Complement + Antigen receptors

Question 1

Which one of the 3 complement pathways is primarily dependent on antibodies for activation?

The two main complement-activating antibodies are ___, which is secreted 1st in an immune response, and ___, which responds to the majority of pathogens

Answer 1

Classical pathway - the antibody has to be bound to the microbe/bug

The two main complement-activating antibodies are IgM, which is secreted 1st in an immune response, and IgG, which responds to the majority of pathogens

Question 2

The 1st protein in the classical complement pathway is the C1 complex, which is comprised of C chains of the ___ protein, a collectin (what’s a collectin?)

What is the significance of the collagen tails and the globular heads of this collectin?

The two proteases in the middle of the complex are ___ and ___

All the molecules are held together by the ___,which allows them to be expressed in their inactive form

Answer 2

The 1st protein in the classical complement pathway is the C1 complex, which is comprised of C chains of the C1q protein, a collectin (colagen containing C-type lectin)

The collagen tails can bind to receptors on the surface of other cells and the globular heads can bind to specific sites on the antibody that’s bound to the bug

The two proteases in the middle of the complex are C1r and C1s

All the molecules are held together by the hinge region, which allows them to be expressed in their inactive form

Question 3

How is the C1 complex able to bind an antibody bound to a microbe?

Between IgG and IgM, which Ab has the strongest binding for the C1 complex? Which IgG has the strongest binding for the C1 complex?

Complement doesn’t get randomly activated by free floating antibodies because ___

Answer 3

When the ab is bound to the surface of the cell, it goes into the stable confirmation which exposes the C1q binding site >> C1 complex binding

IgM, because its a pentamer therefore it has multiple binding sites whereas IgG is a monomer so you’d need more copies to get more binding sites

Note that IgG3 has the strongest binding to C1 whereas IgG2 has the least (IgG1 is in the middle)

C1 complex binds to bound antibody on the bacteria (won’t bind to free floating antibody so you don’t randomly activate complement when the Ab is floating in the blood stream)

Question 4

Describe how C3 convertase forms

Answer 4

When the C1 complex binds, it forces the arms of the C1 arms apart, which puts strain on the C1r and C1s proteases and activates C1r

C1r can activate C1s >> C2 and C4 are clipped into their active forms >> C4b can bind to the surface of the cell and recruits C2a to form C3 convertase (C4 and C2 are cleaved by C1s)

Question 5

Describe the formation of the C5 convertase

What are the functions of C3a and C3b?

Answer 5

C3 convertase clips C3 into C3a and C3b, C3b binds to the cell surface on its own or in ass’n with C3 convertase which changes the specificity of C3 convertase to cleave C5 (thus, C3 convertase becomes C5 convertase upon binding of C3b)

C3b - opsonization; C3a - pro-inflammatory

Question 6

In the MBL pathway, which structure is analogous to the C1 complex of the classical pathway?

___ and ___ are analogous to C1r and C1s of the classical pathway

Answer 6

Mannose binding lectin (made in the liver) or ficolin

MASP 1 and MASP 2

Question 7

Describe the MBL complement activation pathway

Answer 7

MBL or MBficolin binds the mannose on the microbe>> MASP1 activated by shear force >> MASP 2 activated>> C4 and C2 cleaved by MASP2 >> C4b recruits C2a >> C3 convertase formed >> everything else is the same as the classical pathway

**note that MBL or MBficolin are secreted by PRRs**

Question 8

When C3 is cleaved into C3b by the convertase or C3 undergoes autoactivation, a ___ bond is exposed that allows C3b to bind to the surface of microbes

What happens to C3b in the absence and presence of a microbe?

Answer 8

When C3 is cleaved into C3b by the convertase or C3 undergoes autoactivation, a thio-ester bond is exposed that allows C3b to bind to the surface of microbes

In the absence of a microbe, C3b binds water (thioester bond is highly reactive so it’ll bind nearby water molecules quickly)

In the presence of a microbe, C3b will bind carb molecules on the microbe’s surface

Question 9

Describe the activation of the alternative complement pathway

Answer 9

Autoactivation of C3 >> thioester bond exposed and C3b binds to carbohydrate on surface of microbe (if microbe present. If not, it’ll be inactive and bind water) >> inactive factor B cleaved and activated by factor D >> Bb (active factor B) + C3b stabilized by properdin (aka factor P) >> C3 convertase >> more C3 cleaved >> more C3b generated and bound to C3 convertase >> C5 convertase formed >> everything else is the same as in the other two pathways

Question 10

What are the downstream effects of complement activation?

Describe the formation of the ultimate product of C5 convertase activity

Answer 10

Formation of the MAC

Opsonization leading to improved phagocytosis

Induction of the inflammatory response

C5 convertase is formed >> C5 cleaved to C5a and C5b >> C5b binds the surface of the microbe and recruits C6 >> C6 recruits C7 >> C7 recruits C8 >> C8 recruits 10 – 16x copies of C9 >> C9 multimers join and form pore in the microbe’s membrane >> microbial cell death due to osmotic imbalance

(see image below)

Question 11

Macrophages can recognize opsonized bacteria via the receptor ___

What are the functions of C3a, C4a and C5a?

Answer 11

Macrophages can recognize opsonized bacteria (i.e. recognize C3b) via the receptor Complement Receptor 1 (CR1)

C3a, C4a and C5a are pro-inflammatory, C5a being the strongest inducer of the inflammatory response

Question 12

How are the expression of C3 and Factor D regulated? (low/high expression)

Answer 12

C3 is expressed at a high level and isn’t usually doing anything in circulation

Factor D is expressed at very low levels (if you have high levels of this then you have too much complement activation via the alternative pathway which is no Bueno)

Question 13

___ can sequester C1r and s and stops the formation of the C1 complex (stops classical pathway in its tracks)

Answer 13

C1 inhibitor can sequester C1r and s and stops the formation of the c1 complex (stops classical pathway in its tracks)

Question 14

How does Factor I inhibit the complement pathway? What are the functions of Factor H and C4 binding protein?

Answer 14

Factor I is the main protease. It cleaves C3b on the surface of the microbe and makes it inactive; works with the help of Factor H and/or C4BP

Question 15

Explain the slide below

Contrast iC3b with normal C3b

What is the function of C3dg?

Answer 15

Factor I cleaves C3 but requires Factor H, CR1, MCP, or C4BP to supply cofactor activity

If C3b binds to a self cell (or if we’re trying to shut down inflammation), factor I can recruit other factors to cleave C3b into iC3b, which can recruit phagocytic cells but it is NOT able to recruit factor D, therefore iC3b can’t continue with the complement pathway

Further breakdown of iC3b by factor I leads to the formation more cleavage products like C3dg

C3dg further broken down to C3d and g; C3dg can bind to Complement Receptor 2 (CR2) on B cells which activates adaptive immunity

Essentially, this pathway stops the inflammatory response but we’re still activating adaptive immunity (which you will recall is a stronger response anyway)

Question 16

___ inhibits C3 convertase formation so blocks all 3 pathways

(study all the other inhibitors and where they’re expressed)

Answer 16

DAF inhibits C3 convertase formation so blocks all 3 pathways

Question 17

How does the B cell receive its 2nd signal to respond to microbial infection via the complement pathway?

Answer 17

Complement receptor 2 (CR2) can act as the 2nd signal to allow the cells to be stimulated properly (binds to degradation products of C3b) (the 1st being the actual antigen binding to the BCR)

Question 18

Briefly describe the clonal selection theory of T and B cell maturation

Answer 18

During development, B and T cells rearrange their genes which results in each cell expressing one unique, specific receptor

Both undergo selection (recall that T cells undergo positive selection then negative selection in the thymus, and B cells undergo negative selection in the bone marrow) – cells that bind self too strongly undergo clonal deletion via apoptosis

Mature cells will then go to the 2ndary lymphoid organs and wait to be activated

Once activated they become plasma cells or just mature effector T cells

**recall that some defective cells can escape the deletion process, in which case they can then be deleted in the periphery or they can be toned down via anergy**

Question 19

What’s the difference between a BCR and an antibody? (how does each work/what does each recognize)

Answer 19

In naive B cells, the BCR has a transmembrane region on the surface (Ig alpha and beta I’m guessing) and it only recognizes free antigen. When activated, the BCR = the secreted antibody, which can recognize any kind of antigen, free or otherwise

Question 20

T/F: The T cell receptor can recognize whole antigen and binds it directly

Answer 20

Falsehood. The TCR is always on the surface and is never secreted (recall that it recognizes processed Ag presented on MHC)

Question 21

Draw and describe the basic structure of the BCR and TCR (which domains are there? How many? what types of bonds are they linked by?)

Answer 21

The BCR has 2 heavy and light chains linked by disulfide bonds

Heavy chain has 4 IG superfamily domains and light chain has 2 such domains

The TCR is also made up of disulfide linked dimers, each chain has 2 IG superfamily domains

Question 22

In the BCR, the light chain has __ variable domain and ___ constant domain whereas the heavy chain has one variable and ___ constant domains

The V domain has two extra beta strands and these strands are going to form ___

Answer 22

In the BCR, the light chain has 1 variable domain and 1 constant domain whereas the heavy chain has 1 variable and 3 constant domains

The V domain has two extra beta strands and these strands are going to form (one of) the antigen recognition sites (CDR2)

Question 23

What is the difference between affinity vs avidity?

Answer 23

Affinity: binding of ONE of the binding sites to an antigen (specifically the epitope or antigenic determinant)

Vs

Avidity: the overall increased strength of binding that occurs when more than one binding site on the receptor interacts with more than one epitope (essentially avidity is the result of multiple affinities)

Question 24

The most flexible part of the antibody is the ___, which allows for various arrangements of Ab-Ag complexes

There is also flexibility within the Fab and the Fc region. ___ on the Fc region confers the effector function of the antibodies as well as the flexibility in these regions.

Answer 24

The most flexible part of the antibody is the hinge, which allows for various arrangements of Ab-Ag complexes

There is also flexibility within the Fab and the Fc region. The carbohydrate on the Fc region confers the effector function of the antibodies as well as the flexibility in these regions.

Question 25

The 3 broad categories of epitopes are ___, ___ and ___

T/F: A hapten will induce an immune response on its own

Answer 25

The 3 broad categories of epitopes are proteins, carbohydrates and haptens

Falsehood. Another type of epitope is the hapten, which is a tiny benzene ring modified with NO2 groups. If you inject a hapten by itself into an animal, it won’t elicit an immune response so on their own, they are not immunogenic

Question 26

How would you make a hapten immunogenic?

Answer 26

Basically, if you want to make a hapten active, you have to couple it to some molecule (like a protein) that’s going to not only bind the B cell but also recruit T cells

For example: Hapten is coupled to a protein carrier like BSA which stimulates T cells that then activate hapten-specific B cells

Question 27

What are the immunoglobin subtypes?

Answer 27

IgM

IgG

IgD

IgE

IgA

Question 28

___ is the first to be secreted in the immune response, found on most B cells in a transmembrane form, and in the plasma (secreted form) as a pentamer (associated with the J chain)

Which Ig is considered the “workhorse of the body?

___ is co-expressed with IgM on the surface of resting B cells; its function on the cell surface is obscure.

___ is not found in the plasma, but rather bound on the surface of mast cells, basophils, and eosinophils by Fc receptors, and is involved in local inflammatory responses.

___ is secreted across mucosal surfaces and thus is involved in epithelial defense.

Answer 28

IgM is the first to be secreted in the immune response, found on most B cells in a transmembrane form, and in the plasma (secreted form) as a pentamer (associated with the J chain)

IgG is considered the “workhorse” of the body

IgD is co-expressed with IgM on the surface of resting B cells; its function on the cell surface is obscure.

IgE is not found in the plasma, but rather bound on the surface of mast cells, basophils, and eosinophils by Fc receptors, and is involved in local inflammatory responses.

IgA is secreted across mucosal surfaces and thus is involved in epithelial defense.

Question 29

All antibodies have two ___ and ___ light chains (recall their 3:1 ratio unless there’s cancer,in which case it either reverses or it increases)

Answer 29

Kappa and lambda

Question 30

Which antibodies are responsible for neutralization?

___ is/are responsible for opsonization

Which antibody(ies) is/are responsible for NK cell sensitization?

Mast cell sensitization is carried out by ___ antibody(ies)

Which antibodies activate complement?

Answer 30

All except D and E (mainly IgG)

All IgG isotypes except IgG2; IgA

IgG1 + IgG3

Mainly IgE, also IgG1 and 3 (Mast cells like the letter E and the number 13)

Everyone except IgD, IgG4,and IgE (you Don’t Give a 4 if you score an E on complement activation)

Question 31

Which antibody can go across the epithelium?

___ can cross the placenta

Which antibody can diffuse into extravascular sites?

Answer 31

IgA (recall that this is on mucosAl surfaces), and G as well

All IgG

Everyone except IgD

Question 32

IgM and IgA exists as polymers thanks to what molecule? (hint: this is made by the plasma cell and is secreted when the Ab is secreted)

Describe the process of Ab transport across the epithelium using IgA as an example

Answer 32

The J chain

The plasma cell produces Ig and the J chain. Once secreted, the two bind and the J chain binds to the poly-Ig receptor, which is produced by the epithelium

The whole complex of Ig, J chain and receptor are taken up by the epithelial cell and once secreted following proteolytic cleavage, it’s secreted as the Ig, J chain and a piece of the receptor and that’s the active Ab that’ll bind the bug

Question 33

Why does IgG4 not have effector function?

Answer 33

IgG4 exchanges arms in the plasma (no same binding site within the same antibody); has no effector function: it can bind antigen on viruses or bacteria but only to block the bug from interacting with its receptor on whatever host cell so it can’t get into the cell or cause whatever other trouble

Question 34

___ receptor binds IgE on mast cells and basophils

___, ___ and ___ are the IgG receptors, with ___ being the only inhibitory receptor

Activating receptors are ass’d with cytosolic ___s and the inhibitory ones are ass’d with ___s that recruit phosphatases that’ll downregulate immune responses

Answer 34

FcERI receptor binds IgE on mast cells and basophils

FcyRI (CD64), IIB (CD32) and IIIA (CD16) are the IgG receptors, with CD32 being the only inhibitory receptor

(just think in terms of multiples of 16

Activating receptors are ass’d with cytosolic ITAMs and the inhibitory ones are ass’d with ITIMs that recruit phosphatases that’ll downregulate immune responses

Question 35

The ___ recruits kinases for NK cell killing and ___ is involved in opsonization, whereas ___ recruits phosphatases for inhibition

Answer 35

The CD16 receptor recruits kinases for NK cell killing and CD64 is involved in opsonization, whereas CD32 recruits phosphatases for inhibition

Question 36

Explain how maternal IgG can make its way into the fetal circulation

Answer 36

On the surface of the baby’s gut are FcRn (n for neonate) receptors that can bind maternal IgG. Via transcytosis, the receptor-Ab complex is released on the other side to the neonatal circulation. The receptor is also expressed in the placenta and IgG makes its way into fetal circulation by the same process