Mucosal immune system - block D lecture 2

Question 1

what is oral tolerance ?

Answer 1

Oral tolerance (OT) is the term used to refer to the state of unresponsiveness that exists for non-pathogenic antigens present within the gut lumen. This state is NOT a passive lack of response; it is a specifically down-regulated system essential for homeostasis.

Question 2

how do you demonstrate an immune response ?

Answer 2

Demonstrate an immune response, immunise in scruff of neck with soluble antigen in the present of an adjuvant. Then challenge this in the footpad to measure the type of delayed hypersensitivity, will swell in size. If cells taken there would be high levels of antibody and IFN gamma.

If you were to challenge with another antigen, there would be no delayed type hypersensitivity, shows antigen specific.

Feed antigen, repeat after 2 days then challenge in foot pad, then there will be no DTH or antibody and IFN gamma. When challenged with another antigen, there would be no change in response. You would need to challenge with the same antigen that was fed

Question 3

features of oral tolerance induced by ?

Answer 3

Virtually all proteins as well as heavy metals and contact sensitising agents can induce OT, OT can be induced to both humoral and cell mediated immunity. Cell mediated immune responses easier to tolerise than humoral responses as it requires less antigen and lasts longer.

Question 4

are Th1 or Th2 responses easier to tolerise ?

Answer 4

Th1 responses are easier to tolerise than Th2 responses , IgE responses are extremely sensitive to OT, good for using as a therapy.

Question 5

OT on lymphocyte migration ?

Answer 5

OT induces defective lymphocyte migration, as activates the antigen responding T cells in the gut, where once active they do not travel systemically, they stay in the gut.

Question 6

how quickly can OT be induced ?

Answer 6

Tolerance can be induced as quickly as 1-2 days after feeding and lasts ±18 months in the mouse. Antigen persistence is required for maintenance of OT, needs to be present all the time.

Question 7

effector functions (IgE and DTH) most associated with and OT ?

Answer 7

Effector functions (IgE and DTH) most associated with pathology are easiest to tolerise.

Question 8

OT and carrier specificity ?

Answer 8

Carrier specificity uses a hapten and carrier, the carrier can be a large protein such as albumin, and hapten can be the poison ivy or nickel which is pentameric.

If you immunised the mouse with this there would be antibodies against the carrier (albumin) , antibodies against hapten (nickel or poison ivy). Also, there would be antibodies against the conjugate of carrier and hapten.

Tolerance of antibody production can be broken if the defective Th cells are bypassed with an unrelated carrier or LPS

Question 9

factor affecting OT , nature of antigen ?

Answer 9

Active immunity Tolerance

Particulate Soluble

Viable Non-living

Locally invasive Rapidly absorbed ( by epithelail cells)

Peyer’s Patch uptake Mucosal uptake

Question 10

dose and frequency ?

Answer 10

Dose and frequency

low dose versus high dose tolerance

Question 11

genetic background ?

Answer 11

Genetic background have correct MHC phenotype to respond to

coeliac disease is linked closely to HLA-DQw2 locus

Question 12

age ?

Answer 12

Age - young animals are more likely to be primed than tolerised

Question 13

intestinal flora ?

Answer 13

Intestinal flora - germ free mice are resistant to OT

Question 14

nutrition ?

Answer 14

Nutrition -poor nutrition decreases OT

Question 15

immunological status ?

Answer 15

Immunological status - can you tolerise immune animals?

Question 16

mechanisms of OT , clonal deletion ?

Answer 16

Clonal deletion

T cells responding to oral antigens are deleted in same way as selection in thymus.

Question 17

clonal anergy ?

Answer 17

Clonal anergy

T cells exposed to antigen in absence of costimulatory molecules do not respond , T cells know antigen is there by MHC presentation but no danger signal or co stimulatory molecule so no activation.

Question 18

regulatory T cells ?

Answer 18

Regulatory T cells

T cells exposed to antigen in presence of TGF-b and/or IL-10 are polarised to become Treg

TGF-b found in high levels in epithelial cells in gut

Question 19

why does oral tolerance occur ?

Answer 19

Oral tolerance to proteins protects against inappropriate responses to food antigens and commensal bacteria

Question 20

what happens to food we digest ?

Answer 20

Once ingested protein passes through the stomach where it is exposed to gastric acids and enzymes , it then enters the small intestine (SI) and is exposed to pancreatic enzymes. Once in the SI , there are long finger like extension that allow efficient absorption of protein. Protein can be absorbed two ways : either across epithelial cells or by the Peyer’s patch

Question 21

how does epithelial uptake of protein antigens occur ?

Answer 21

epithelial cells- lamina propria

lymphatic system and circulation

Question 22

epithelial cell route ?

Answer 22

the first way is the most important, protein is taken up by epithelial cells, once in the lamina propria it is absorbed by the High endothelial venules , these blood vessels take the protein from the small intestine and merge into the portal vein into the liver where it is processed and enters the circulation through the spleen before coming back to the liver.

Question 23

lymphtic system ?

Answer 23

The other route is entering the lymphatic system where it drains from the gut into the mesenteric lymph node where it leaves via other lymphatic vessels into the thoracic duct into the thymus and goes back into the circulation or by other lymphatics back to the gut

Question 24

what does intestinal processing generate ?

Answer 24

Intestinal processing generates tolerogenic protein, it changes the antigen ingested. Immunological intact protein is absorbed from gut

Question 25

depth explaination of clonal deletion ?

Answer 25

Clonal deletion - remove reactive T cells

Seen when there is a high antigen dose

Mechanism of tolerance of self reactive T cells

Uncommon after peripheral tolerance

Very high doses can induce apoptosis of responding T cells but lower doses do not do this

Tolerance is normal in Fas deficient mice , this is what causes apoptosis

Tolerance cannot be induced in TNF-R1 KO mice , shows TNF-R1 is required for apoptosis.

Question 26

in depth explanation of clonal anergy ?

Answer 26

Clonal anergy

Low dose of antigen over a longer period of time

Highly soluble, monomeric, lacks inflammatory signals

Basis of peripheral tolerance to many self Ag

Can be reversed by addition of IL-2 to activate them and limiting dilution analysis.

Cells proliferate prior to induction of tolerance and give similar numbers of specific T cells, primed at beginning with antigen but after tolerance they no longer proliferate

Inappropriate antigen presentation/ costimulation?

Question 27

can Ag specific cytokine production occur in the abscence of cell proliferation

Answer 27

yes

Question 28

how can tolerance be induced ?

Answer 28

olerance can be induced with peptides, enhanced availability of antigen to APC

Question 29

how is OT prevented ?

Answer 29

OT is prevented if upregulate APC functions are activated for example by LPS

Question 30

what can epithelial cells selectively present Ag to ?

Answer 30

Epithelial cells may selectively present Ag to CD8 regulatory cells lymphocytes surveying the muscosal surfaces.

Question 31

what can unusual population of APC in PP and LP induce ?

Answer 31

Unusual population of APC in PP and LP induce tolerance in naïve recipients.

Question 32

what can local tolerance presentation induce ?

Answer 32

Local presentation of Ag would induce local tolerance.

Question 33

intestinal DC which lack CD80 and CD86 do ?

Answer 33

Intestinal DC lack CD80 and CD86 and cannot respond as lack co stimulatory molecules.

Question 34

what augments and increases OT ?

Answer 34

IL-2 - stimulate production of regT cells

IL-4/IL-10

Anti-IL-12 as they supress Th1 response

LPS

IFN-b

Multiple emulsions

Question 35

what decreases OT as inflammatory cytokines ?

Answer 35

IFN-g

IL-12

Anti-MCP-1

Anti-gd

Cyclophosphamide

Graft versus host disease

Parasite infection

as this provides danger signals so D T cells are activated

Question 36

when does clonal anergy occur ?

Answer 36

Clonal anergy induced when antigen presented in absences of costimulatory antigens

Question 37

bystander supression ?

Answer 37

Associated with feeding of low doses of Ag, OT cells suppress proliferation of unrelated Ag-specific cells - factor is soluble, TGF- b1. T cells induced by OT secrete regulatory cytokines following challenge by OT Ag - suppress inflammation locally. In human autoimmune diseases there are multiple autoantigens (e.g., in MS, there is reactivity to MBP, PLP and MOG) you only need to induce OT to one protein to tolerise all.

Question 38

transplant and bystander suppression ?

Answer 38

In this circumstance, the mouse is tolerised, then the transplant occurs, the transplant is still rejected. However, if there is immunisation around the transplant area, this is successful as the regulatory T cells are supressed which would respond to the MHC mix match. This is bystander supression.

Question 39

role of CD4+ T cells in OT ?

Answer 39

Depletion of CD4 prevents OT , this shows they play a role in OT. Tolerance can be transferred by CD4 cells

Preferential upregulation of T cell subsets

Th2, increased, Th1 decreased

Th1 and Th2 dependent cytokine and antibody responses can be tolerised by different regimes.

IgE is VERY susceptible to OT but IL-4 KO can be tolerised.

CD4 Th3 cells, like Th2 cells produce IL-4 and IL-10 but also produce TGF-b

Question 40

role of gd T cells in OT ?

Answer 40

OT can be transferred with gd CD8+ T cells intraepithelial lymphocytes. Only suppresses IgE and not IgG. Diabetes in NOD mice can be prevented by transfer of tolerised CD8 gd T cells.OT can be prevented or abrogated by depleting gd T cells. OT cannot be induced in defeicinet TcR KO mice

gd T cells may suppress conventional Ag specific ab T cells in an idiotype specific manner. Emphasises role of gd T cells in intestinal homeostasis.

Question 41

is OT induction the result of switching the T helper response ?

Answer 41

OT can be induced normally in anti-IL-4 treated or IL-4 KO mice , shows this is not due to switching.OT is normal in IL-5 and IL-6 KO mice , suggesting these do not play a role.

IL-10 treatment can restore tolerance to microflora , IL-10 KO mice develop IBD in response to gut microflora.

IL-10 and IL-4 are upregulated following OT. OT is normal in IL-10 depleted mice and anti-IL-10 cannot abrogate induced OT. Some experiments that support and refute this , what experiments were they high dose or low dose of antigens, what were the factors.

IFN-g is suppressed by OT but is preceded by priming of Ag-specific IFN- g response. OT of IgE is dependent on IFN- g. OT is normal following IFN- g depletion and in IFN- g R KO mice.

Question 42

where is TGF beta abundant and what is it’s role ?

Answer 42

TGF-b is abundant in gut

mediator of epithelial cell differentiation

IgA class switching

Immunosuppressive

Question 43

why is bystander supression dependent on TGF beta ?

Answer 43

Bystander suppression dependent on TGF-b

TGF-b associated with prevention of colitis

TGF-b secreting CD4 and CD8 isolated from PP and MLN following OT

TGF-b produced by Th3 T regulatory cells, macrophages, enterocytes

Question 44

regulatory T cells ?

Answer 44

Early studies suggested that CD8 regulatory or suppressor T cells mediated OT as the induction of tolerance could be prevented with cyclophosphamide. IL-10 and TGF-b enhanced following oral tolerance. Blocking TGF- b blocks oral tolerance

Treg cells secrete IL-10 and TGF- b.

Question 45

low dose tolerance ?

Answer 45

Low doses of antigen , the APC ( epithelial or dendritic) will lack the co stimulatory molecules and will produce TGF beta and IL-10 to induce reg T cells , T reg cells themselves will also be producing TGF beta and IL-10.

Tr1 will produce IL-10 and TH-3 produce TGF beta, both of these will supress the responses of antigen specific cell and induce the responses to the antigens that are not the same as the inducing antigen (by stander).

Question 46

myasthenia gravis ?

Answer 46

Myasthenia gravis is a reaction to the acetylcholine receptor, by feeding this protein there is prevention of the autoimmune disease in the model.

Some are in clinical trials, but none are in clinical use now

Question 47

peanut allergy and OT ?

Answer 47

Small number of peanut proteins and upped the dose to restore oral tolerance without the anaphylaxis reaction, needs to be done under medical supervision.

Question 48

when can OT be a problem ?

Answer 48

Developing oral vaccines

Oral vaccines against mucosal pathogens would be desirable as they are direct immune response where it is needed, however if oral tolerance then vaccine is not effective.

Oral vaccines mean you don’t need needles expensive, painful) …HIV and hepatitis

Development of recombinant vaccines that could be incorporated into transgenic plants e.g. bananas.