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last time we talked about class I and class II pathways. now we're going 
to start out by a quick review - any questions about why there are two 
pathways and how it helps us deal with different pathogens?

the class I/II thing is NOT completely esoteric: has real effects.
remember: class I pathway was the pathway that involved antigens or 
viruses or bacteria that happen to be IN the cytoplasm - and in the 
cytoplasm they're broken down by proteosomes, a component of cells used 
to break down proteins in many contexts, and they are broken into 
peptides which are transported into the ER, where the peptides bind to 
class I MHC, and the bound peptides go to the surface of the cell.
Class II pathway differs in that it involves class II being made in the 
ER like class I, but in contrast, now there is the invariant chain in the 
peptide binding groove of class II, and this chain helps to target the 
class II molecule to the endosome, and then once in the endosome, Class 
II binds peptides. the petides are from bacteria which are phagocytosed 
and broken down in an endosome.

so: cytosolic pathogens (endogenous pathway) are degraded in the 
cytoplasm, and bind to MHC I and you get a CD8+ T cells. the antigen 
presenting cell is killed. extracellular pathogens (exogenous pathway) 
and toxins are degraded in aidified vesicles, and are bound to class II 
in the endosome, and CD4+ T cells respond, activating macrophages to kill 
intracellular pathogens/and or/ activates B cells to secrete Ig to 
eliminate extracellular bacteria/toxins.

--
moving on....
before we get into the various T cells and their function, it's really an 
issue of how T cells develop. WHAT IS a T cell? has TcR, has CD4 and CD8, 
but where does it get these characteristics? it gets them in the thymus. 
Originally they develop from stem cells in bone marrow. they leave as PRE 
T cells and migrate to thymus. DiGeorge syndrome: no thymus: patients 
have no T cells.

what happens in thymus?
well, *a lot*. 
think of the structure of the thymus, briefly. The thymus has many 
lobules and is broken into two main regions: cortex, outer portion; and 
the inner medulla. so PRE T cells come into cortical region and migrate 
into medulla and eventually leave the thymus as mature T cell. On 
histologic preparation, looking at the thymus, it's darker staining where 
there are more lymphocytes.

going back...what is most important thing to happen in thymus? adding TcR 
to cell. ok, so you've rearranged the TcR - is that a rearrangement that 
has knowledge of the host or is it random? it's random. SO, given that 
scenaria, after the T cells rearrange, they have potential to recognize 
self proteins. so you have to ALSO get rid of self-recognizing T cells to 
avoid autoimmune problem (negative selection). ALSO have to teach T-cells 
to recognize MHC. T-cells only recognize antigen presented by SELF-MHC 
molecules. and it's in the THYMUS that they learn about self MHC, and 
there is a POSITIVE SELECTION process which selects T-cells that CAN 
recognize self MHC. furthermore, they start expressing CD4 and CD8 in the 
thymus.

think of it: pre T cells enter thymus. TcRs rearrange. then their's 
positive and negative selection. so of all the T cells entering thymus, 
only about 5% leave as working T cells. 95% undergo apoptosis!

now...we know T cells don't recognize antigen presented by NONSELF-MHC. 
This is important. This discovery was made by Doherty and Zinkernagel in 
1975, who used virally infected target cells, and they found T cells only  
interacted w/them if they expressed Self MHC . Doherty (a vet) won the 
Nobel prize this year.
This MHC restriction is important. you can see it experimentally...

MHC RESTRICTION EXPERIMENT:
tells you that T cells do learn to recognize self MHC.

if you have a (red) F1 mouse, with MHCa and MHCb (eg, offspring of MHCa 
mouse and MHCb mouse). You take bone marrow from this mouse and put it 
into irradiated recipients. You give the yellow irradiated mouse (which 
no longer has its own bone marrow or T cells) bone marrow from red mouse 
- making it a bone marrow chimera, and also you do this to the blue 
irradiated mouse. the stem cells from the red mouse repopulate the yellow 
and blue mice, so now the yellow and blue mice have T cells. Now, will 
the T-cells recognize MHCa and b, or what? if the yellow mouse is MHCa 
only, these new T cells will ONLY recognize MHCa, and will not recognize 
MHCb, because the T cells don't see it in the thymus. you take t cells 
out of yellow mouse and put in culture with APCs. if the APCs have MHCa 
the T cells will respond, but not if in with MHCb APCs. Even though, in 
red mouse, these cells WOULD be able to see b...and the stem cells from 
which these yellow mouse t cells derived are precursors for T cells 
which, in the red mouse, can see MHCa and b. The same thing happens with 
the blue MHCb mouse...those T cells only recognize antigen presented by 
MHCb expressing cells.

[my back is *killing* me. i think i need to see a *real* doctor, not 
these student health NPs. I've about maxed out the effectiveness of 
naproxen sodium and ibuprofen over the past few weeks, and the pain is 
getting down into my leg again.]

so...in THYMUS:

1. rearrange TcR
2. selection - positive and negative
3. aquisition of CD4 or CD8
(4. apoptosis)

ok. what about acquisition of CD mols?
the pre-t cells are CD-. as they mature in thymus they begin to express 
CD3 and the alpha-beta chain, and then starts expressing BOTH CD4 & CD8. 
as it differentiates, there's +/- selection and most cells die. the ones 
that live become two groups: CD4+ and CD8+. so, the TcR has to learn to 
recognize MHC, and must be linked to CD4 or 8, since these are the 
molecules that see MHC. why they stop expressing one or the other is not 
clear, but in the end the T cells only express CD4 or CD8. [note: 
alpha-beta is the TcR protein]

again, thinking of the thymus...immature T cells are in cortex, are 
double negative thymocytes, and they differentiate as above, and finally 
become mature CD4-CD8+ or CD4+ CD8- cells and are released from thymus.

at the same time as CD4 and CD8 start being expressed, you have the 
rearrangement of TcR genes - quite similar to Ig gene rearrangement.VDJ 
rearrangement in beta chain and VJ rearrangement in alpha chain.. FIrst 
the beta chain rearranges, then subsequently you have the rearrangement 
of the alpha chain - so at that point you have rearranged TcR, and CD4 
and CD8, and so you have a double positive cell. note when the alpha 
chain rearranges it deletes the delta region of the gene. the delta 
rearrangement will occur earlier on, before beta rearrangement, and those 
gamma delta T cells are exported to the periphery and never express CD4 
or CD8 [note: he's not going to ask us real detailed questions about this 
but we should understand concept...]

so we have double positive cells ready to undergo positive selection. 
first the TcR is rearranged, then is exposed to class I and II on surface 
of cortical epithelial cells. They apparently get a positive signal if 
there is some binding there. if they DON'T recognize the self MHC, they 
die. the cells that are selected to live continue developing and migrate 
through thymus, and at a later stage they are susceptible to being 
deleted if they respond to self MHC.

positive selection: they get a signal that they have to get in order to 
continue. eg, they must bind self MHC to live and go on. if they do not, 
they die.

negative selection: they get a signal which induces death.

one experiment which tells you that this really occurs in the thymus:

transgenic mice which have T cells that ALL express the SAME TcR. how? 
you take a T cell, clone it, isolate genes for TcR, and inject them, and 
make transgenic mouse, yada yada. 

the TcR genes you put in are already rearranged, so that inhibits further 
rearrangement of TcR genes. now, in Ig lectures, we talked about how once 
you have productive rearrangement, you don't do it again, and so each B 
cell has a single specificity. well, same with T cell. Each cell normally 
expresses ONE receptor with one specificity. Now, these transgenic mice 
have no endogenous rearrangement due to this inhibition. SO all the t 
cells in these mice have the same TcR and recognize the same Ag.  so you 
create a mouse with T cells recognizing a peptide from ovalbumen. it can 
ONLY recognize this peptide. this animal has all these T cells 
recognizing the same Ag. if you let them develop, they have normal 
looking thymus. if you inject into thymus the peptide the t cells are 
specific for, the T cells all start dying. all of their T cells will be 
deleted because of negative selection.

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leaving T cell development in the thymus, now... after a quick 
review...this is an important factor in preventing autoimmunity. T cell 
is most important cell in immune response. 

Cortical region is where they go first, and they migrate down into 
medulla, dropping in number due to +/- selection. apoptosis is occuring. 
apoptotic cells are eating by macrophages pretty quickly. it's very 
important, this apoptosis, not only in thymus to get rid of bad T cells, 
but also for downregulating the immune response. many effector cells have 
seriously toxic effects, and are downregulated by apoptosis.

within the thymus, really important things happen. you get the two 
population of T cells developing...the double positive cells migrate into 
medulla and become positive for only ONE CD type.

TcR rearrangement is RANDOM and not induced by antigen in any way.

now moving on...
WHAT DO T CELLS DO??? we're going to spend the next couple of lectures on 
this topic.

FIRST...HOW DO T CELLS FUNCTION? lets go back and remember what are major 
subsets of T cells?

1. alpha-beta: 
	-CD4+: these recognize MHC II, give help to Bcells, activate macrophages

	-CD8+: recognize MHCI, are "cytotoxic" T cells, "CTL"

note: CD4 cells MAY be cytotoxic under certain circumstances, and CD8s 
may be helpers. but in GENERAL it is as above.

What do these cells do? Two main things:

they release SOLUBLE MEDIATORS eg CYTOKINES, LYMPHOKINES
We used to just call them lymphokines, and the monocyte factors 
monokines, but so many are produced by BOTH cells that we call them all 
CYTOKINES. They can also have a physical contact with cells - critical 
for cytotoxic activity is physical interaction between T cell and target 
cell.

GENERAL CHARACTERISTICS OF CYTOKINES:

there are many many different cytokines that are produced, and every year 
there are a few new ones discovered, a new protein made by a T, B or 
macrophage that has significant effects. It's hard to keep up, for 
immunologists even. but they have certain general characteristics, and 
we'll go over those, and then we'll get a list of particularly important 
cytokines.

a. Extensively pleiotrophic: have many different effects. one cytokine 
can do  
   many different things. Sometimes it will do one thing at low levels 
and the 
   opposite at higher levels.
b. Redundant: many cytokines have same effect.
c. Local effects: contrast w/hormones, which act at far reaching sites. 
   cytokines react within their own microenvironment. Now, sometimes you 
have 
   cytokines circulating in the serum, and this is usally an indication 
of a 
   severely out of control toxic immune response.
d. Short half life: usually about 30 min. varies per cytokine. the 
message for 
   the cytokine is turned on and then right back off again.
e. receptors often have more than one chain: cytokines act like hormones 
in 
   that they must bind with a receptor. cytokine has no specificity, just 
binds
   to its receptor, like hormones.
f. single copy genes: almost all cytokines are single copy genes. so we 
make 
   transgenic animals and knockout the gene for a particular cytokine and 
see
   what functionality is lost.
g. related genomic structure: all have related genomic structure.

on page 220-221 of the purple immuno book is a list of cytokines. we're 
going to go over some important ones. all cytokines are proteins

IL = interleukin
TNF = tumor necrosis factor
IFN = interferon
GF = growth factor

CYTOKINE	 PRODUCER	  TARGET		     PRIMARY FUNCTION
----------  ----------- --------------- ------------------------------
IL-2		T cells		T and B cells	  T Cell growth factor, B cell gf.
									      really gets immune system initiated.
										  makes cells upregulate IL-2 receptor
 										  and respond better. This cytokine 
										  really drives proliferation and 
									      expansion of T cells.

IL-4		T, (mast)     T/B    	    B/T cell GF, isotype switch (esp IgE)
IL-5		T cells	    T/B, eos		B cell GF, Eosinophil GF 

[skip 6,7,8,9]

IL-10		T, macro	  T, macro      Downregulate immune response
IL-12		macro, +/-B    T cells		T cell GF, induces IFN-gamma production
										stimulates T-H1 cells
IFN-gamma	T, NK		T, macro		macrophage activation, upregulation of
										class I and class II molecules
TNF			T, macro	T, macro	    kills target cells 	   



Try to recall these cytokines and how they function in immune response.
Next two hours we'll exclusively discuss functions of T cells, how they 
do things, etc.
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