class 6, total lecture 7

Types of humoral immune responses and functional diversification of antibodies

2/08

antibody functional diversity by isotype class switch recombination

last class - isotype switching and mechanisms of how its done

Ig heavy chain class (isotype) switching

why we make diff isotype

bc of their functions

antibody class: immunoglobulin M

• IgM - first Ab on surface of a B cell (BCR)
• IgM secreted as a pentamer Ab
  • IgM molecule w/ 5 antibodies held together by J chain
  • present as pentameric form
• IgM is the main class without T cell help (T cell independent, TI)
• IgM is best in complement mediated killing of pathogens

IgM always used as first antibody - doesn't matter if response is T dependent or independent

• have lower affinity bc have not undergone processes of B cell maturation to increase affinity
• IgM antibodies made have lower affinity bc has not undergone any processes to make binding site better

in T cell independent immune response, where there is no germinal center reaction, the antibodies remain lower affinity bc those do not go through the same maturation for enhanced affinity

IgM best in complement mediated killing

• antibodies act mostly as extracellular protective molecule
• typically, antibodies catch pathogens when they are not in cells
• thus, antibodies catch pathogens more as a whole, when this happens, IgM attaches to pathogens + recruits complement protein C31Q. Complement recruited to IgM molecule that is bound to molecule
• so this helps immune response earlier on, bc complement adds to it

antibody class: IgA

• IgA is a monomer and a dimer
• serves as opsonin for neutrophils, eosinophils, and macrophages
• IgA dimer is the main Ig secreted at the mucosal surfaces (ex: lungs, intestines, etc.)

How does structure affect function?

IgM is BULKY

- very limited to reach everywhere in body

- drawback – its bulky size = less ability to go thru membranes

Now IgA:

- most protective antibody molecule in our mucosal membranes (areas exposed to world)

- GI, lungs, genetalia, etc.

- secreted + present in mucus

IgA dimer is transported through epithelial cells to be secreted at the mucosal surfaces

• IgA is secreted through epithelial cells via poly-Ig receptor
• IgA is secreted in the mother's milk

Mechanism for IgA

- when produced, has ability to bind receptor on epithelial cells (lining of lung/gut), where there is polyIg receptor

- IgA bind to receptor -> get internalized, pass thru cell, and IgA can pass thru the cell

= transport protein (IgA) from 1 side to the other

For IgA molecule to be present in all the palces it is, it binds to poly-Ig receptor to get secreted outside cell on the other side

antibody class: IgG

• IgG is a monomer
• is most compact of Abs
• there are 4 subclasses of IgG (1-4)
• serves as an opsonin
• activates complement
• sensitizes NK cells
  • + mediates antibody dependent cellular toxicity (ADCC)
• most abundant in the blood and at mucosal surfaces
• crosses placenta

most versatile

small + compact

produced only after Ig class switching (during the germinal center reaction)

• during this time, there is also affinity maturation through somatic mutation
• thus, these IgGs always have higher affinity

has most compact structure

comes in subclasses

functions examples:

• can serve as opsonin - target pathogen becomes more attractive for phagocytes
• can activate complement - IgG constant region can bind complement proteins + activate the complement system (which ultimately leads to formation of protein complex that pokes holes in biological membranes)
  • complement leads to death of target pathogens
• recruits + activates NK cells - important for killing of infected cells

important to protect fetus from infection, even after birth (give baby passive immunity from mother)

multiple functions

antibody class: IgE

• IgE is a monomer
• primarily induced by IL-4 (cytokine)
• triggers release on granular contents from mast cells, eosinophils, and basophils
• respond to non-pathogenic substances; pollen, peanut, etc -> allergies

- to take care of large pathogens, like parasites

- binds to certain types of innate cells (mast cells)

- sensitizes mast cells and get them ready to be active vs target

- molecule that first is on surfaces (skin, etc. bc where parasitic infections occur) in very small quantities

- once pathogen, more IgG is made

antibody classes are selectively distributed in the body

map for where they are in the body

some antibodies recruit specific cells (like how IgE recruit mast cells + eosinophils)

Which antibody isotype can cross the placental barrier and is passed on to the fetus?

Which antibody isotype is designed to be secreted via the epithelial cells at the mucous membranes?

pause for activity

answer:

IgG, IgA

effector mechanism of humoral immunity

functions of antibody classes

antibody functions

1. neutralization of microbes + toxins
   
   • get pathogen before it enters
2. opsonization and phagocytosis of microbes
3. antibody dependent cellular cytotoxicity
   • using NK cell
4. Can bind bacteria directly and recruit complement (antibody molecules + complement together can form immune complexes) (big significance clinically bc the complexes can grow so big that cannot be filtered / can clog filtering system in kidneys that can lead to kidney dysfunction)
   1. lysis of microbes
      • via complement
   2. phagocytosis of microbes opsonizes with complement fragments (ex: C3b)
      • via complement
   3. inflammation
      • via complement
      • important inflammatory mechanism. Are often involved in autoimmune diseases

Ab blocks penetration of microbe through epithelial

without antibody:

• microbe get thru epithelial barrier cells

with antibody:

• antibody blocks penetration of microbe thru epithelial barrier

= antibodies protect from extracellular pathogens

Microbes outside epithelial surfaces can bind to structures on microbes, which are used by microbes to attach to epithelial cells

- ex: spike protein on covid causes covid 19

Bind to it, and prevent pathogen from causing harm and entering

which 2 antibody types work against microbes that try to penetrate epithelia?

IgG and IgA

Abs block microbes from infecting neighboring cells

IgG and IgA

these antibodies block binding of microbe and infection of cells

antibodies protect from extracellular pathogens

IgA and IgG

- generally protect vs extracellular pathogens

- same concept. Neutralized

FcRs-mediate destruction of Ab-coated pathogens

Receptors on innate cells can be motivated/activated by antibodies to destroy pathogens

Fc receptors

Constant regions of antibodies – called Fc regions

Abs mediate phagocytosis of pathogens by opsonization

1. opsonization of microbe by IgG
2. binding of opsonized microbe to phagocyte Fc receptors (FcyRI)
3. Fc receptor signals activate phagocyte
4. phagocytosis of micropbe
5. killing of ingested microbe

Phagocyte has receptors on surface

1st (Fc) receptor binds constant region on the antibody

Fc receptor is specific for the constant region

And binding site of antibody keeps microbe attached

èGets phagocyte to internalize the pathogen that is attached to antibody

This is not just physical action of antibody bindig – phagocyte grabbing

- things changed in phagocyte

activation of phagocytes and phagocytosis of pathogens requires Fc receptors

free immunoglobulin does not cross link Fc receptors

-> no activation of macrophage, no destruction of bacterium

aggregation of immunoglobulin on bacterial surface allows cross linking of Fc receptors

-> activation of macrophage, leading to phagocytosis and destruction of bacterium

notes:

• Free floating antibodies do not bind well to phagocyte
• But when bound to pathogen, they bring Fc receptors together to activate the phagocyte – now bc has pathogen, it can bind well to phagocyte
  • - cause intracellular signal transduction in the phagocyte and inside the phagocyte chemically changes
  • -> phagocyte becomes activated -> phagocytosis

Ab dependent cellular cytotoxicity

the IgG receptor Fcy R can recruit NK cells to kill infected cells

• IgG bind to surface antigen of antigen
• -> go to NK cell which binds the antibodies
• -> lead to killing of the cell w/ surface antigens

now bound to antigen on target cell that is infected

• when cells infection
• constant region recognized by NK cell -> forms holes and kills the infected cell

activation of NK cells requires Fc receptor cross-linking

Fc receptors on NK cells recognize bound antibody

-> cross-linking of Fc receptors signals the NK cell to kill the target cell (via degranulation of NK cells)

Bind foreign antigen on our own cells

- recruit NK cells and activate them

Process happens fast

Ab neutralize toxins by blocking toxin from binding cellular

pathologic effects of toxin

• antibody blocks binding of toxin to cellular receptor

Another function of antibody

- neutralize a toxin (poison, can be from anything)

- toxins can be internalized by many cell types

- cause intracellular biochemical processes to be disrupted and cause cells to be dysfunctional, etc.

ex: toxins that cause paralysis of certain organs, etc.

can bind to circulating molecule

without immune cell participation (can do the job alone)

IgE binds to FcER1 and mediates degranulation of mast cells and eosinophils

Some cells express Fc receptors that bind only certain constant regions

ex: mast cells and eosinophils express FcepsilonR1 which ONLY binds IgE

-> bind thru Fc receptor

- bound antibody targets allergen/pathogen

- brings multiple Fc receptors together = Fc receptor cross linking

Common thing in immune cell activation is clustering of receptors. Happens often. Just like the T cell indepndent IgM cross linking I think?

IgE binding to FcER1 and crosslinking mediates degranulation of mast cells

resting mast cell -> activated mast cell

resting mast cell contains granules containing histamine and other inflammatory mediators -> multivalent antigen crosslinks bound IgE antibody, causing release of granule contents

Antigen come, granules start secreting – doing degranulation

- how mast cell look after degranulation

degranulation of mast cells and eosinophils results in the expulsion of parasites

mast cells/eosinophils attack parasites

Mast cells bound to IgE specific to this parasite

Mechanism of getting rid of huge parasites

Releases histamine – mast cells

Fc receptors signal and the activation of innate immune cells

there are Fc receptors specific for each Ab class

How do diff antibody isotypes enhance their functional diversity?

pause for activity

Ab neutralize toxins by blocking toxin from binding cellular

Carried out by antibody protein molecules by themselves

Cannot do this if have specific antibodies

binding of C1q to Ab initiates activation of the classical complement pathway

pentameric IgM molecules bind to antigens on the bacterial surface and adopt the "staple" form

-> C1q binds to one bound IgM molecule

-> binding of C1q to Ig activates C1r, which cleaves + activates the serine protease C1s

IgG molecules bind to antigens on the bacterial surface

-> C1q binds to at least 2 IgG molecules

-> binding of C1q to Ig activates C1r, which cleaves + activates the serine protease C1s

Antibodies can bind to targets that have multiple binding sites -> recruit binding + activation of complement

Important: IgM (despite poor affinity) overcomes the deficit by having multiple binding sites -> create complex that is good for activating complement

Same thing achieved by IgG.

HE WILL ASK ABOUT ON TEST:

2 functions of antbodies that are independent of other cells

answer: 1.Neutralize toxins 2.Activate complement

which 2 Ab functions can be performed without the help of other immune cells?

pause for activity

*** EXAM QUESTION ***

1- Toxin neutralization

2. Complement fixation (activation)

inhibition of BCR and FcR signaling

Immune system response can be destructive

Don’t want it to go on for longer than needed, would cause more disease than pathogen would

- excessive/prolonged immune response is BAD and cause many problems

Then how do u calm active immune response?

- mechanisms to turn off response – we will discuss this in terms of B cells

FcRs activate innate cells

ITAM, immunoreceptor tyrosine based activation motif

Fc receptord activcate innate cells bc they have ITAM In their cytoplasm

- very potent

B cells express FcR that inhibit B cell activation

ITAM, Immunoreceptor Tyrosine Based Activation Motif

ITI(?)M, Immunoreceptor Tyrosine Based Inhibition Motif

Antibody bound to target

Specific clone of B cell also has same specificity

So these floating complexes w/ Fc come back and bind Fc receptor on B cell

Antibody bring antigen to bind to original B cell receptor, and antibody is bound to Fc receptor

- Fc receptor carries inhibitory motif (ITAM)

usually tyrosine kinase is necessary for activation in the cells by phosphorylation, initiating signaling cascade to activate B cell

ITIM motif has activity to recruit phosphatase – disrupts the process of activation and phosphorylation is STOPPED

Presence of Fc gamma receptor on B cells bind to antibodies the B cell made(??? Idk what he was saying),

When they find B cells, they attenuate process of activation

- temper/reduce b cell actvation and can no longer be activated

FcR mediate Ab feedback inhibition of B cell activation

BCR activated motifs

FCR has inhibitory motifs?

Idk

How prevent excessive inflammation

Excessive inflammation causes most of damage

distribution and functions of immunoglobulin classes

• Antibodies of different classes operate in distinct places and have distinct effector functions.
• Transport proteins that bind to the Fc regions of antibodies carry particular isotypes across epithelial barriers.
• High-affinity IgG and IgA antibodies can neutralize bacterial toxins.
• High-affinity IgG and IgA antibodies can inhibit the infectivity of viruses.
• Antibodies can block the adherence of bacteria to host cells.
• Antibody:antigen complexes activate the classical pathway of complement by binding to Complement.
• Antibodies inhibit BCR activation signaling through inhibitory Fc receptors