to productively rearrange H chain (maternal and paternal chromosomes) and four opportunities to productively rearrange L chains (paternal and maternal k and l loci). Human B cells usually rearrange DH and JH segments on both chromosomes simultaneously. DH can also be read in any reading frame, so all D-J rearrangements are productive. Estimates are that only about half of developing B cells make productive H chain rearrangements. These successful pre-B cells divide to make clones of B cells (the large pre-B cell stage) that can proceed to L chain recombination.During the small pre-B cell stage, light chain V-J joining usually occurs first for k chain. If rearrangement is productive, k chain is made and the cell becomes an immature B cell expressing membrane IgM(k) BCR. B cells are able to repeat V-J joining several times if the first attempts are nonproductive; this process is called light chain rescue. If k genes are not successfully rearranged on either chromosome, l genes are rearranged. Success leads to production of IgM(l) BCR. If neither k nor l is productively rearranged, the cell undergoes apoptosis in the bone marrow. Only a minority of human pre-B cells fail to become mature B cells.

4)Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival.Artemis is a member of the metallo-β-lactamase superfamily of proteins Artemis was identified as the protein mutated in patients with SCID associated with radiosensitivity (RS-SCID). The majority of Artemis mutations that cause RS-SCID are located within its highly conserved N-terminal domain termed as the catalytic core of the protein, with its C-terminal region shown to be dispensable for V(D)J recombination on plasmid substrates.

Importance :Artemis is the newest player in VDJ recombination and double strand break repair.The lymphoid-specific components of the recombination machinery are RAG-1, RAG-2, and terminal deoxynucleotidyl transferase (TdT). RAG-1 and RAG-2 together constitute the recombinase. TdT, although not essential for catalysis, plays an important role increasing diversity by mediating the incorporation of nontemplate-dependent nucleotides. The nonlymphoid-restricted components identified so far are DNA-PKcs, Ku70, Ku80, XRCC4, ligase 4, and the most recently identified Artemis. All these proteins are involved in DNA double strand break repair as well as VDJ recombination. These nonlymphoid-specific components are likely to participate in the processing and joining steps of VDJ recombination, but their specific architectural or catalytic roles in the reaction remain largely unclear. Two other nonlymphoid-specific components, HMG1 and HMG2, have been implicated in VDJ recombination. In vitro experiments showed that these proteins increase the efficiency of cleavage by RAGs .