---start 1.17.97 immuno----
phil scott: pscott@vet.upenn.edu

development of monoclonal Ab. important for research and diagnosis in 
human and veterinary medicine.

one thing you can see is if you immunize an animal against an antigen, 
you get a B cell response and a lot of antibodies against many of the 
epitopes on that Ag. if you immunize against a PATHOGEN, you get Ab 
against many Ag AND many epitopes of the Ag. The antisera can have cross 
reactivities. if you want to dx for a particular pathogen, in the past 
you'd make an antisera, but you had the potential for cross reactivity. 
This was a big problem from a diagnostic point of you. you want an Ab 
specific to only the pathogen you're looking for. 

so you isolate B cells making Ab and clone them so you have many of ONE B 
cell, and it will make ONE antibody with ONE specificity. One B cell, one 
vote - get it?

so if you take a single B cell and make it expand, you can harvest the 
monoclonal Ab- antibody from the ONE B cell clone cell line.

the problem was, could you take the B cell adn grow it in bulk culture 
and keep it alive? the lymphocytes don't grow in culture long term, 
though. they die. people worked on this for many years to get them grow, 
but it never worked. in the 1970s there were two people, milstein and 
kohler (very famous people. george kohler just died) who got the Nobel 
prize for solving this problem. conceptually, what they did was say "if B 
cells can be cloned and they have this specificity, how can we make them 
grow? fuse them to a tumor cell line!" so they would become immortalized, 
and have the capacity to continuously grow. so you take b cells and fuse 
them with tumor cells giving them capacity for long term growth; so now 
we can make monoclonal antibodies.

[slide]antigen injected into mouse or hamster or whatever. you take 
spleen aspirate and culture the spleen cells, which contain B 
cells.you'll have many b cells making polyclonal antibody. you then take 
tumor cells that are NOT making Ab (nonsecretor myeloma cells) and you 
put them together in a tube with polyethylene glycol to fuse them 
together. so now you have a fusion of b and tumor cells; still a 
polyclonal mixture. now you need to get out the b cells that have fused 
with the myloma cells and isolate them specifically. now, the myeloma 
cells themselves are sensitive to 3 chemicals (hypoxanthine et al), and 
the B cells are resistant. so the FUSED cells have the resistance 
phenotype, and the tumor cells don't, so you add that to the culture, and 
you get only the fused cells growing (b cells unfused also die, 
remember). so now you have a culture of fused B/myeloma cells. but it's 
still polyclonal. so next you have to clone these cells in 96 well 
plates. you dilute them out with a variety of support cells and growth 
factors and you clone down so that each well has a single B cell, and you 
let them grow up. then you reclone them to make sure it's a single b cell 
line. then you take the supernatant from the grown cells, which contains 
monoclonal Ab, and you test the specificity. so if you immunize with a 
particular Ag, you'll get maybe 10 monoclonal cell lines. then you test 
the Ab to see what molecules are recognized by it. so if you wanted to 
make a test for a pathogen, you look for monoclonal Ab that reacts ONLY 
with a part of this one pathogen and not with anything else. so this was 
done in the 70s and since then have been very important in diagnostics 
and dissecting out functions and proteins etc.

so that's how you make monoclonal b cell line. you quite can't do this 
with T cells, but you can make monoclonal T cell lines similarly and you 
can test the clones for reactivity, but they're not that useful since 
they make no product that you can measure.

question: you're trying to get down to 10 different monoclonal lines?
answer: well, it varies. you could end up with more or less, and then you 
would look at them. and some of them may be recognizing the same epitope 
or whatever. and you use western blots or something to look at the 
epitopes or what have you.

Q: is it possible to find out which epitope on the Ag the monoclonals 
recognize?
A: definitely. you find out is it seeing protein, or CHO or lipid? you 
can remove CHO and lipids from Ag and see what happens. you can cleave a 
protein and see what parts it recognizes. this can be arduous and you 
don't always do it.

Q: what about the mutations that occur when you're making Ab? 
A: once you clone the cells they don't occur. for the most part the 
ability to mutate and change isn't really something that happens in 
vitro. it happens more in vivo.

[:( everyone's talking again. hard to hear.]

if you culture these cells for a long time, you can have changes occur; 
the cells may become nonsecretors, etc. there are ways to fix this.

Q: what was that about monoclonal T cells?
A: well, there's interest in making T cells that have one specificity 
also. when you immunize with Ag, multiple epitopes are recognized by T 
cells similarlly to B cells. So there's interest in making monoclonal T 
cells. less interesting diagnostically, but interesting theoretically. 
you can make single clones similarly to b cell cloning as above.

AN exam is coming up. after the exam we'll discuss T cells. Exam's on 
friday. That's in one week. arg argh argh argh. If you want, read about T 
cells over the weekend after the exam. We're going to talk about what t 
cells are, what they do, what they make, how they work, and the core of 
the in vivo immune response.

Q:could you go over competitive radioimmunoassays? i don't get the 
concept?
A: concept....it's that if you have a - what are you looking for? you're 
looking for presence of a particular antigen, how much Ag is there. you 
have an Ab that reacts to that Ag. so you take the Ab and the Ag and you 
radiolabel the Ag. now ifyou have a rxn with Ab, Ag and Ab will bind, and 
you can precipitate out the AbAb complex. that will have a certain amt of 
radioactivity within it.
eg, you mix the Ag and Ab, and a percent of Ag will bind Ab, and some Ag 
will stay free. so you get some Ag/Ab complex, and some free Ag. if you 
then add NON labelled Ag, or "cold" Ag, into the mix, there will be LESS 
radioactivity BOUND, because of competition. As you increase the amt of 
cold Ag, you get decreased binding to the hot Ag. Now, you know how much 
cold Ag you add. you set up a standard curve to tell you at any 
particular concentration (eg, you add serum with unknown amount of 
antigen) how much binding has occured, so you can tell what concentration 
of Ag is in you patient's serum.

---break---

dr weber

reminders: monday: discussion groups. recall in the first packet of 
handouts is a set of study questions. you should be able to answer them 
by monday. for the first hour monday go to the group you're assigned to. 
after that you can pick a group to go to. also, since it's the last time 
we meet as a group (monday), it's nothing to get concerned about. for the 
exam we're using rm 13 and MDL 11. but come to rm 13 first, and sit down, 
and be calm. 
questions on the exam: expect about 30:30:30:10 ratio of questions for 
the instructors. multiple choice, short answer, etc. ok?

TOday, we're going through the complement system. it evolved as part of 
the innate mechanism; long before acquired immune system came about. the 
classical pathway, amplification pathway, alternate pathway, inhibition  
molecules on normal cells,will all be discussed. we have a handout about 
this, summarizing the textbook chapter. it's not complete however.

ok so..in terms of terminology, in the classical pathway you usually find 
capital C. In alternate pathway, you see factor E, factor B, etc. so it 
it's not a C, it's alternate. Also, the complement proteins, as they 
interact with each other you get split products a and b, a is smaller and 
b is larger. C3a is smaller than C3b.

classical pathway:
easiest to activate with IgM (or IgG close together)
most common way to activate this pathway is through Ag/Ab interaction. So 
you get an Ag/Ab complex. IgM and IgG are best at activating complement. 
IgM is better than IgG because it has 5 Fc portions. It has to be 
distorted on a cell surface to activate complement, though. FREE IgM 
isn't going to do it, because it is in a planar form, the Fc portions 
aren't sticking out th right way. a single molecule of IgM will activate 
complement pathway, though.IgG would have to be fairly closely 
appositioned on the pathogen, however. the C1q molecule is made of 6 
rodlike structures, and at least two of them have to interact with Fc 
portion of Ig molecules.  SO it's more easily activated by IgM, but IgG 
works. remember acute phase proteins? they can activate this pathway too 
- CRP (c reactive protein) and MBP(mannose binding protein). MBP 
resembles C1q, and it binds mannose residues on the surface of the 
bacterium, and it attracts Ig molecules, which trigger the pathway.

note r and s are special associated enzymatic proteins. not a big deal.

why is C4 after C1? well, they were named in order of discovery.
C4bC2b* is a C3 convertase. why? it can break C3. So now you have C3 
getting activated...
C4b2b3b is C5 convertase

note: C3a contains anaphylatoxins as does C5a, and C3a is product of C3. 
C5a is very potent anaphylatoxin.
C5b has minimal physiologic significance, as does C4a.
C6789: membrane attack complex. forms hole in membrane. once C5,6,7,8 
activated, you get like 10-14 C9s polymerizing into a pore in the 
membrane.

About the pore structure: has structural homology to perforins. When 
eosinophils attach to membranes, and release granules, some of those 
proteins also act this way. this mechanism is highly conserved, in other 
words. much homology among these molecules that use this pore-making 
mechanism to kill a cell.

                                                C3a             C5a
                                                /               /   
Ag-Ab--->C1qrs*---C4--->C4b---C2--->C4bC2b*----C3---[C4b2b3b]*--C5[6,7,8,
9] 
                                               /                    *
                                               C3b                 MAC
ALTERNATE PATHWAY                         /---/   \
Ok, once C3 is activated you get         /         B
amplification/alternative pathway.   /---           \
C3bBb is C3/C5 convertase.          / amplification! \D
          					       /                 |
C3b + factor B-->C3bB             /                 /
                   \+factor D      \               /
                    \               \             /
                     ---- ----------C3bBb*--------                
                                      |
						this molecule acts like C4bC2b, splits C3.

*inhibitory spots

now, what about stimulation of alternative pathway without Ag/Ab 
interaction?
well, gram negative organisms contain ENDOTOXIN in their cell walls. 
These bugs often hang out in intestines. ENDOTOXIN == lipopolysaccharide 
== LPS. So gram - organisms big suspects for triggering this pathway, but 
some gram +, some yeast, some rickettsia and some viruses can also do it 
and we don't always know what it is on their surfaces that does it. IgA 
aggregates can do this well. Snake venom (cobra) can also activate 
alternate complement pathway.

so, there's some degree of natural activation of complement in the 
plasma, followed by inactivation. Now, C3 is most abundant molecule in 
the plasma and it has a spontaneous trigger, so there's always a little 
bit of C3a and C3b in the plasma too. So what happens? it interacts 
somehow with the endotoxin or whatever...

macrophages, neuts, etc, have C3b receptors. 

the anaphylatoxins cause vasodilation, increased vascular permeability, 
edema, they draw in lots of inflammatory cells (chemotaxis of WBCs). this 
is beneficial up to a point, but if it becomes a systemic reaction you 
have a problem. your blood pressure will plummet...
but, locally, edema occurs, lymph drainage occurs, more antigens are 
taken back to LNs, local pathogens are phagocytized, so this is good, 
locally.

now if it becomes systemic, this production of anaphylatoxins, you end up 
in septic shock. septic shock is bad. you get massive peripheral 
vasodilation/blood pooling, you have a hypovolemic shock component, and 
the LPS will trigger tremendous # of macrophages to produce things like 
IL1, TNF, etc. these cytokines have the abiility to cause increase in 
adhesion molecules on endothelium of small vessels, so when these are 
produced, they upregulate the adhesion mols on endothelium, which will 
bind neutrophils, causing them to release products, so you release 
clotting factors, activate the coagulation system, (he's saying this in a 
confusing way, but you end up with DIC, ok?) so you spontaneously form 
clots and yet you use up all the clotting factors so you bleed 
unstoppably.

ok, now, most bacteria don't have inhibitory molecules to prevent the 
stimulation of the complement pathway all the way down to MAC formation. 
so they can't inhibit the activation of proteins taking place on the 
surface of the bacteria. so that begs the question; what protects the 
normal cells from this pathway? in pathway above there are * noted. these 
are sites where inhibitors work.

C1 inhibitor prevents spontaneous triggering of C1. Once C1qrs is formed, 
C1 inhibitor can split r and s off, and pathway will fail. some people 
are born with autosomal dominant C1 inhibitor mutation, so the inhibitor 
doesn't work. this leads to increased incidence of classical pathway 
stimulation, which we call "hereditary angioedema" because they get 
production of anaphylatoxins. this can be fatal due to tracheal and 
pulmonary edema,etc. it's very unpleasant. this is now treated by giving 
people C1 inhibitor. 

another point to have inhibitory molecules is at C3 convertase level. 
Here we have Decay Accelerating Factor DAF which causes the molecule to 
deca and C4Binding Protein C4BP prevents binding of C2
                              
further there is an I molecule that causes C3 to be catabolized more 
quickly.
there are some cofactors (factor H, membrane cofactor protein) but 
they're not that important. next step down the line - at C5,6,7 - low 
density lipoproteins (LDLs) and vitronectin prevent portions of the MAC 
from binding. and CD59 prevents the pore formation and the assembly of 
the 10-14 C9 molecules that make up the pore. SO normal cells aren't 
lysed by the complement pathway readily due to these inhibitory 
molecules.  there are also molecules inhibiting the C3 convertase of the 
alternative pathway by causing it to dissociate.

are there any bacteria which can evade this mechanism? eg, once it starts 
to grow on the surface? well, some have abundant polysaccharide or other 
capsules. you still get C3 deposit on the bug, but it's not on the 
membrane, it's on a capsule. so MAC forms a pore in the capsule, but not 
the membrane, so it won't kill it...furthermore, some bugs have "learned" 
to use some of the complement receptors to get IN to the cells. eg they 
attach like yeast attaches to Cr2 which is on macrophags, and then gets 
into the cell. some viruses can enter the cells by attaching to some of 
these proteins on the cell that normally deal with complement activation. 
there are always exceptions to the rules....


look at handout for better diagram and explanation of action of 
inhibitory molecules. you don't have to remember EVERYTHING but you 
should be able to figure out where major inhibitory points are, i guess.

ok. septic shock is a major pathological event associated with this 
pathway.

also: clearance of Ig complexes: most of the time you make Ag/Ab 
complexes, they're big enough to be removed by reticuloendothelial system 
and they're metabolized and cleared out. BUT if the complex is small - eg 
toxin/antitoxin complex, or soluble Ag complex, they can deposit in 
places where you don't want them to go! Especially in the kidney, skin, 
joints...then you get bad problem- Immune Complex Dz. that will be the 
subject of another set of lectures. but when the complexes are deposited, 
the complement system is activated and you make a lot of inflammatory 
response in those regions, so you get rheumatoid arthritis, glomerular 
nephritis, skin lesions, etc.


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