Objectives and Vocabulary for Week 4

Question 1

Understand how developmental steps in the bone marrow lead to the generation of mature B cells with functional B cell receptors

Answer 1

So, before anything even starts the B cells begin as hematopoietic stem cells (HSCs) which give rise to all other cell types. When cells first begin they are sequestered into bone marrow islets which contain stromal cells. These stromal cells secrete FLT3L which mulipotent progenitors bind to via FLT3. Signaling through FLT3 allows for the differentiation by several methods. Levels of IL-7R on the surface of the progenitor cell increases, and the stromal cell produces Il-7 which is essential for the growth and survival of lymphocytes. At this point in development, the B cell is transforming from a hematopoietic stem cell to an early and late pro-B cell.
IL-7 signaling also promotes the expression of E2A—which is specific for B cells—which cooperates with PU.1 to induce early B cell factor (EBF) which then promotes Pax-5 expression and leading to eventually surface expression of CD19 and Igα. Igα is an essential component in the formation of the heavy chain. At this stage of development the cell has become a Pro B cell. As a rule, Pro B cells are where Ig rearrangement begins.
Noted by the expression of RAG1/2, Igα, Igμ, λ-5, and BLNK, V(D)J rearrangement for the heavy chain takes place in the pro-B cell. Once the heavy chain is expressed on the surface of the cell along with surrogate light chains is the cell considered a pre-B cell.
One the pre-B cell receptor—composed of the heavy chain and surrogate light chain—is completed signals through the pre-BCR which is mediated by a downstream gene Btk will then signal the cell to move onto the next stage, the immature B cell stage. Thus, the pre-B cell stage marks the first checkpoint in the development of B cells due to the fact that it marks successful recombination and expression of the heavy chain. Without a properly formed heavy chain, the Pre-B cell will enter apoptosis. The rearrangement can occur on either one of the alleles present, and infact if one allele successfully recombines before the gene segment on another allele recombines the other allele’s recombination will cease in a process referred to as allelic exclusion. (Also, these H chains formed are also referred to as μchains.)
Once the H chain has been successfully recombined, many large pre-B cells begin to proliferate and produce many cells with an identical and these cells are referred to as small pre-B cells. Later, VJ recombination takes place and light chain rearrangement takes place at one allele at a time and also exhibits allelic exclusion, eventually presenting the formed light chain on the surface of the cell. Thus marks the formation of an immature B cell.
Once the immature B cell has been formed the time for the second checkpoint in B cell development occurs. To ensure that the antigen formed by said developing B cell isn’t harmful to the body, the bell cell comes into contact with a self antigen and is tested to see how high its avidity for said antigen is. If the avidity is high, receptor edition and the expression of a new light chain takes place and the new heavy and light chain pairing are tested for self avidity. If this avidity is too high the second time, the cell is subject to apoptosis. Another possible consequence is that a B cell interacts with a self antigen with low avidity and will have reduced receptor signaling becoming an anergic B cell.

Question 2

Identify the cytokines, transcription factors and cell surface molecules that regulate B cell development

Answer 2

IL-7, Pax5, E2A, EBF, FLT3, sigma 5, muIG, pre-BCR, Btk, CD10, CD19, CD34, Ig alpha and beta, BLNK, VpreB, Igm, and IgD are all some sort of thing mentioned above.

Question 3

Discuss the two checkpoints in B cell maturation and how signaling at these checkpoints generates B cells with functional B cell receptors

Answer 3

The two checkpoints in B cell maturation involve the Pre-B stage, and the Immature B cell stage. These are two checkpoints due to the fact that the point of the Pre-B stage is the proper formation of the heavy chain found on immunoglobulins/antibodies. If a heavy chain is not properly made or is not made at all then the lymphocyte in question is essentially useless and should be destroyed as it could either pose some harm to the host’s body or do nothing. After proper formation of the heavy chain the cell can continue onward to develop into an immature B cell, which is where VJ recombination takes place and where the light chain is made. Light chains are incredibly important due to the fact that they play a role in combination diversity for antibody recognition as well as the fact that light chains play an important structural role in terms of binding to epitopes in a three dimensional space. Without proper formation of a light chain, or formation of a light chain to a self antigen, the host organism in question will suffer some ill effect.

Question 4

Discuss the three mechanisms in place that contribute to tolerance induction in B cells.

Answer 4

Central tolerance uses three methods to ensure that mature B lymphocytes and their clones won’t attack the body in three ways. Clonal deletion occurs when BCR bind strongly to self antigens, and due to this the cell receives a strong signal through IgM and dies at the immature stage.
Clonal anergy occurs when BCR binds soluble self antigens, BCR is downregulated and IgM cannot be activated. These anergic B cells have a limited life span, and can no longer be activated. Finally, clonal ignorance occurs when BCR binds soluble self antigen with low affinity, the cell receives a weak or no signal and the cell still exists and in many cases does not encounter antigens while it is in circulation.

Question 5

Central lymhpoid organs

Answer 5

Specialized tissues where lymphocyte development takes place in Bone marrow for most B cells and Thymus for most T cells

Question 6

Peripheral lymphoid organs

Answer 6

Specialized lymphoid tissues where mature lymphocytes reside, and circulate in from the blood—peripheral organsare the lymph nodes, spleen, mucosallymphoid tissue (peyer’s patch).

Question 7

Stromal cells

Answer 7

Connective tissue cells of an organ found the loose connective tissue. They are often associated with the uterine mucosa and the ovary as well as the haematopoietic system and elsewhere. They are essential for B cell development, and they form specific adhesive contacts and provide cytokine signals that control proliferation and development. These compounds are FLT3, and IL-7R.

Question 8

Common lymphoid progenitor

Answer 8

The earliest lymphoid progenitor cells which give rise to T cells, B cells, and natural killer cells.

Question 9

Early Pro B cell

Answer 9

When a hematopoietic stem cells receive signals from bone marrow stromal cells to begin B cell development. Cytokines induce TdT, RAG-1/2, and the cells undergo D-J joining on the H chain chromosome to become early pro-B cells. These cells express DJ rearranged H chain genes, germline L chain genes, absence of surface immunoglobulin, expression of RAG recombinase, TdT expression, surrogate light chains, Ig antibody expression, and expression of Btk. They also express CD34, CD45

Question 10

Late Pro B cell

Answer 10

Joining of a V segment to the D-JH completes the late pro-B cell stage. These cells become pre-B cells. They are characterized by the presence of VDJ rearranged H chain genes, germline L chian genes, absence of surface immunoglobulin, expression of RAG recombinase and TdT, expression of surrogate L chains, Ig antibody expression, expression of Btk. The cells also express CD45R, CD19, CD40.

Question 11

Pre B cell

Answer 11

When a Pro B cell successfully rearranges and produces an intact μ chain—where VDJ rearrangement ceases and the cell becomes a pre B cell. Surrogate light chains are present in the pre-B receptor, surrogate L chains resemble actual L chians, but are the same on every pre-B cell. Once this stage is complete, the light chain has undergone V-J rearranging.

Question 12

Immature B cell

Answer 12

During this process of cell development, VDJ and VJ rearrangement has occurred, IgM has been expressed on the cell surface, and have not yet undergone antigen specific checks for self antigens.

Question 13

FLT3:FLT3L

Answer 13

FLT3 is an adhesive cytokine signal on multipotent progenitor cells that controls proliferation and development which binds to FLT3L on stromal cells. This signaling through FLT3 is needed to differentiate to the common lymphoid progenitor.

Question 14

IL-7:IL-7R

Answer 14

IL-7R is produced by B cells. IL-7 is a produced by stromal cells which is essential for the growth and survival of lymphocytes. When these bind together, Pax-5, E2A signaling is promoted.

Question 15

Surrogate L chain:

Answer 15

Two non-variable proteins that associate with Ig μ heavy chains in pre-B cells to form that pre-B cell receptor. The two surrogate light chain proteins include the V pre-B protein, which is homologous to a light-chain V domain, and λ5, which is covalently bonded to the μ heavy chain by a disulfide bond

Question 16

Clonal Deletion

Answer 16

A mechanism of lymphocyte tolerance in which an immature T cell in the thymus or an immature B cell in the bone marrow undergoes apoptotic death as a consequence of recognizing a self antigen.

Question 17

Clonal Anergy

Answer 17

A state of antigen unresponsiveness of a clone of T or B lymphocytes experimentally induced by recognition of an antigen in the absence of additional signals (costimulatory signals) required for functional activation. Clonal anergy is considered a model for one mechanism of tolerance to self antigens and may be applicable to B lymphocytes as well.

Question 18

Clonal Ignorance

Answer 18

A form of lymphocyte unresponsiveness in which self antigens are ignored by the immune system even though lymphoctyes specific for those antigens remain viable and functional

Question 19

Allelic exclusion

Answer 19

The exclusive expression of only one of two inherited alleles encoding Ig heavy and light chains and TCR beta chains. Allelic exclusion occurs when the protein product of one productively recombined antigen receptor locus on one chromosome blocks rearrangement of the corresponding locus on the other chromesome. This property ensures that each lymphocyte will express a single antigen receptor and that all antigen receptors expressed by one clone of lymphocytes will have identical specificity. Because the TCR alpha chain locus does not show allelic exclusion, some T cells do express two different types of TCR.

Question 20

Kit

Answer 20

A gene which encodes c-kit, or CD117, which is a receptor tyrosine kinase protein. CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells as well as other cell types. Altered from of this gene is associated with cancer. CD117 binds to stem cell factor, also known as c-kit ligand, which activates signal transduction molecules that propagate the signal in the cell, playing a role in cell survival, proliferation, and differentiation.

Question 21

SCF

Answer 21

Stem cell factor, a cytokine that binds to the c-KIT receptor (CD117) which can exist as a transmembrane protein and a soluble protein. It plays a important role in hematopoiesis, increasing the survival of HSCs (hematopoetic stem cells)

Question 22

E2A:

Answer 22

a transcription factor that plays a major role in determing tissue-specific cell fate during embryogenesis, like muscle or early-B-cell differentiation. E2a expression is promoted by IL-7 signaling, and cooperates with PU.1 to induce EBF, early B cell factor (EBF)

Question 23

EBF

Answer 23

A cell type specific transcription factor that is expressed at all antigen-independent stages of B lymphocyte development, and various experiments suggest that EBF plays a specific and important role in the transcriptional control of B-cell differentiation at a stage before Ig gene rearrangement but after commitment of cells to the B-lymphoid lineage. Early B cell factor promotes the expression of Pax-5.

Question 24

Pax-5

Answer 24

A transcription factor that leads to B cell specific expression of CD19, and Igα

Question 25

Pre-B cell receptors

Answer 25

Heavy chains with a surrogate light chain indicates the status of the cell as a Pre B cell and it signals through Pre-BCR signals to move onto the next step, the Immature B cell stage. This acts as the first of two important checkpoints to ensure that the B cell is properly developing

Question 26

VpreB

Answer 26

A gene which encodes CD179A, and makes up part of the surrogate light chain

Question 27

λ5

Answer 27

Associates with VpreB to from the surrogate light chain.

Question 28

Igα

Answer 28

A signaling protein which is required for functional receptor complex formation

Question 29

Igβ:

Answer 29

A signaling protein which is required for functional receptor complex formation

Question 30

Central tolerance

Answer 30

A form of self-tolerance induced in generative (central) lymphoid organs as a consequence of immature self-reactive lymphocytes recognizing self antigens and subsequently leading to their death or inactivation. Central tolerance prevents the emergence of lymphocytes with high-affinity receptors for the self antigens that are expressed in the bone marrow or thymus

Question 31

Flow cytometry

Answer 31

A method of analysis of the phenotype of cell populations requiring a specialized instrument (flow cytometer) that can detect fluorescence on individual cells in a suspension and thereby determine the number of cells expressing the molecule to which a fluorescent probe binds, as well as the relative amount of the molecule expressed. Suspensions of cells are incubated with fluorescently labeled antibodies or other probes, and the amount of probe bound by each cell in the population is measured by passing the cells one at atime through a fluorimeter with a laser generated incident eam