B-Cell and T-Cell Maturation Dr. Diebel 5/13/14

Question 1

B-Cell Development

Answer 1

Start out as stem cells w/ IL-3 guiding them toward the lymphoid progenitor line. Bone marrow stromal cells and signals that guide the B-cell development. Starts out w/ early proB where DJ H chain recombination occurs and the start of V DJ H chain recombination. Next is proB or preB and here V DJ H chain recombination completes, clonal expansion and VJ L chain recombination occurs. Finally immature B when negative selection occurs. After this they now have the ability to leave the bone marrow, go through transitional phases 1,2, etc and become mature B cells w/ IgM and IgD.

B cell development occurs in the bone marrow and does not require contact w/ antigen. The initial BCR complex includes: mIgM, and the signaling chains Ig alpha and Ig beta (CD79a and CD79b).

If the cells don’t encounter antigen within a few weeks they will die by apoptosis.

Question 2

proB Cells

Answer 2

Earliest stage of antigen-independent B cell development and can be divided into three groups based on the expression of TdT and B220 (CD45R)(receptor for growth and differentiation).

1. Early proB express TdT alone
2. Intermediate express both TdT and B220
3. Late express B220 (remains expressed on the surface throughout the remainder of B cell ontogeny) and have down regulated TdT

As the cells progress through the proB stage they rearrange their heavy chains and begin to express CD43 (leukosialin), CD19 (BCR co-receptor, works w/ CD21 and CD81), RAG 1 and 2.

proB also express c-kit which binds to stem cell factor expressed on bone marrow stromal cells which induces the proB cells to proliferate and differentiate into preB cells.

As proB enters preB they down regulate TdT, RAG 1, RAG 2, and CD43. (c-kit and CD34 are basically gone in late preB which allows the B cell to be released from the stromal cells)

Question 3

preB Cells

Answer 3

Divided into two groups.

1. Large mitotically active preB cells
2. Small non-dividing preB cells

Both groups of cells express IgM heavy chains in the cytoplasm and preB BCR complex on their surface.

The large preB cells have successfully rearranged their Ig Heavy chains and as they progress to the small preB group they being to rearrange their Ig light chain genes and up regulate RAG 1 and RAG 2.

preB cells express IL-7R and are stimulated to divide and differentiate using IL-7 (growth function released from stromal cells).

Question 4

Immature B Cells

Answer 4

Final stage of B cell development. At this point the B cells have successfully rearranged their light chain genes and express IgM. The RAG 1 and RAG 2 expression has been down regulated. As the immature B cells develop even more they start to express both IgM and IgD on their surface (gradient).

After exiting the bone marrow B cells go through a phase known as transition phase.

Question 5

Cytokines required for B cell development

Answer 5

Common lymphoid progenitors (CLP) are responsive to IL-7 which promotes B cell lineage development at the proB cell stage.

Blys signaling through its receptor BR3 is important for the survival of pre-immune B-cell stages from transition stage onwards.

IL-4, IL-3 and low molecular weight B growth factor (L-BCGF) are important in initiating the process of B cell differentiation.

Question 6

E2A and EBF

Answer 6

Transcription factors in B cell development that coordinately activate early B-cell genes

Question 7

Pax5

Answer 7

Transcription factors in B cell development that ensures development to B-cell lineages by restricting transcription of lineage inappropriate genes and activating expression of B lineage signaling molecules

Question 8

Sox4 and LEF1

Answer 8

Transcription factors in B cell development that promote the survival and proliferation of proB cells

Question 9

IRF4 & IRF8

Answer 9

Transcription factors in B cell development that terminate pre-BCR signaling by IRF4 and promote differentiation to small pre-B cells

Question 10

Bcl-6

Answer 10

Transcription factors in B cell development which is required for germinal B cell differentiation and generation of memory B cells

Question 11

Blimp-1

Answer 11

Suppresses Bcl-6 expression and is required for development of Ig secreting cells and maintenance of long lived plasma cells.

Question 12

Surface Ig receptor expression is present during what stages?

Answer 12

No expression is present during pre-pro, early pro, or late pro.

Pre-BCR is present during large pre cell stage.

Decreasing levels of pre-BCR is found during small pre cell stage.

IgM is present during immature B stage.

Question 13

c-kit is expressed during what cell stages?

Answer 13

Low amounts of c-kit are found during early and late pro cell stages.

Question 14

CD25 is expressed during what cell stages?

Answer 14

+/- during late pro and is present from then on.

Question 15

CD19 is expressed during what cell stages?

Answer 15

Every stage except pre-pro.

Question 16

B220 (CD45R) is expressed during what cell stages?

Answer 16

Expressed during every stage.

Question 17

Immunodeficiency XLA

Answer 17

Leads to a block at the proB cell to large preB cell transition. The defect is mostly caused by a mutation in the gene encoding the enzyme Bruton’s tyrosine kinase (Btk) which is a key enzyme involved in signal transduction downstream of the pre-BCR and BCR. In these patients there is very few circulating B cells and negligible serum immunoglobulin.

Question 18

Common variable immunodeficiency (CVID)

Answer 18

Impacts the later stages of B cell development and manifests itself in reduced serum Ig, memory B-cells, class switch recombination and B cell activation. Mutations in CD40 ligand on T cells, B cell surface receptor CD19, activated T cell costimulatory molecules ICOS and TACI (another receptor for Blys) have all been identified in CVID patients.

Question 19

Negative selection

Answer 19

Occurs in the bone marrow (clonal deletion). If immature B cells that express mIgM recognize self-antigen:
1. Some of the immature B cells undergo apoptosis.
2. Some of the immature B cells undergo editing of light chain genes to produce a different light chain that does not recognize self-antigen.
Not all self-reactive B cells are eliminated in the bone marrow because not every self-anitgen is expressed in the bone marrow.

Question 20

Transitional Immature B cells

Answer 20

T1 expresses IgM only. Negative selection in PALS.

T2 expresses both IgM and IgD. Positive selection in follicle.

Question 21

The three major populations of B cell in the periphery

Answer 21

1. Follicular (B-2) B cells

These are the cells when we normally think of B cells. They are found in the secondary lymphoid organs. Formed from precursors in bone marrow, somatic hypermutation, requires T cell help, produces high level of IgG, surface IgD found on naive B cells.

2. B-1 B cells

These are found in the peritoneal and pleural cavities and self-renewing. Somatic hypermutation does not occur, no T cell help is needed, express high levels of IgM, little or no memory, little or no surface IgD. CD5 is found on the surface.

3. Marginal zone B cells

Found in the marginal zones of the spleen and are long lived or might be self-renewing. Primarily IgM and some IgG. In general not much is known about these.

Question 22

T-independent B cell activation

Answer 22

A small number of antigens can activate B cells w/o MHC II restricted T cell help. These can be divided into two groups TI-1 and TI-2 based on the manner in which they activate B cells.

TI-1 predominately bacterial cell wall components (LPS of gram (-) is a major stimulant).

TI-2 predominately large polysaccharide molecules w/ repeating antigenic determinants (e.g. Ficoll, dextran, polymeric bacterial flagellin, and poliomyelitis virus).

Many TI antigens are PAMPS that can be recognized by TLR

CD5 receptor

TI antigen produce IgM about a day earlier than TD IgM.

Question 23

B cell response to type 1 TI antigen

Answer 23

B1 B cells can bind to LPS either through TLR4 or BCR

TLR4=nonspecific (polyclonal activation) and can stimulate both mature and immature cells.

BCR=specific (clonal)

Only IgM is produced in response and no memory is formed.

The B cell-co-receptor complex consists of CD21, CD19, and CD81 binding to complement. (C3D)

Question 24

B cell response to type 2 TI antigen

Answer 24

Can only stimulate mature B cells through cross linking of BCR (specific clonal activation). Mostly IgM is produced in response to this situation.

CD4 + Th2 T cells can be involved to produce cytokine for a full B cell response that can include class-switching. (very low level of IgD produced)

Question 25

B/T cell surface molecule interactions

Answer 25

CD28 (CTLA-4)(eventual down regulator)–CD80/86

CD40L–CD40

LFA-1–ICAM-1/3

CD2–LFA-3

Th2 makes IL-2, IL-4, IL-5 to push B cell proliferation and differentiation. (Th1 can help a little bit w/ IL-4 and IFN-gamma but Th1 mainly and the one that initiates)

Question 26

Primary and secondary antibody responses

Answer 26

Naive B cell

Lag period after antigen is 4-7 days, peak response 7-10 days, IgM predominates in the early primary response, TI and TD antigens, low antibody affinity and cells are short-lived.

Memory B cell

Lag period after antigen is 1-3 days, peak response 3-5 days, antibody response is 10 to 1000 times higher than primary response, IgM predominates (IgA in mucosal), primary thymus dependent and long-lived.

Question 27

B cell development in germinal centers

Answer 27

Ig hypermutation–if produce a BCR that no longer binds antigen it is removed for fear of being able to bind self.

Bcl-2 is a key protein that allows B cells to be long lived

***Look up/review-not much on slide

Question 28

Development of T cells

Answer 28

Development takes about 3 wks. Early thymocyte development takes place in the thymic cortex. During this period they are DN and then at the end turn into DP then go through positive selection then move to medulla and go through negative selection before entering blood and peripheral tissues.

NOTCH a transcription factor produced by thymic stromal cells commit T cells to the lineage and w/o you would have no T cell differentiation.

Question 29

Double negative thymocytes

Answer 29

DN1
C-kit (CD117)++, CD44+ (required for localization to thymus), CD25- / located in the bone marrow to thymus / migration to thymus

DN2
C-kit (CD117)++, CD44+, CD25+ / located sub capsular cortex / TCR gamma, delta, beta chain rearrangement, T-cell lineage commitment

DN3
C-kit (CD117)+, CD44-, CD25+ / sub capsular cortex / expression of pre-TCR, Beta selection

DN4
C-kit (CD117)low/-, CD44-, CD25- / sub capsular cortex to cortex / proliferation, allelic exclusion of beta chain, alpha chain locus rearrangement begins, becomes DP thymocyte

As transition from DN to DP the alpha chain matures.

Question 30

Signals required for activation of naive T cells

Answer 30

1. TCR/MHC
2. Costimulatory interaction (B7/CD28)
3. Cytokine signaling (e.g. APC paracrine releasing IL-12, T cell autocrine releasing IL-2)

IL-2 switches from low to high affinity receptor (keeps it in check so don’t activate to trace amounts randomly in body). This happens after 24 hours. IL-2 expression increased 100 fold.

Question 31

Clonal anergy

Answer 31

If T cell becomes APC and TCR/MHC bind but not B7/CD28 the cell becomes desensitized (random activation could have taken place w/ self) to activation and ultimately dies.

Question 32

Superantigens

Answer 32

Recognizes component of Beta chain TCR and alpha chain MHC. When coupled also brings in CD28 (no B7 on APC needed for activation). Results in non-specific T cell activation and cytokine storm.

Question 33

Th1/Th2 polarizing factors

Answer 33

Th1

• Cell-mediated immunity. Most viruses, some bacteria and fungi
• IL-12
• T-Bet
• IFN-gamma

Th2

• Humoral immunity particularly extracellular parasites
• IL-4
• GATA-3
• IL-4, IL-5, IL-13

Question 34

Induced Treg regulation and function

Answer 34

Polarizing cytokines–IL-2, TGF-beta

Master transcriptional regulator–FOXP3

Effector cytokines–IL-10 TGF-beta

Question 35

Th 17 regulation and function

Answer 35

Polarizing cytokines–IL-1, IL-6, IL-23, TGF-beta

Master transcriptional regulator–RORgamma

Effector cytokines–IL-17A, IL-17F, IL-22

Question 36

Th2 regulation and function

Answer 36

Polarizing cytokines–IL-4

Master transcriptional regulator–GATA3

Effector cytokines–IL-4, IL-5, IL-13

Question 37

Tfh regulation and function

Answer 37

Polarizing cytokines–IL-6, IL-21

Master transcriptional regulator–Bcl-6

Effector cytokines–IL-4, IL-21

Question 38

Th1 regulation and function

Answer 38

Polarizing cytokines–IL-12, IFN-gamma, IL-18

Master transcriptional regulator–T-Bet

Effector cytokines–IFN-gamma, TNF