---start immuno 1.27.97---
Dr. Phillip Scott
898 1602
T Cell Biology -->handout

now we're going on into different material. lectures for the next 6 hrs 
will be on T cells and T cell function, beginning to put together how 
immune system really functions after exposure to a pathogen. then dr 
weber will take the material we've already covered and will apply it to 
clinical situations.

Make sure to read the handout which is NOT comprehensive, and also read 
ch 9,10,11, and 12 after reading the handout.

today: how do T cells get made? what makes them? what structures are 
involved in determining the function of a T cell? how do these structures 
interact and lead to effector functions?

one thing that is the case in immunology and esp cellular immuno, we do 
experiments to figure out what we think is happening, and then make 
theories about what's happening. we need to understand - we're just 
starting 4 yrs of vet school, and by the time we are out, some of these 
theories will change. but the FACTS will not change...only the 
INTERPRETATION of the facts. so some things we don't really know the 
answer to because we just aren't sure how things work in vivo yet. immuno 
is not just taking cells out of animals and looking at them...you need to 
understand how things function in the living animal system, and so we 
guess a lot.

so.
WHAT IS A T CELL?
a lymphocyte which comes from the bone marrow and matures in the thymus.
why does the T cell go to the thymus to mature? to be discussed later.
how do we know a t cell when we se it? It has a T cell receptor (TcR), 
and it has other surface markers which are specific to T cells. You can 
NOT tell the difference between B and T cells by microscopy. You need to 
look at surface proteins or something. "B cells are irrelevant, T cells 
are most important, that's what you should learn in this lecture" ha ha.
SO if you take peripheral blood from an animal you have to look for Ig on 
the surfaces of B cells, and other stuff on T cells.

WHAT IS THE OTHER STUFF?

TcR: recognizes antigen in association with MHC II. 
CD4
CD8
CD28

these markers define the types of T cells, and each has a specific 
function. So there is a relationship between the boring old structure and 
the exciting neat-o function of the T cells. So this is 
interesting....function follows form, if you will.

So we're going to discuss T cells, and the difference between Cell 
Mediated immunity (CMI) and humoral immunity (HI).

The whole B cell/Ig deal is HI.
The T cell response is CMI

these two arms of the immune system have been known for years, and for 
years HI was considered most important. This is because of two things: 
Meshnikoff, who put forth the idea that cells were important in 
controlling infxns - eg macrophages eating pathogens, etc, and another 
group who found in the serum there were factors that would kill bacteria 
(immunoglobulins, complement) - and everyone was trying to find these 
factors....but, they didn't look at the cell mediated immunity.

late 1960s-1970, it was shown that if you took animals you could immunize 
them against bacteria (Listeria) and you could immunize a mouse, and then 
transfer the immunity to another mouse, challenge it with live Listeria, 
and get protection...and you could do this using CELLS ONLY - eg, 
transferring serum didn't help, had to transfer cells. Now, L. 
monocytogenes is an INTRACELLULAR PATHOGEN, if i recall correctly, so 
that would explain this, I think. (ooh, he just confirmed that :)).

in any event...this experiment was important in establishing the field of 
CMI. and subsequent to this T cells and subsets of T cells were 
discovered. Molecular biology came along and it became possible to clone 
cells and products of cells. SO this field has grown immensely in past 20 
yrs.

T CELL RECEPTOR (TcR)

T cell has the TcR - each T cell has about 30,000 or so on its surface, 
depending on if activated or not. Structurally, this molecule is very 
similar to immunoglobulin. it's a member of the Ig supergene family, and 
has a structure similar to the Fab region of Ig, it has a binding region 
which can bind antigen like the Fab binding sites do, but it's UNIVALENT 
and it's MEMBRANE BOUND> it is not a secreted molecule. it primarily 
functions as a membrane bound molecule- not to say it can't be removed 
from the T cell... but AFAWK, it's only active in membrane bound form.

now, the Ig molecule and TcR molecule are structurally similar, and wrt 
genetic organization they are also similar...you have variable regions, 
joining regions, and constant regions as before. we aren't going to go 
into the genetics of the TcR because almost all of the rules from Ig also 
apply to TcR. two major chains - alpha and beta - and they form the 
mature TcR. But, there's no spliced variable form of mRNA for a secreted 
form of TcR of course. so alpha and beta join to form TcR. We call this 
abTcR, and it is the best defined TcR. there are others - gamma delta T 
cells (they have gdTcR) - we know much less about these, and about their 
function and where and how they function, but we should know they exist. 
Two major classes of T cells: ab and gd.

Just as immunoglobulin has the ability to rearrange genes to create 
diversity, the same is true for t cells and TcR. we're not going into the 
calculations - it's just good to have some concept of the possible 
diversity.

Ig 10^16, and about the same in TcR - a HUGE numer of potential combining 
sites that can be generated.

so, the variable region of TcR is coded for similarly to that in Ig, and 
determines specificity of the T cell and if it can bind to a particular 
Ag/MHC combo. but, this variable region does'nt function on its own, at 
all. the primary reason it doesn't function on its own is because hardly 
any of it reaches into the cytoplasm - so it can't SIGNAL the T cell 
after binding Ag. You need a larger cytoplasmic tail to have signalling 
capability - and so you have the CD3 molecules, composed of 5 types of 
molecules: epsilon, delta, gamma, zeta, and nu. don't need to know those 
names, just need to know that CD3 associates w/TcR and is vital to 
function of TcR. THe CD3 molecules are involved in signalling when TcR 
binds Ag. zeta chain is of particular importance in the signalling 
process.

The TcR is not found on the surface of T cells w/o CD3, probably because 
in order to export TcR to membrane, it needs to be associated w/CD3. the 
whole complex of how a T cell recognizes Ag involves the TcR and CD3 
molecules. One CD3 on either side of the receptor.

WHAT ELSE IS THERE ON THE T CELL SURFACE??

a whole lot of stuff, we're only going over a few of the most important 
things.
we've already discussed the TcR/CD3 complex. 
two other molecules: CD4 and CD8 - T cells normally have one or the other 
of these on their surfaces - not normally both. these are important in 
signalling.

there are other molecules: CD45 et al...we don't have time to discuss 
these.

what function do CD4 and CD8 molecules serve?
-signalling
-dictate how the T cell will recognize Ag. Huh? well, the CD4 or CD8 
molecule binds to MHC I (CD8) or MHC II (CD4). REPEAT: CD4 binds MHC II 
and CD8 binds MHC I.

so you have ab T cells and gd T cells.

the ab T cells can be divided into CD4+ and CD8+ groups, which you can 
identify via antibody tests. The CD4+ bind class II, and the CD8+ bind 
class I (so the CD number multiplied by the MHC number is always 8! easy 
way to remember).

Dr Scott wants to know if we know about MHC. SOmeone said we learned 
about it in histology. hmmm. well,there's this diagram on the board which 
is from the book - pg 177 - showing gene of mouse MHC. this is an 
important series of genes that was first defined by the fact that they 
dictated if you could transplant tissue from one individual to another. 
so during transplantation experiments it was shown that the 
histocompatibility complex was the major determining factor when figuring 
out if two individuals could share tissue. now, at the time of discovery 
of transplant, etc, no one understood function of molecules coded for by 
these genes. but we know now that they dictate antigen recognition, which 
makes sense if you think about it a bit, yes? the products we're going to 
talk about are the class I and class II molecules. So, class I and class 
II are membrane bound molecules - these are known as IA and IE (in the 
mouse) (class II) and K, B, and L (class I, mouse). each species has 
similar structured molecules. in many species they have been cloned and 
are well defined. THIS IS IN THE BOOK - CHECK IT OUT. realize that the 
class II product is formed from several genes, and that the class one 
molecule is formed by one gene product plus another protein called 
beta2-microglobulin, which is NOT formed by the MHC region, but is 
another thing entirely. This should become clear as you look at the book.


so..what are the characteristics of MHC? there are many different genes 
involved in coding these molecules...at least 4 coding for Class II and 3 
for class I, so it's polygenic (that just means several genes are 
involved)...so there is a lot of variability, and lots of antigens can be 
recognized. but what really creates the diversity is the fact that each 
of these gene products varies in each individual, and that 
variability/polymorphism means that in a given species, in a given 
population, you have individuals coding for different types of molecules 
(alleles) at these loci. So each person in this room has slightly 
different MHC. Eg, each gene can code for many different types of 
molecules. Now, all of us have identical CONSTANT regions of our TcRs, 
but the variable regions are quite different. Now, with MHC, the products 
coded for turn out different in each individual - NOT constant. so, they 
can then bind different antigens for presentation. so the polymorphism 
leads to the whole population being able to respond to different 
antigens.

so there's polygenic characteristic- many genes coding for products.
there's polymorphism - genes produce many alleles, different gene 
products

and these combine to provide much diversity.

confused yet? good. let's go on to how they bind antigen, and maybe that 
will make things clear.
[slide]- class I molecule. recall 3 genes code for this molecule, but all 
make homologous molecules. the structure is best discussed by looking at 
some slide i can't see from here :(.

there's a peptide binding cleft on top, and a beta 2 microglobulin under 
it on the right, and on the left is an alpha3 chain, i think. the peptide 
binding cleft is where the antigen binds. the peptide binding site is 
made of alpha one and alpha two chains. i think. it's kinda fuzzy. so, 
you can see, the molecule has been crystallized, and when we discuss MHC, 
this is what we're talking about. now, the variability in these 
molecules, if you go back to the fact that there are different alleles at 
each region, in a population, individuals will have differences in their 
alpha one and alpha two regions, which is where you get peptide binding.  
if you take many alleles from a population, you find that there are 
differences in AAs at various parts of molecule and that ends up being in 
the binding site, usually. Now this is NOT like TcR variability, because 
that is generated by gene rearrangement in the T cell. But, individuals 
do not generate MHC variability. there is no rearrangement. the 
polymorphism is dictated by your parents having different alleles in that 
region. you will, as an individual, have only one type of class I product 
for each of those genes. but it's important to understand it's dictated 
from a species level, not an individual level. if you look at class II 
molecules, it is very similar. an alpha and beta chain combine to form 
mature molecule, with a peptide binding cleft,etc.

[slide]- globs of protein. if you look down at a molecule as a t cell 
might, if you do that with a peptide, you can see the MHC with peptide in 
its cleft, and you can see that the shape is different...there is no 
longer a cleft, there is stuff in there. if a t cell sees this, it will 
recognize it.

---break----

so: class I and II PRESENT ANTIGEN to T cells. T cells do not recognize 
native Ag - only processed Ag as presented by I and II. These class I and 
II molecules present the Ag in their peptide binding clefts.

In MHC I peptides are normally about 9 AAs. Why? because of the structure 
of MHC I, the binding cleft holds only about 9AAs. Class II molecules 
bind longer peptides - from 9-15+ AAs. this is because the MHC II binding 
cleft is open on either end, so longer peptides can fit in there. this 
should beg the question...the T cell has a lot of variability wrt what it 
recognizes - 10^16 possibilities, right? now, MHC....there are two class 
II molecules in the mouse, IE and IA. with those two class II molecules, 
assuming codominant expression so you have four, and then you think of 
all the possible peptide combinations that exist, and you have such 
limited MHC, how does this work? how can so few MHC bind such a great 
range of peptides? well, the MHC must not have such exquisite specificity 
as do the Ig Fab regions or the TcRs. So, these MHCs don't bind every AA 
in the peptide. they only bind ANCHOR RESIDUES. if you look at a class I 
MHC with a bound peptide....you have 9 AA in the peptide. MHC is only 
noticing TWO of the AAs - say, position 2 and 9 - and these are called 
anchor residues. what that means is that that particular class I molecule 
can bind many peptides, as long as it has a particular AA at site 2 and 9 
- and everything else can be different. so class I and II can bind whole 
groups of peptides that are quite dissimilar but which have the correct 
anchor residues.

now, there are 3 genes coding for class I, and each class I will 
recognize different anchor residues, and each allele is different as 
well, so as an individual you have 3 from mom and 3 from dad so you can 
bind 6 classes of peptide; and as a population, because of alleles, the 
whole POPULATION can recognize many many more peptide classes. so any 
pathogen is broken into peptides, and t cells will only see those that 
bind to class I or II. and in a particular pathogen there will only be a 
few peptides that fit the bill. at times, you may end up not being able 
to bind to peptides from a particular pathogen because you don't have the 
right MHC -- too bad for you. but probably enough members of the 
population can respond to ensure survival of the species in the face of 
an epidemic, or whatever. in MHC II the anchor residues are different, 
not 2 and 9, but we don't have to know which residues they are :).

the binding is hydrophobic forces, van der waals forces, electrostatic 
forces - not covalent bonding.

reminder: class I molecules are present on almost all nucleated cells, 
class II present only on a certain subset of cells.

brief tangent: re: peptide binding to class II: SUPERANTIGENS. We know a 
normal antigen binds in the peptide binding cleft and is recognized by 
binding region of TcR. now, there's a class of Ag called SUPERANTIGENS 
which are very clinically important and which are an interesting 
exception to these rules. these are proteins that are derived from 
bacteria, protozoa, or viruses. if you add them to T cells instead of a 
small response you might normally have you get a LARGE T cell response, 
and so these are called SUPERANTIGENS. No one understood why this 
happened, but it was important because they could induce a systemic toxic 
response, where all the T cells were responding, which is bad, because a 
massive t cell response can make you ill. so these bacterial products 
would induce clinical signs of shock: eg, food poisoning - staphylococcus 
produces a superantigen which causes a systemic response like this. see, 
instead of binding in the peptide groove, they bind to external part of 
MHC II. Furthermore, they bind in the CONSTANT region of TcR, not the 
variable region. so all T cells would respond! so a protein product of a 
bacteria, aka toxin, or viralproduct or what have you, can do this. 
why do superantigens develop? probably not chance! not sure of exactly 
why these develop, but we may be enlightened in microbiology. but there 
is evolutionary pressure for class I and II to develop polymorphism, and 
there is pressure for pathogens evolve and become more successful as 
well.

so. we are down to the portion of the handout where we are going to start 
discussing T cell Ag recognition and APCs. REmember - T cells only 
recognize peptides - because only peptides bind to MHC I and II. CHO do 
not bind to class I and II.

So. Antigen Presenting Cells: APCs
we're talking about those that express MHC II and present Ag to CD4+ T 
cells.

macrophages
B cells
dendritic cells

class I is expressed on most nucleated cells, and class I expressing 
cells are sometimes APC and sometimes not. 

so, generally speaking, if you look at APCs as defined above, you can 
understand they must have MHC I or II. MHC I is seen by CD8+, btw.
having MHC isn't enough. T cell must see MHC + peptide, and ALSO must see 
"costimulatory molecules" - of which there are many. There is an 
interaction between C28 on the T cell and B7 on the APC.

again: need recognition of Ag/MHC complex, AND costimulatory signal.

WHY do you need second signal? well, to guard against autoimmune disease 
for one...

what exactly happens if a T cell recognizes costimulatory molecule 
without seeing TcR? well, nothing happens. it doesn't do anything. 
similarly if it sees TcR w/o seeing costimulatory signal, the T cell can 
be anergized - meaning it will not be able to respond to Ag at all ever, 
or it could be deleted - it could undergo apoptosis. so if your T cell 
sees ONLY Ag/MHC w/o costim, it's  not long for this world.

so how might autoimmunity develop? well, if you have aberrantly high 
upregulation of costimulatory molecules? so - what regulates expression 
of costimulatory molecules? because if this occurs, you could see T cells 
responding to self peptides, since costim mol being expressed 
inappropriately...

so...there are those dendritic cells, macrophages, and B cells thought of 
as traditional APC. why are they APCs?
they have certain characteristics....

B cell: excellent antigen specific receptor (Ig), and express MHC - 
constitutive, increases on activation +++ to ++++. Has inducible 
co-stimulator ability, - to +++. present toxins, bacteria, viruses. 
located: blood, lymphoid tissue


macrophages: +++phagocytic, so good at antigen uptake as well. MHC 
expression inducible by bacteria and cytokines, - to +++. costimulator 
inducible - to +++. present extracellular and vesicular bacteria. found 
in lymphoid tissue, CT, body cavities

dendritic cells: +++phagocytic, ++++ constitutive MHC 
expression.constitutive costimulator delivery as well ++++. present 
viruses and allergens. found in lymphoid tissue, CT, epithelia

so: antigen uptake, MHC II expression, and costimulatory factor 
expression, and location are KEY factors in APC determination.

DENDRITIC cells are probably the quintessential APC for initiating immune 
response - they are phagocytic, they are constitutive expressors of MHC 
and costim factors, and they are in the right places.

finally, for the next 12 minutes we'll talk about what exactly antigen 
processing is, and what's going on inside these APCs. 

Ag enters cell, and something occurs, leading to expression of the Ag on 
cell surface in combination with MHC. there are two major pathways which 
determine whether or not T cells recognize antigen that is class I or II 
bound. PAGE 189 fig 10.5 has good representation of these pathways. the 
class I or II pathway is determined by the pathway of how class I and II 
develop within the cell.

so, look at class I presentation....
you have live virus in the cell. virus is hanging out in cytoplasm. virus 
is broken down in cytoplasm, and proteins are broken into peptides by the 
PROTEOSOME (which clips proteins into peptides of 9 AA length) and the 
peptides are transported by TAP transporter into the ER where class I is 
being formed, and here the peptide is bound to class I and also beta 2 
microglobulin is bound to class I, and then the whole shebang goes to the 
surface of the cell and waits for a T cell to come along and see it. it 
will be recognized by CD8+ cells of course.
the class I pathway is aka the endogenous pathway. this is called 
endogenous pathway in the book, but it may be better to think of it as a 
pathway which requires cytoplasmic antigen.

now...class II pathway
aka exogenous pathway. uses dead virus or intrevesicular pathogen. is 
taken up by cell in a vesicle - an endosome - and it stays in the 
endosome and is broken into peptides.  meanwhile, class II is forming in 
ER. it's exported from ER into the endosome, where it binds peptide, and 
then the whole shebang goes to cell surface. TcR will recognize it - CD4 
molecule will bind class II.

now, if class I binds peptides in ER, why doesn't class II? well the 
invariant chain is bound to class II in the ER, and it stays with it on 
the way to the endosome, then it gets clipped off, exposing the peptide 
binding groove so it can bind peptide in the endosome. the invariant 
chain is involved in transport of MHC II to endosome, and prevents 
binding to peptide in ER.

EXOGENOUS: "from the outside" - some confusion - you may have 
intracellular bacteria which live in endosomes, eg TB, but they are still 
EXOGENOUS even though it's live intracellular bacteria....they will 
stimulate CD4+ cells. 
the difference is whether or not the organism is CYTOPLASMIC. class 
I/CD8+ pathway requires CYTOPLASMIC pathogen.

---end---