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Flashcards in B cells and antibody production Deck (49):
1

whats the basic structure of Ab?

- 2 identical light chains Constant and variable domains (VLCL). Encoded by rearranging variable, joining and constant elements (VLJLCL)
- 2 identical heavy chains Constant and variable domains (CHVH). Encoded by rearranging variable, joining, diversity and constant elements (VHDHJHCH)


2

what are antibodies?

Soluble plasma proteins
Monomers, dimers or pentamers
ISOTYPE - IgA IgD IgE IgG IgM
Effector functions: fix complement - neutralise - block - opsonise - interact with cells etc.

3

what are B cell antigen receptors?

- Cell surface-bound Ig of any isotype.
- Only found on B cells
- Always a monomer
- No effector functions
- Senses the antigenic environment of the B cell
- Connects the extracellular environment with signalling pathways
- Activates B cell effector function
- Only one antigen specificity per B cell

4

how are Immunoglobulins bifunctional?

- Conserved enough to interact with small number of specialised molecules.
- Allow changes to interactions during an immune response.
- Allow extensive variation between antibodies to match the infinite number of potential antigens but retain specificity throughout the immune response
- requires the protein to be both conserved and infinitely variable

5

what is the Immunoglobulin fold?

- a β barrel of 7 (CL) or 8 (VL) strands connected by loops and held together with a disulphide bond

6

The domains all look the same how can domain structure account for bifunctionality?

Examine genes encoding VH & VL domains

7

how does Organisation of Ig VH domain genes Maximise diverse specificity?

1. COMBINATORIAL DIVERSITY - VH1 domains made from any combination of V gene with a D gene and a J gene
2. JUNCTIONAL DIVERSITY - Imprecise breaking and joining of DNA when V, D and J genes recombine

8

how is the Ig heavy chain gene rearranged?

- RNA splicing joins the CH domains.
- Imprecise breaking and rejoining of DNA between the V and D genes and the D and J genes creates diversity of sequence and specificity

9

How does diversity arise from Ig light chain gene rearrangements (κ and λ)


1. any V gene being able to join to any J gene
2. deliberate errors in breaking and rejoining DNA
3. insertion of new nucleotides between the join

10

what are Hypervariable sequences in Ig?

1. COMPLEMENTARITY DETERMINING REGIONS (CDRs)
Distinct regions of high variability suggested region of antibody interacted with antigens.
2. More conserved parts acted as a FRAMEWORK (FR), on to which the hypervariable regions were suspended

11

how does Organisation of Ig CH chain genes Maximise effector function?

- the ability to switch to, one of several isotype-defining C genes
- Each CH chain domain is encoded by a separate exon

12

Summarise Bifunctionality of antibodies explained
by domain structure

- Fc- Structurally conserved end Effector functions
- Fab- Structurally diverse end Antigen recognition
- Light chain C domains κ or λ
- Heavy chain V domain
- Heavy chain C domains 3 x α, δ, γ, or 4 x μ, ε
- Papain cleavage sites- 2 x Fab 1 x Fc
- Domain structure allows diverse specificity with conserved Fc-dependent effector function





13

Why are they called ‘B’ cells?

1954 - Bruce Glick, Ohio State University

Functional studies of the bursa of Fabricius - a lymphoid organ in the cloacal region of birds

14

what was Bruce Glick's experiment?

- Bursectomy – no apparent effect
- Bursectomised chickens re-used to raise anti-Salmonella antibodies
- Bursectomised chickens did not make anti-Salmonella antibodies
- The Bursa therefore the organ which antibody producing cells developed
- No bursa of Fabricius in mammals - bone marrow

15

how is Bone Marrow a maturation & differentiation microenvironment for B cells

- Regulates construction of antigen receptors
- Ensures each cell has one specificity
- Checks and disposes of self-reactive B cells
- Exports useful cells to the periphery
- Is a site of antibody production

16

how do Stromal cells nurture developing B cells?

1. Contact between stromal cells and developing B cells
2. Secrete CYTOKINES at each stage of differentiation

17

how do Cytokines and cell-cell interactions regulate differentiation?

Different cytokines and cell-cell contacts are required at each stage of differentiation






18

how is The stage of differentiation is defined by Ig gene rearrangement

Stem cell- Germline unrearranged
Early pro-B- DH to JH
Late pro-B- VH to DHJH
Large pre-B- VHDHJH, PRE-B CELL RECEPTOR expressed





19

what are the Consequences of pre-B cell receptor ligation?

1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
3. Ensures only one specificty of Ab expressed per cell
4. Expands only the pre-B cells with in frame VHDHJH joins

20

what is ALLELIC EXCLUSION

Expression of a gene on one chromosome prevents expression of the allele on the second chromosome

21

what is the Evidence for allelic exclusion of Ig genes?

1. one allotype is inherited from each parent
2. Allotypes identified by staining B cell surface Ig with antibodies
3. Suppression of IgH gene rearrangement after pre-B cell receptor ligation prevents two specificities of antibody per B cell

22

why is Allelic exclusion important?

prevents autoimmunity
Suppression of IgH gene rearrangement ensures only one specificity of Ab per cell –may prevent pathogens provoking undesirable responses

23

why is allelic exclusion needed to prevent ‘holes in the repertoire?

1. Two specificities of Ag receptor per cell – anti-brain Ig AND anti S. aureus
2. Infection with S. aureus due to self tolerance
3. ‘Hole’ in the repertoire of B cell specificities allows infection to proceed unchecked.

24

what are the Consequences of pre-B cell receptor ligation?

1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
3. Ensures only one specificty of Ab exparessed per cell
4. Expands only the pre-B cells with in frame VHDHJH joins


25

why are Large pre-B cells need in frame VHDHJH joins to mature?

Only B cells with potential to make a B cell receptor can pass this stage

26

why is Heavy and light chain rearrangement wasteful?

- Two “random” joins – only a 1:9 chance of a rearrangement being in frame
- One “random” join only a 1:3 chance of a rearrangement being of frame
- Only a 1:27 chance of an in frame rearrangement per B cell
- Out of frame rearrangements arrest further B cell maturation

27

how do B cells have more than one chance to
rearrange heavy chains

1. DH-JH or VH-DJH On first chromosome 14
2. DH-JH or VH-DJH On second chromosome 14


28

why is Heavy and light chain rearrangement wasteful?


- Only a 1:27 chance of an in frame rearrangement per B cell
- Out of frame rearrangements arrest further B cell maturation

29

what are Lymph nodes

A secondary lymphoid organ in which lymphocytes and antigens encounter each other

30

describe Lymphocyte recirculation through 2° lymphoid tissue

Non-lymphoid cells
Pass through the blood vessels in the lymph node and continue arterio-venous circulation
Lymphoid cells
Adhere to and squeeze between HIGH ENDOTHELIAL VENULES (HEV), then percolate through the lymph node and exit via the efferent lymphatic vessel

31

how do Cells with antigen receptors check for recognition of self antigens

EXPORT
DELETION
ANERGY
STALLED MATURATION
RECEPTOR EDITING

32

explain Germinal Centre formation

1. Antigen drains in lymph and on cells into afferent lymphatic.
2. Ag activated B cells increase CCR7 expression and move to CCL19/21 rich T/B zone border
3. GERMINAL CENTRE forms – a transient structure of intense proliferation
4. B cells released by germinal centre differentiate into plasma & memory cells
5. Mature B cells leave blood via
HEV and home to the B cell follicles - CXCL13/CXCR5 dependent

33

whats the difference between Follicular dendritic cells
and DCs?

FDC- Probably develop from mesenchymal precursors
DC- Bone marrow derived
DC are the only antigen presenting cell that can present antigen to naïve (as opposed to memory) T cells.
DC are crucial to establishing the primary immune response

34

explain Germinal Centre formation

1. Antigen drains in lymph and on cells into afferent lymphatic.
2. Ag activated B cells increase CCR7 expression and move to CCL19/21 rich T/B zone border
3. GERMINAL CENTRE forms
4. B cells released by germinal centre differentiate into plasma & memory cells
5. Mature B cells leave blood via HEV and home to the B cell follicles - CXCL13/CXCR5 dependent

35

describe FDC bead formation & release

- Veils on antigen-bearing DC surround the FDC beads.
- Immune complexes transferred from DC to FDC beads.
- B cells also involved in transport of immune complexes to FDC
- Immune complexes bind to and are taken up by B cell
surface immunoglobulin

36

explain Germinal Centre formation

1. Antigen drains in lymph
2. Ag activated B cells and move to T/B zone border
3. GERMINAL CENTRE forms
4. B cells released by germinal centre differentiate into plasma & memory cells
5. Mature B cells leave blood via HEV and home to the B cell follicles

37

describe FDC bead formation & release

- antigen-bearing DC surround FDC beads.
- Immune complexes transferred from DC to FDC beads.
- B cells also involved in transport of immune complexes to FDC
- Immune complexes bind to and are taken up by B cell
surface immunoglobulin

38

how do T cells help B cells survive?

1. Signal 1 antigen & antigen receptor
Signal 2 delivered via CD40 and cytokines in the germinal centre

2. B cells are inherently prone to die by apoptosis

3. Signals 1 & 2 upregulate Bcl-XL, which rescues B cells from apoptosis

4. Signal 2 from T cells allows the B cell to survive
5. T cells control the CLONAL SELECTION of B cells

39

where do T cells control B cell clonal selection?

germinal centre

40

what is affinity maturation?

1. Cells accumulate mutations in their CDR
2. are selected or neglected on the basis of antigen binding affinity
3. affinity ‘matures’ throughout the response

41

how do T helper cells control affinity maturation?

1. if a cell has a higher affinity for antigen, it will
-bind more antigen (receive more signal 1)
-present more antigen to T cells (solicit and receive more signal 2)
-express more CD40 (receive more signal 2)
-express more cytokine receptors (receive more signal 2)

2. Receipt of more signal 1 and 2 gives the cell a selective advantage i.e. it is rescued from apoptosis and is clonally selected.

3. Cells with lower affinity receptors are neglected and die by apoptosis

42

how do T cells help B cells survive?

T cells control CLONAL SELECTION of B cells
1. Signal 1 antigen & antigen receptor
Signal 2 delivered via CD40 and cytokines in the germinal centre

2. B cells are inherently prone to die by apoptosis

3. Signals 1 & 2 upregulate Bcl-XL, which rescues B cells from apoptosis

4. Signal 2 from T cells allows the B cell to survive

43

how do T helper cells control antibody isotype switching in the germinal centre?

1. Diverse effector function arises from ability to switch isotype-defining C genes

2. Class Switch Recombination (CSR) is T cell dependent and takes place in the germinal centre

44

what are Switch regions?

Repetitive regions of DNA upstream of C regions

45

how do switch regions work?


1. Switching similar to V(D)J recombination.

2. Occurs after B cell activation by antigen and help from T cells in germinal centres

46

what is Switch recombination?

At each recombination constant regions are deleted from the genome
An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1

47

what is an ALLOTyPE?

a polymorphism in a conserved region of Ig

48

what are High endothelial venules?

specialised properties to allow lymphocytes and no other types of cells into the lymph node

49

what is the GERMINAL CENTRE?

a transient structure of intense proliferation