B cells structure, signalling and repertoire diversity Flashcards

Question

Tell me about the primary and secondary protective antibody response and how they differ in kinetics, magnitude and isotype

Answer 1

The primary response is slower, peaking at 7-14 days compared to 3-5 days in the secondary response Weaker (less antibody is produced) and is characterised by IgM antibodies compared to IgG antibodies, or other class-switched isotypes in the secondary response

Answer 2

BCR cross-linking is required to generate a signal

Answer 3

The Src family kinases Lyn, Blk and Fyn as well as the Syk and Blk tyrosine kinases

Answer 4

1. The **maturation** state of the cell (e.g., immature, mature). 2. The **nature of the antige**n (e.g., size, valence, solubility, foreign or self). 3. The **magnitude and duration of BCR signalling** (influenced by the affinity of antigen binding). 4. **Signals from other receptors** (e.g.CD40, TLR, IL-21 receptor, BAFF-R, CD45, CD19, CD22 and FcγRIIB1) are not parallel, separate pathways. They crosstalk.

Answer 5

A state of unresponsiveness to self-antigens

Answer 6

Tolerance refers to the mechanisms that control self/non-self-discrimination which protect an individual from anti-self-immune attack by self-reactive lymphocytes.

Answer 7

Autoimmune diseases involve the failure of the elimination and inhibition mechanisms that maintain self-tolerance by the immune system. Autoimmune disease is characterized by the destruction of host cells by autoantibodies and/or self-reactive T cells.

Answer 8

In both the primary and secondary lymphoid tissue

Answer 9

In the bone marrow

Answer 10

* **clonal deletion** * **receptor editing** * **Anergy (non-responsiveness)**

Answer 11

Clonal deletion of immature high-affinity self-reactive B cells by apoptosis if self-antigen is multivalent and immobilised (e.g., expressed on cell membrane)

Answer 12

Receptor editing: a different light chain pairs with the heavy chain in immature B cells to eliminate self-specificity when the self-antigen is multivalent and cross-links BCR. **Predominant mechanism**

Answer 13

Anergy (non-responsive): self-reactive immature B cells exposed to large amounts of soluble, weakly cross linking, low valence self-antigen in the bone marrow migrate to the periphery where they are anergic However, not all self-antigens are represented in bone marrow (e.g., post-transitionally modified proteins found only in the periphery or are tissue specific molecules)

Answer 14

1. Clonal deletion by apoptosis if the antigen is strongly cross-linking. 2. Anergy- cell survival but unresponsive to soluble antigen. 3. Receptor editing- L chain and rare IGHV rearrangement. 4. Physical separation of cells from antigen prevents cellular activation 5. Generation of regulatory cells: * B-cell suppression via Treg effects on T cells * B-cell suppression via Breg cells (IL-10 and TGF-beta production)

Answer 15

If mature peripheral B cells encounter multivalent self-antigen in the absence of T cell help, they do not receive co-stimulatory signals and fail to enter the primary follicle and become trapped in the T cell zone and die by apoptosis.

Answer 16

if mature peripheral B cells recognize soluble self-antigen in the absence of T cell help, they produce few surface IgM receptors and become anergic. They express Fas and are killed by Fas-L expressing CD4+ T cells. This is called activation-induced cell death (AICD).

Answer 17

* Healthy individuals have mature, recirculating self-reactive lymphocytes. * 55% of bone marrow B cells were found to be polyreactive/autospecific. * 24% of naïve B cell antibody specificities in the peripheral blood were found to be polyreactive/autoreactive. * 7% of circulating naïve B cells are anergic, an indication that they are possibly self-reactive. * \>50% of IgD antibodies were found to be autoreactive. * 31% of IgG memory cell antibodies were weakly autoreactive. Most autoreactivity in this subset is due to somatic hypermutations.

Answer 18

Autoimmunity occurs in ~3-8% of populations in developed countries (e.g., cold agglutinin disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus and certain types of diabetes). Autoimmunity is the result of the combination of genetic predisposition and environment. The physical nature of the self-antigen (soluble vs membrane bound, size, valence, affinity) is an important determinant of outcome.

Answer 19

* Plasma membrane * Cytoplasm * Nucleus

Answer 20

Pre-existing inactive molecules are activated by **phosphorylation.** There is a balance between phosphorylation (kinase activation) and **dephosphorylation** (phosphatase activation).

Answer 21

**Cytoplasm** * Phosphorylation cascade * Calcium release * Inactive transcription factors are activated. * Activated factors translocate into the nucleus.

Answer 22

Alteration of transcriptional programme.

Answer 23

Increased expression of BCR associated kinases is associated with a shorter treatment-free interval in some diseases. Signaling therefore is a therapeutic target in some diseases.

Answer 24

Immunosuppressive drugs (small molecule compounds). Immunotherapy (monoclonal antibodies).

Answer 25

**SYK inhibitors** SYK-mediated B-cell receptor signalling inhibitors have shown positive results in the treatment of allergy, autoimmune diseases and B cell lineage malignancies.

Answer 26

**BTK inhibitors** Ibrutinib- drug

Answer 27

**Inhibitors of calcineurin-dependent activation of NFAT** Cyclosporin A: Inhibition of B cell proliferation following ligation of surface Ig Tacrolimus: Induction of apoptosis following B-cell activation

Answer 28

**ERK pathway inhibitors** 1. The ERK pathway is deregulated in approximately one-third of all human cancers. 2. The ERK pathway has been a focus for drug discovery for almost 15 years with Ras, Raf and MEK as the main targets.

Answer 29

1. Btk is a cytoplasmic tyrosine kinase required for normal B cell differentiation. 2. Btk is activated following signalling through the BCR and pre-BCR. 3. Btk is located on the inner side of the plasma membrane where it is phosphorylated. 4. Btk phosphorylates PLCg2 leading to calcium flux and activation of the NF-kB and NFAT pathways. 5. Ibrutinib is a small molecule drug that binds and inactivates BTK. It is the most broadly used inhibitor in B cell lymphoma (CLL, MCL, MZL, ABC-DLBCL)

Answer 30

**Cytoplasm:** 1. Ras is a monomeric G protein which is inactive when bound to GDP. Ras-GDP (inactive). 2. DAG recruits RasGRP (guanine nucleotide releasing protein) to membrane where it activates Ras. Ras-GTP (active) 3. Ras-GTP binds Raf (a MAP kinase kinase kinase). MAP = Mitogen Activated Protein 4. Activated Raf binds to MEK (a MAP kinase kinase). 5. Activated MEK phosphorylates ERK (a MAP kinase). **Nucleus:** 6. Phosphorylated ERK enters the nucleus. 7. The transcription factor AP-1 is induced leading to transcriptional activation of several genes

Answer 31

* **Immunoglobulins are made only by B cells.** * **Immunoglobulins are bivalent having a “Y” shaped structure.** * **The BCR is made up of surface immunoglobulins which binds cognate antigen and the CD79a/b component which sends intracellular signals to the cell nucleus.** * **BCR signalling pathways are therapeutic targets.**

Answer 32

* **The genetic basis of antibody variable region diversity** * **The genetic mechanisms involved in class (isotype) switching** * **The mechanisms of variable region somatic hypermutation**

Answer 33

Humans have **23 pairs** of chromosomes (46 chromosomes in total). Three pairs of chromosomes are involved in antibody production: 1. **Chromosome 14- heavy chain locus** 2. **Chromosome 2- kappa chain locus** 3. **Chromosome 22- lambda chain locus**

Answer 34

There are approximately 20,000 genes in a human B cell. Approximately 10¹² different immunoglobulins can be generated by the B cell population at any one time.

Answer 35

Because the DNA encoding antibody polypeptide chains is arranged as separated gene segments which recombine.

Answer 36

**In the bone marrow** in the absence of antigen: 1. Heavy chains rearrange first (one chromosome 14 then the other if the first rearrangement is not productive) 2. Light chain rearrangement takes place next (usually kappa followed by lambda). **IGHD --\> IGHJ, IGHV --\> IGHD-IGHJ = heavy chain** **IGKV --\> IGKJ (kappa before lambda) = light chain** **B cells are an accident waiting to happen because the DNA is cut into fragments and pasted together**

Answer 37

**Combinatorial diversity (1): gene segment selection** There is a reservoir of multiple V(D)J gene segments that are randomly assembled. **Combinatorial diversity (2): H and L chain pairs** The association of different variable heavy and light chains to form antibody recognition sites.

Answer 38

**Each recombination signal sequence (RSS) consists of:** **1.Heptamer- 7 bp 5’CACAGTG3’ (well conserved).** The palindromic heptamer has at least two roles: i) enhances binding of the recombination activating proteins (RAG) to the RSS ii) specifies the site of DNA cleavage. The first three nucleotides of the heptamer (5′-CAC3’) are particularly important. **2.Spacer- 12±1nt or 23± nt.** The spacer is variable in sequence. Spacer lengths are important. The primary role of the spacer is to properly align the heptamer and nonamer. **3.Nonamer- 9 bp 5’ACAAAAACC3’ (conserved).** The A (or T) rich nonamer is a sequence-specific binding site for the nonamer-binding domain of RAG1 and functions to anchor the RAG proteins to the DNA.

Answer 39

**The RAG1 core** contains a well-defined nonamer-binding domain and a heptamer-binding domain responsible for interactions with both the heptamer and RAG2. Zinc finger region B (ZnB) is thought to be important for RAG2 interactions. **The RAG2 core** is crucial for DNA cleavage activity. It interacts with RAG1 and enhances the DNA-binding affinity and specificity of the RAG complex, although RAG2 has little or no DNA binding activity on its own.

Answer 40

The dominant factor determining IGHV segment utilization is that RAG1/2 cleaves individual RSS with different efficiencies. IGHV4-34 (VH4 is from that family, 34 is the 34th from the constant region?) has the most efficient RSS of all human IGHV genes and hence is over-represented in the developing and naïve B cells (6-8% in healthy people). (Yu K et al. JBC 277:5040, 2002.)

Answer 41

RSS with 12 bp spacers (1 turn of DNA helix) usually pair with RSS with 23 bp (2 turns of DNA helix) spacers. This is known as the 12:23 rule.

Answer 42

1. that IGHV–IGHV rearrangements do not occur. 2. inclusion of the D gene segment. 3. VDJ joining is restricted to biologically productive events. 4. ensures that a pair of RSS control the activation of the catalytic activity of the recombination activating proteins (RAG) preventing RAG induced genomic instability.

Answer 43

Nucleotides are gained and lost at the junctions (junctional diversity). Approximately 2 out of 3 rearrangements are non-productive due to frameshifts. **Palindromic (P) nucleotides** are short inverted-repeat sequences resulting from uneven cleavage of dsDNA and filling in by DNA polymerase. Nucleolytic activity removes nucleotides. **Terminal deoxynucleotidyl transferase (TdT)** inserts non-templated nucleotides. Up to 20 nucleotides can be added. These sequences are not encoded in the germline. Enzymatic activity is the major source of **CDR3 diversity**. The CDR3 nucleotide sequence is characteristic of each B cell

Answer 44

1. **DNA recognition and cleavage** 2. **End processing and joining**

Answer 45

**1. DNA recognition and cleavage:** * Lymphocyte specific recombination activating proteins (RAG-1 and RAG-2) protein complexes randomly bind gene segments and bring together the gene segments to * be recombined and cut double-stranded breaks in * the DNA exactly at the junction of the * gene segment and its RSS. **2. End processing and joining** * Require in addition to RAG1/RAG2, general dsDNA repair enzymes of the non-homologous end joining (NHEJ) pathway (e.g. Artemis, TdT, DNA ligase IV).

Answer 46

involves error-prone DNA repair involves activation-induced cytidine deaminase (AID).

Answer 47

deletions (4-7%), duplications (1%) Frequency of mutation: 1 x10-3 per base pair per cell generation. (~106 more frequent than by spontaneous mutation).

Answer 48

**Mutation hotspots:** RGYW (R=A or G; Y=C or T; W=A or T). bCDR contain more hotspots than FR in the germline sequence. **Outcome:** Affinity maturation usually resulting in increased affinity of antigen binding due to antigen selection.

Answer 49

First discovered in **1999**. Is specifically induced in activated B cells. 198 amino acid protein that initiates both **somatic hypermutation** (SHM) and **class switch recombination** (CSR) by producing U:G mismatches in DNA. Different domains of AID are involved in SHM and CSR. AID catalyses the **deamination (-NH2) of cytidine to uridine** producing a U:G mismatch in DNA.

Answer 50

SHM and CSR are supported by a pathway involving uracil DNA glycosylase (ung) which removes uracil from DNA. The gapped DNA may be a substrate for error-prone repair.

Answer 51

Uracils are converted by UNG and apyrimidinic endonuclease (APE1) into single stranded nicks in the DNA on both strands

Answer 52

1. Generation of stop codons in in-frame sequences stops protein production. 2. Codon insertions/deletions (multiples of 3 nucleotides) which do not disrupt an in-frame sequence create diversity and increase affinity of Ag binding. 3. Insertions or deletions that are not multiples of 3 result in an out of frame sequence which generates a nonsense peptide. 4. Generating broadly neutralizing antibodies can be protective. (e.g., anti-HIV). 5. Generation of auto specific antibodies which can cause disease. 6. DNA damage during SHM and CSR may account for B cells being prone to malignant transformation (e.g., lymphoma).