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Tolerance: specific unresponsiveness to one specific epitope or antigen 
if someone is tolerant to  one epitope, he can still respond fine to 
other epitopes. this can really only be occuring at the T cell or B cell 
level since it is specific.

hyporesponsiveness is on a broad scale, all responsiveness is decreased.
whereas tolerance is very specific.

why did immune system develop tolerance mechanism, esp to self ag?
at T and B cell levels, as B cell Ab combining sites and TcR form, 
inevitably, some cells will respond to self epitopes. at thymus and bone 
marrow, as well as in periphery, mechanisms exist to get rid of self 
reactive T and B cells.
so realizing that we've already gone over central tolerance w/thymus, a 
quick recap...

in thymus there are positive and neg selection

pos selection: both CD4 and CD8 T cells recognize epitopes in context of 
self. all those cells that can do that (recognize epitopes w/MHC) will 
expand. but as they mature, in cortex and a bit in medulla, there is neg 
selection for cells recognizing self epitopes, 'cause mphages and B cells 
present w/MHCII self epitopes. T cells which recognize them get 
functionally deleted.  So ultimately you have only T cells which respond 
to foreign Ag. But what is this regulated by and is it really complete? 
well it depends on the concentration of the self Ag being presented in 
thymus, and the affinity of the TcR for the Ag. If the self epitope or 
auto-Ag is bound w/high affinity to TcR, the cells will be deleted. if 
the Ag presentation is in very low concentration, eg, "cryptic" epitopes, 
the T cells, which may also have a relatively weaker affinity for htat 
epitope, may sneak out of the thymus, andyou end up w/autoreactive T 
cells in the periphery. why do they not regularly respond to self 
epitopes? as T cells migrate, the self epitopes they are usually directed 
against are in low concentration, and the T cells may not be in a tissue 
where the Ag are located, or the Ag may not be presented appropriately. 
eg, if Ag is presented on thyroid cell, in absence of costimulatory 
signals, even if being shown w/MHCII eg during infxn, T cell will likely 
not respond. also many of the tolerant T cells don't encounter self 
epitopes in any significant concentration because the Ag are sequestered 
- not in normal circulation pathway. Eg, brain MBP, thyroglobuline, eye 
lens protein, sperm products. also there is a lack of adhesion molecules 
in these areas. Most people feel thymus cells are deleted there or don't 
respond due the factors just mentioned.

can a T cell out in the periphery develop tolerance outside of the 
thymus? if youhave an autoreactive T cell, and it makes contact with Ag 
and MHC w/o costimulatory molecule, you kind of deactivate the cell 
without deleting it. so if T cell encounters self w/o costimulatory 
molecule it becomes ANERGIC - functionally useless and unable to respond. 
what are costimulatory molecules? a molecule in the membrane hanging out 
near the ag/mhc complex.

a B cell with downregulation of Ig production isn't going to have 
costimulatory molecules (what???)

normally, if you have a foreign Ag presented by mphage w/mhcII, after 
binding w/TcR and costim, you get first and second signal allowing T cell 
to expand and proliferate. but if self ag is presented, youwill get first 
signal, but there is no costimulatory molecule to give second signal, so 
IL2 is never produced, IL2 receptors don't form, and T cell remains 
anergic.

this t cell tolerance can develop centrally (usually the cells are 
deleted then) or peripherally (cells are present but anergic eg 
functionally defective). so you don't usually have attack of self 
tissues.

if you orally present an Ag through GI tract, you often end up with 
tolerance. if you present that same Ag through the skin, you often get an 
immune response.
why? what is different about the APC in different places?

it's thought that the APC cells in GI tract don't have costimulatory 
molecule.

note: tolerance is "the final frontier" of immunology so we're not going 
to learn all about it in this class, lots isn't known yet.

tolerance and self:
potentially any protein in the body can be recognized by T and B cells, 
but normally self proteins are not.

some of the proteins are sequestered- in brain, testis, lens of eye. T 
cell won't see it.
some are in low concentrations and ignored-
maybe as long as TH cells are tolerant,not making cytokines, B and T 
cells won't respond. need IL2 and IFNg to drive T cell expansion and need 
IL4 IL5 for B cell response.
MHCII hasvery restricted distribution, so unless self Ag is expressed on 
an MHCII+ cell, no recognition takes place. normally only "professional 
APC" eg dendritic cells, interdigitating cells, activated Mphages and 
activated B cells can do this.
Now, sometimes cells can be triggered to express MHCII and then all hell 
breaks loose and you can see autoimmune dz occur in these situations.

what about B cell tolerance? we knwo that this takes place largely in 
marrow, but you can also make B cells anergic in marrow or periphery. if 
you have developing immature B cells in the marrow, with IgM only so far, 
if that IgM has high affinity to a cell membrane associated self Ag, 
those cells will be deleted via apoptosis and also via IgM receptor 
downregulation which causes the B cell to die in about 3-4 days.

It is clear that there are B cells that leave the marrow that can respond 
to self, however, as in thymus the deletion isn't perfect. so now if they 
get out, why don't they respond to self ag? the autoreactive B cells 
don't respond 'cause the TH cells are tolerant and so the B cells don't 
get T cell help, or any stimulation. there are no cytokines from the T 
cells.

as long as B cells encounter Ag in low concentration in absence of Th 
cells, the B cell becomes anergic, again meaning functionally incapable 
of responding. an anergic b or t cell is present but cannot respond. one 
problem w/tolerant B cells...anergy can be overridden, and anergic B cell 
can be triggered into responsiveness apparently via crosslinking 
receptors on their surfaces by polyclonal activators like LPS 
(lipopolysaccharide) etc. this can trigger then nonspecifically to make 
autoAb. also via overstimulation, can get overproduction of cytokines by 
active T cells, and this can override B cell anergy as well, causing B 
cell to make autoAb leading to local destruction of tissue. one safeguard 
to all this is - B cells can be rendered anergic and even deleted in the 
germinal centers of the LNs. this gets rid of autoreactive B cells. in 
germinal center is high proliferation of immature B cells, which are 
making IgM receptors. what else goes on? affinity maturation. this can 
imply that you get development of very hgh affinity binding sites to self 
epitope, which you do not want.

so the germinal center is a safeguard, because you get deletion of cells 
with high affinity for self epitopes (see handout. i was sneezing and 
missed his explanation).

are both t and b cells tolerant of self?

serum Abs to almost all body constituents can be found. almost everyone 
has low levels of serum autoAb. as long as complement isn't fixed, no big 
deal.

single B cells- if you take them, and expand them with LPS, you will find 
B cells with receptors for self Ag and in about the same concentration as 
to foreign substance.

B cells can also be stimulated with mitogen and fused w/myeloma cells, 
and youcan make monoclonal Ab to self.

this means probably there are some B cells not tolerant of self, while 
most are probably tolerant. but nonspecific triggering of B cells CAN 
promote autoAb production. 

AQUIRED IMMUNOLOGICAL TOLERANCE TO FOREIGN AG

a state of unresponsiveness to one or more antigenic determinants, either 
via cell mediated or humoral immunity. 

1. natural chimeras: dizygotic cattle twins. these were the first animals 
in which we discovered and looked into tolerance. what's happening here 
is that through the placenta there is a constant exchange of stem cells 
during developement. so the fetuses end up as chimeras, with continuous 
presence of cells of a different genetic background within their bodies. 
when the developing immune system is exposed to genetically different 
cells, they become totally tolerant and this is permanent, and later you 
can do transplants between these animals w/no problems. here exposure of 
an Ag will actually make the other fetus totally tolerant.

2. experimental chimeras

conditions favoring tolerance induction.

1. immature lymphoid system, eg fetus, newborn - note that this varies 
between species, when appropriate time is. some species ok at birth, 
others only in utero.

2. in mature animals, possible only after irradiation or chemotherapy. 
must give Ag during the treatment period; give Ag in non-immunogenic 
form.
as long as chimerism persists, animal will be tolerant. if you do this 
for one epitope, eg BSA or whatever, you have to make sure to give it in 
non=immunogenic form so it isn't presented or phagocytosed well, and you 
have to do it during the tx period (radiation/chemo) - right after that.

3. inherent immunogenicity of the molecule: bovine gamma globulin (bgg) 
low vs diptheria toxin (high). BGG may be low immunogenicity because it 
is similar to everyone else's GG, but diptheria toxin isn't like anything 
else and is highly immunogenic. very hard to make animal tolerant to DT.

4. form of Ag: aggregate vs soluble. if you give Ag in aggregate form, eg 
complexed, that is much more readily phagocytosed and presented, so you 
don't want to do that. soluble Ag is usually poorly digested or 
presented. role of Ag processing is important. go back to albumen 
molecule. if you have a serum alb molecule sitting there for a few days, 
if you spin the serum down and take the supernatant off and the bottom cc 
out, you'll find that inthe top was the soluble albumen, and in the 
bottom was aggregated albumen. not only are concentrations different, but 
the bottom is clumped and stuff. and you can induce tolerance w/the 
soluble kind, but not the bottom kind. so aggregates are usually 
antigenic, while soluble Ag usually able to induce tolerance.

5. route of administration: oral, IV: role of Ag processing/presenting 
cells. if you can induce tolerance by feeding the offending epitope, that 
would be great to treat autoimmune dz. oral Ag presentation frequently 
does lead to tolerance induction likely due to different APCs in 
different locations and the lack of a costimulatory molecule.

6. antigen dose: induction of tolerance at opposing extremes of dosage. 
can occur at very low or very high level of Ag. 
you can induce tolerance by using a given epitope in varying 
concentrations. you  take groups ofanimals and  they get injected with 
different concentration of antigens, there's a low zone area and a high 
zone area, notice that in primary immune response, those animals getting 
low dose don't show much immune response. then they start developing one 
in the high dose area - but then at the REALLY hgh dose area, response 
isn't as high.

then months later, a second challenge with a single dose of a middle 
range amount of Ag, you see that animals who were injected with low 
concentrations of antigens have developed "low zone tolerance" and 
animals who were injected with high conc Ag have developed "high zone 
tolerance".



what has been worked out is, how quickly to b and t cells get tolerized, 
and what Ag concentration is necessary to do this?
T cells get tolerized very quickly, it lasts 100-150 days, and then it 
drops off. why do you get a breakdown of tolerance? the thymus is 
producing a few new T cells and they will not be tolerized. B cells take 
about 7-10 days to tolerize, need higher Ag concentration, and it doesn't 
last very long at all, only about 30-40 days. why does it drop off? b/c 
bone marrow is constantly making lots of new b cells, unlike thymus which 
decreases its function over time.

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a summary:
T Cells are tolerized w/in hours, takes only low conc of Ag, and 
tolerance is long lasting - months

B cells tolerized slowly - several days. takes high concentration of Ag. 
tolerance lasts only a few weeks due to rapid renewal of B cell 
population. rarely get complete B cell tolerance. to maintain tolerance, 
need to keep giving tolerogenic epitope in correct form.

LOW ZONE tolerance affects only T cells.
HIGH ZONE tolerance affects T and B cells.

Mechanism of tolerance for mature T and B cells

T cells 0 lack of second costimulatory molecule after Ag expossure...TcR 
+ MHCII+Ag w/o costim --> anergy
missed the B cell part, he's going too fast.

if you give BSA to newborn animal, the animal will be 
tolerant/nonresponsive to that Ag. this is why newborns can't be 
vaccinated right away. can you break tolerance down w/crossreactive ag?

if you have a BSA tolerized animal, and then you give it HSA, you wiill 
see a response, because it isn't tolerized to BSA. now, if you give it a 
challenge of BSA, suddenly it WILL respond because the two epitopes are 
similar, and there is crossreactivity. that's one way of breaking down 
tolerance.

another way of triggering the breakdown of tolerance - you have HGG 
tolerant animal, and you give HGG - there is no response of course. if 
you give HGG WITH LPS, you will get a response - because LPS causes 
nonspecific B cell activation.LPS is a polyclonal stimulator which will 
stimulate anergic b cells to respond.

termination of tolerance:

with time, responsiveness returns: need continuous administration of 
tolerogen to or periodic rechallenge to maintain.

injection of crossreactive Ag will break down tolerance.

injection of crossreactive Ag MAY terminate tolerance to the body's own 
molecules. if you inject an animal with eg thyroglobulin of another 
species, it will make Ab against foreign thyroglobulin as well as its OWN 
thyroglobulin, because the molecules are so similar and have shared 
epitopes.

administration of chemically altered Ag can also break down tolerance.

Mechanisms of Autoimmunity

1. self reactive clones not eliminated? we've said that thymus is 
normally good at deleting all self reactive clones, but a few sneak out. 
since autoimmune dz runs in families, there seems to be genetic 
predisposition for it, and maybe those individuals with that 
predisposition have a problem with negative selection being inefficient, 
so they liberate more self reactive clones into the periphery. 


2. failure of suppressor Tcell function? this is under contention. 
basically folks thought there may be a regulatory or suppressor T cell 
which downregulates response of TH cells and B cells. but we don't have a 
phenotype for these cells. haven't been able to capture and grow in 
culture any T suppressor cells. but there are some interesting exp'ts. if 
you make animal tolerant to given epitope, then take the animal's T cells 
and put them into a new recipient (inbred strain) and then have the new 
recipient challenged, there isn't a response - due to "infectious 
tolerance" - and they think maybe the transferred cells suppress the 
function of the non-tolerized cells in the host animal. so maybe there 
are suppressor cells, or maybe instead of a new class of T cells, these 
transferred cells may function by giving some cytokines to downregulate T 
and B cells - TGFbeta (transforming gf) woud be that cytokine.

3. breakdown of idiotype-antiidiotype network? the idea is that if you 
have a self Ag, because the specificity of interaction at binding site, 
the anti idiotype would be making an Ab fitting into that same site, and 
if you consider the anti idiotype mechanism, once you have one Abthere, 
you can make an anti-Ab, etc etc...so there can be a network of idiotypes 
directed against a combining site, regulating normal production of Ab, eg 
against self epitope, but if this breaks down, then maybe you see 
autoimmune dz. but problem: once you get beyond first antiidiotype, it's 
hard to show anything experimentally. have to take it on faith. note: the 
ANTIIDIOTYPE is the mirror image of the epitope on the Ag, and binds in 
the Ag binding site on the Ab.

4. tolerance broken down by molecular mimicry? postulate: microbes try to 
escape recognition by mimicking self Ag. 

4% of monoclonal Abs to dns/rna viruses crossreact with host epitopes 
expressed on uninfected tissues.

Abs to coxsackie B4 virus Ag also reacts with cardiac muscle tissue. 
(causes myocarditis)

peptide of hepatitis B virus polymerase induces CNS tissue injury similar 
to that induced by MBP (shared epitopes)

protein gliadin (wheat gluten) shares epitopes with E1B protein of human 
adenovirus AD12. gliadin believed to activate coeliac dz - small 
intestinal inflammation. (Abs attack tissues  infected with that virus, I 
guess?)

if you have foreign antigenic epitope presented w/MHCII to T cell 
(w/costim mol) you get a response, yes? notice that some self Ag may look 
very similar to the foreign Ag. so a cell responding to the foreign 
epitope may also respond against your own cell.

assume you have an individual tolerant to own self epitope. you ahve a 
tolerant B cell - an anergic B cell - that normally would not respond to 
self thyroglobulin, because it gets no T cell help. BUT, if presented 
with a cross reactive foreign Ag, eg another species' thyroglobulin, what 
happens is, the autoreactive B cell combines w/foreign Ag, binds T cell 
(NON tolerant) and does get T cell help, so makes autoantibodies (because 
Ab to foreign epitope are ALSO autoantibodies).

under what circumstances do some microbial Ag get presented w/MHCII by 
APC? if you  have individual that normally woudln't have high affinity to 
a given epitope, if it does'nt have high affinity and if B cell isn't 
presenting Ag well, T cell won't respond well and won't be triggered. can 
override this w/high concentration of microbial antigen which is then 
presented in higher quantity, so T cells mature and clone out, and then 
they respond much more readily to APC presenting an epitope, and it no 
longer needs B7 and CD28 - needs other adhesion molecules. increased 
affinity of T cell not due to change in TcR, but due to an overriding of 
low affinity cells by high Ag concentration, causing them to clone out 
with lower requirement for functional activity - so they end up more 
responsive, with fewer requirements for activation. so once you get a 
primed one, if self Ag is presented, it can be triggered much more 
readily because of lower requirements for activation.

how about if you asked why certain tissues do not normally present Ag? 
take something like a pancreas. we make insulin and have islet cells and 
normally insulin isn't presented on the islet cells..why? normally, the 
islet cell doesn't have MHCII, that's why we remain tolerant to insulin 
and cells making it. BUT if you put IFNg gene next to insulin promoter 
gene, the islet cell starts making IFNg whenever it makes insulin. that's 
bad. because with the IFNg, certain tissues can express MHCII under 
stimulation of IFNg. so now you have the self epitope being presented 
w/MHCII. then you get TH cells destroying islet cells. further, if you 
graft in other islet cells, they will be destroyed also. also you have to 
ask why simply having MHCII will trigger the T cells. are there also 
costimulatory molecules? normally no, they do not. at least not B 7. but 
they may make other minor ones. but assuming they don't...you have these 
islet cells w/self epitope in contact w/MHCII - let a few of those cells 
die, and an APC will phagocytose it, and present it to the TH cells WITH 
costim molecules, and you have the same effect. now, all you need is 
focal infxn, viral or bacterial, and when normal T and B cells respond, 
they'l produce IFNg, and that could locally upregulate MHCII production 
and set off this whole thing.

diagram of evasion of controls on self reactivity will be given to 
transcriber.
autoimmune dz can be based on B cells making Ab or on T cells directly 
acting on self epitopes on self cells. what is necessary for either B or 
cytotoxic T cells to cause dz is Th cell HELP. need them to drive 
cytotoxic T cell rxns and B cell Ab production. normally Th cells are 
held in check by either T suppressor cells or by regulatory cytokines, 
depending who you ask (cytokines likely), prostoglandins, transforming 
growth factors, TNF, macrophage products. some Th2 cells are inhibited by 
Th1 cells and vice versa. so normally the Th cells are not going to 
provide the help, unless the regulatory mechanisms are overridden. 

this can occur by exposure suddenly of a sequestered autoantigen, and 
presentation of such by APC. eg, lens protein of eye is normally 
sequestered, and immune system never sees it. if you ahve an eye injury, 
with inflammatory rxn, some people after a few weeks get a horrible 
inflammatory rxn in other eye "sympathetic ophthalmia" - trauma causes 
release of self Ag, and self reactive cells start attacking other eye.

also can have inappropriate MHC expression, as described above w/islet 
cells. usually caused by local infection or trauma/inflammation.

or you can have failure of the normal suppressor/regulatory stuff.

causes of autoimmunity:

multiplicity of factors involved

1.genetic predisposition: MHC gene complex implicated

2. exogenous agents: viruses, bacteria. autoimmune dz may occur after an 
infection - due to molecular mimicry/cross reactivity w/self Ag.

3. self components become immunogenic: modified by drugs, exposed from 
sequestration. parts of cell membrane may be altered by drugs (or 
infection) but this isn't usually a big cause, because the effect is 
usually fleeting. so you can have virus or drug attach to RBC and you can 
get hemolytic anemia because of it, but it's not autoimmune - Ab is 
against the thing SITTING on the cell, not the cell. and it stops when 
you clear the virus or drug.

4. physiological factors: aging, hormones. in humans, females are more 
prone to develop autoimmune dz, and estrogens are implicated. also with 
aging there is more autoimmune dz. why would age be a factor? well, you 
could have a decrease in function of suppressor cytokines so you lose 
normal inhibitions on Th cells.

probably no single causal chain, but complex network of interactions.


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