Antibodies and B-cells Flashcards
B-cell development in adults
bone marrow
b-cell development in fetus?
yolk sac and liver
6 phases of B-cell selection
- Making competent B-cells in bone marrow
- Negative selection
- Positive selection
- Searching for infection
- Finding infection
- Attacking infection
Markers on stem cell–> pro-B-cell
Stem cell= CD34
Lymphoid Progenitor= CD34, CD7, CD10,
B-Cell precursor= IL-7Ra, CD10
Induction of B-cell development
Stromal cells in bone marrow release IL-7
B-cell first makes?
heavy chain of antibody
Heavy chain production
Early pro-B cell= DJ recombination on BOTH chromosomes
Late Pro-B= V-DJ recombination one one chromosome. Success= inhibition of other segment.
50% cells undergo apoptosis
How many chances for IgH rearrangement?
2
Pro B cells vs Pre-B cell?
Heavy chain of antibody (mu antibody) is formed in Pre-B-cell. Associates with surrogate light chain
Pre-B cell needs to?
make light chain to become Immature B-cell
Light chain rearrangement
Kappa chain rearrangement (tried on both chromosomes) followed by lambda if both kappas fail
90% of cells undergo apoptosis here.
Allelic exclusion
Occurs in pro and pre-b-cells (heavy and light chain)
makes sure Ab only have one specificity. (two heavy chains don’t pair together). If one allele successfully does recombination, it prevents the rearrangement of the other alleles.
Difference between heavy and light chain rearrangement
heavy chain rearrangement has the diversity region, limiting number of recombination attempts. In light chain synthesis, each allele kappa and lambda get 5 tries before they die.
Pro-B-cell–> pre b-cell
pre b-cell ==> immature b-cell
addition of heavy chain (with temporary surrogate chain)
addition of light chain
Immature B-cells undergo
negative selection before they can leave the bone marrow.
Negative selection
Immature B-cells with self-reactive antibodies to MULTIVALENT self antigens cannot leave bone marrow. Light chain rearrangement starts up again= RECEPTOR EDITING (assuming it has more combinations). If it can’t figure it out, apoptosis.
Anergy
Self reactive B-cells to MONOVALENT antigens express high IgD and lower IgM so they’re useless. They leave the bone marrow, circulate, but quickly die.
Naive B-cells need?
activation by antigen. Go to lymph node via HEV.
How do B-cells get into the lymph nodes?
CCL21 attracts b-cell to HEV
CCL21 and CCL19 attract b-cell into lymph node
CCL13 into primary follicle
Immature B-cell to mature B-cell?
In the primary follicle (of 2 lymphoid tissue), follicular dendritic cells secrete BAFF= b-cell activating factor. BAFF binds b-cell and makes it mature. Still not active.
Path of B-cell through lymph node?
enters afferent blood vessel==> HEV in paracortex==> moves to outer part (primary follicle) and interacts with follicular d-cell. Mature B-cell exits via efferent blood vessel and circulates
B-1 Cells
Make in the fetal bone marrow. Pretty weird, not much is understood about them. Part of early response= considered part of innate immune system.
B1-B2 cells reaction with antigens?
B1= poly reactive- not specific to one antigen. High chance of cross reactivity. B2= somatic hypermutation makes them very specific.
Memory cells?
B2. B2= “classical” b-cells= memory or plasma.
create many lymphomas and leukemias
B1. Do not need T- cell activation.
IgM vs IgG on B1/B2?
B1= IgM»IgG B2= IgG»IgM
Primary location of B1/B2?
B1= peritoneal and pleural cavities B2= Secondary lymph organs
B-cell tumors in bone marrow
Lymphoid progenitor= Acute lymphocytic leukemia
Pre-B-cell= pre-b-cell leukemia
plasma cell= multiple myeloma
Peripheral B-cell tumors
Germinal center b-cell= Hodgkin’s lymphoma–found in lymph organs
B-cell activation requires. Where does this happen?
Occurs in lymph node
- Finds antigen (presented by follicular dendritic cell)
- finds T-cell
B-cell: antigen interaction
Ag binding
Bcell CR2: C3d Ag =phosphorylation of CD19==downstream signaling.
B-cell CD19 and CD81 mediate interaction
Cytoplasmic signaling into b-cell?
Ig-alpha and Ig-beta have longer cytoplasmic domains that participate in downstream signaling once IgM has bound antigen.
purpose of CR2 on B-Cell
lowers threshold for antigen so that fewer IgM molecules need to bind. . binds C3D.
Steps of B-cell activation (from inactive mature B-cell)
Proliferation, somative hypermutation & affinity maturation, isotype switching, plasma/b-cell development
Proliferation of B-cells occurs?
In germinal centers of lymphoid follicles (activated b-cell proliferation)
germinal center mantle zone has
non-activated b-cells
After B-cell finds antigen from DC in primary follicle, it?
goes into T-cell area to find a t-cell match. Then they go into the medullary cords for proliferation. Then they move into the primary follicle for expansion= germinal center.
Follicular dendritic cells have two crucial jobs
- Release of BAFF to make mature B-cells
2. Activate B2 cells (antigen specificity)
Follicular Dendritic cell lineage
fibroblast
After proliferation, B-cells undergo…
somatic hypermutation
Somatic hypermutation & affinity maturation
Somatic hypermutation of hypervariable region of new centrocytes changes antigen specificity. If Ab can still bind dendritic cell, it gets survival factors. If it can’t it dies.
B-cell: t-cell interaction?
B-cell part :: T-cell part (another name because it wouldn’t be medicine if things only had one name)
CD40 (BCR):: CD40L (T-cell ligand)
MHC II (with Ag) :: TCR (CD3)
B7(CD 80):: CD28
What happens after somatic hypermutation?
Isotype switching. dependent on cytokine signaling.
IFN-gamma= IgG2a, IgG3
TGFb/IL5= IgA, IgG2b
IL-4= IgE, IgG1
IL-4 secreted by?
TH2 cell
Centrocyte (b-cell)==> plasma cell requires
IL-10. Plasma cells then go to the bone marrow
Centrocyte + ___ = memory b-cells
IL-4 (TH2 cells)
TI-1 antigen
T-cell independent antigen. Polyclonal activation of B lymphocytes.
Ex: LPS (binds TLR4)
DNA= TLR9
TI-2 antigen
repeated epitopes cause significant cross linking = B-cell activation. Antigen (polysaccharide) is not processed. No CD4 activation. Can activate B1 and B2 cells.
Can we make vaccines to TI2 antigens?
Yes! Stick polysaccharide on a protein. B-cell specific for polysaccharide so that the B-cell processes it. Antigen (polysaccharide + protein) is broken down and polysaccharide is broken down and presented to T-cell= => activation of B-cell ==> formation of plasma cell that makes Ab for polysaccharide
How does IgA reach destination?
IgA= mucosal, must cross epithelium.
Polymeric Ig receptor transcytosis into mucus with or without IgA. Since it’s polymeric it can take IgM but usually IgA»>IgM. IgM + secretory component (the receptor) secreted into mucus together.
IgG transport
IgG= into extravascular space (across endothelial cells), into fetus (across placenta)
Brambell Receptor= FcRB
IgA types
IgA1= 26AA. IgA2= 13 AA= less flexible, but more stable to proteases
Dimeric IgA
Secretory component covers hinge to protect from proteolytic degradation. J-chain connects two IgA’s.
IgA1 vs IgA2 (location, structure)
IgA1= longer hinge region. More flexible, more susceptible to proteolytic degradation. More common in upper GI and Respiratory tract where there’s less bacteria.
IgA2= shorter hinge. More common in colon because can tolerate high levels of bacteria.
Brambell receptor
= FcRB. Moves IgG into ECM. Active transport.
First Ab fetus makes? Ab levels at 0 and 9 months?
IgM.
0 months= fetal IgM= maternal IgG»_space; fetal IgG
9 months= fetal IgM>IgG> IgA. no more maternal IgG
Purpose/Functions of Antibodies
- Neutralization
- Opsonization
- Complement Activation
Neutralization
Ab binds antigen to prevent it binding to host cell.
Ex: IgA binds influenza.
Bacterial toxins
AB receptor on antigen
A- active
B- binding.
B binding allows A to poison cell.
Neutralization inhibits B-binding to host cell.
Fc Receptors
Link innate and adaptive immunity. Innate immune cells express Fc receptors in order for adaptic immune cells to bind.
Fc(gamma)R- binds what? found on which cell(s)? plays a role in?
Bind IgG
Expressed on neutrophils, macrophages, B-cells, follicular cells, Dendritic cells and NK cells
Plays a role in: opsonization, NK ADCC
Fc(epsilon)R- binds what? found where? plays a role in?
Binds IgE
Found on mast cells and basophils. Plays a role in allergic reactions. Binding==> cross linking==>degranulation.
Fc(gamma)R1
high affinity receptor for IgG1 and IgG3
Receptor on macrophage binds CH2 domains of the IgG ab==>activation of phagocytosis.
Main Fc on neutrophils and macrophages? NK cells?
Neutrophils and macrophages= Fc(gamma)RI (CD64)
NK cells= Fc(gamma)RIII (CD16)
This IgG plays an anti-inflammatory role. Ratio of these two is what determines outcome of allergic response.
IgG4: IgE
NK Antibody-dependent cell- mediated cytotoxicity
Fc(gamma) RIII= CD16 binds antibody bound to antigen.
Ex: Lymphoma has CD20 R, which binds Ab Anti-CD20. NK cell sees AntiCD20 and binds with its Fc(gamma)III R and elicits cell death.
IgG with greatest Fc binding?
IgG in greatest concentration?
IgG3- longest hinge= greatest flexibility.1/2 life= 7 days
IgG1- most prevalent, 21 days
Primary immune response produces?
low affinity IgM Ab
Secondary immune response produces?
IgM+ naive B-cell that binds antigen is inhibited by Memory B-cell for that antigen. Therefore, secondary immune response results in production of high affinity IgA, IgG, and IgE. No time wasted on affinity maturation and stuff.
B-lymphocytes cytokines in the bone marrow
IL 3,4,7
Total surface receptors of a mature B-cell
IgM, IgD, class II MHC, CR1, CR2, Fc, CD19, CD20
When macrophages secrete IL10…
TH2 response= B-cell proliferation, anti inflammatory response.
What regulates light and heavy chain recombination?
RSS= recognition signal sequence. 12 bp space must combine with 23 bp spacer. 12= V 23= J
What does RAG do
during recombination, RAG bring V+J together.
RAG recombinase cuts out the middle part and joins VJ
Diversity is added by?
TdT= N-nucleotide addition. Random addition of bases.
switching between IgM and IgD
= RNA splicing. VDJ regions remain the same so the antigen specificity is unaltered.
Membrane vs secreted IgM from?
alternative splicing
Alternative splicing does 2 things
- IgM vs IgD
2. transmembrane Ab vs secreted Ab
Somatic hypermutation
AID (activation induced cytosine deaminase) adds uracil randomly. Uracil is a mistake (working with DNA) so it is replaced randomly with another base.
Hypervariability regions
regions of IgM/IgD light and heavy chains with high variability. 2 weeks after immunization, CDR1 has highest variation.
CDR1, CDR2, CDR3= hypervariability regions. Determine Ag binding specificity.
AID mediates 2 things
Activation induced cytosine deaminase.
- Somatic hypermutation
- Isotype switching
Isotype switching AID
AID targets switch regions and results in new Ab production
IgM structure/Function (4)
secreted as pentamer (usually). No hinge region, 4HC domains
- Most important mucosal Ab.
- First Ab made during primary immune response
- Activation of compliment cascade
- Agglutination
How does IgM make up for it’s low affinity to antigens?
Pentamer= lots of binding spots= higher avidity
IgG structure/ Function
Four subclasses, Activate complement (IgG3>1>2) NOT IgG4! Opsonization via Fc receptor binding
IgG subclass structure comparisons
IgG3= longest hinge, highest flexibility, increased susceptibility to proteases= shortest 1/2 life
Monovalent IgG? What induces this?
IL-4
Monovalent= binding only ONE antigen.
IgG4- can dissociate at hinge, and combine with another IgG4 with different antigen specificity, thereby reducing the ability of IgG4 to bind with two antigens. Decreased effectivity= dampened immune response= ANTI INFLAMMATORY
Bivalent antibody
All of them. Except IgG4. And IgM is supervalent (pentamer= 10 binding spots)
IgA– what cytokine promotes IgA
- Structure
- Functions
- Weak to? (ex of pathogens and why it’s weak)
IL-5
- dimer w/ J chain that increases stability
- Does not activate compliment effectively. Works to neutralize bacteria in intestinal and respiratory tract by cross-linking to prevent their entry.
- S. Pneumonia, Hemophilus influenza, m. meningtiidis make proteases that cleave IgA. (ie resistant to Iga)
IgE
- Normal serum concentration
- Structure
- Function
IL-4
- Very low in normal people. Marker for allergies (US) and parasites (aka HELMINTH–in 3rd world countries)
- Extra CH (constant heavy domain) and no hinge region
- Binds FcEpsilon receptor on mast cells and basophils to induce cross linking and degranulation= release of inflammatory mediators.
IgD
- Serum concentration
- Structure
- Main location
- Very low
- same as IgG, 2 heavy chains (3Hc, 1Hv) 1 light chain. Hinge present
- IgD secreting plasma cells in URT. Function isn’t really known because knockout mice don’t show symptoms. Assumed to help protect URT, though.
Heaviest Ig
Most prevalent Ig
Heaviest: IgM- pentamer= 970kDa
Highest concentration: IgG1
Best antibodies for neutralization?
IgG1,2,3,4 and IgA
Best Ab for opsonization?
IgG1, IgG3
Activators of NK cells? (ab)
IgG1, IgG3
Activation of mast cells? (which Ab)
IgE
Activation of complement system? (which Ab)
IgM, IgG1, IgG3
Epithelial transport of Ab?
IgA»>IgM
Placental transport of Ab?
IgG1> IgG3, IgG4»IgG2
Diffusion into tissue (extravascular sites)? (Ab)
IgG’s>IgA>IgE»_space;>IgM (depends on if it’s a pentamer)
Pentamer IgM cannot diffuse
FYI: Air can, from zone 17-23
Ig Genes are rearranged a lot during a B-cells life cycle….What mechanisms are reversible?
- IgM IgD switching- Why? It’s via RNA splicing, not DNA
2. Secreted vs surface Ab on B-cell- again, RNA splicing, not DNA recombination
What promotes IgG1/IgG3 class switching?
IFN-gamma
How does B-cell down regulate T-cell activity
This is from core:
B7 (CD80/86):: CTLA-4
No idea what all that really means…
