1. Immunologic Tolerance

2. Contents Part Ⅰ Introduction
Part Ⅱ Development of Immunologic Tolerance
Part Ⅲ Mechanism of Immunologic Tolerance
Part Ⅳ Immunologic Tolerance and Clinic Medicine

3. Part Ⅰ Introduction
Definition: A type of specific unresponsiveness to an antigen induced by the exposure of specific lymphocytes to that antigen, but response to other antigens normally.
Tolerogens: antigens that induce tolerance

4. General features of Immunologic tolerance
Tolerance is antigenic specific and results from the recognition of antigens by specific lymphocytes.
Normal individuals are tolerant of their own antigens(self antigen)----- Self-tolerance.
Foreign antigens may be administered in ways that preferentially inhibit immune response by inducing tolerance in specific lymphocytes---antigen induction.

5. Immunologic features of tolerance
It is an antigen-induced, active process
Like immunologic memory, it is antigen specific
Like immunologic memory, it can exist in B cells, T cells or both
Like immunologic memory, its easier to induce and lasts longer in T cells than in B cell
Induction of tolerance is very similar to induction of an immune response.
Immunologic features of tolerance:
Tolerance is different from non-specific immunosuppression, and immunodeficiency. It is an active antigen dependent process in response to the antigen. Like immune response tolerance is specific and like immunological memory, it can exist in T-cell, B cells or both and like immunological memory, tolerance at the T cell level is longer lasting than tolerance at the B cell level.

6. Difference of Immuologic tolerance & immunodeficiency, immunosuppression
Immunodeficiency: any condition in which there is deficiency in the production of humoral and /or cell-mediated immunity---non-specificity to Ag
Immunosuppression: The suppression of immune responses to antigens. This can be achieved by various means, including physical, chemical----non-specificity to Ag

7. Part Ⅱ Development of Immunologic Tolerance

8. 1. Induction of immunologic tolerance to antigen in fetal period and neonate period
Owen first observed immunologic tolerance to allogenic antigen in fetal period in 1945

9. cattle of dizygotic twin
Need for tissue and organ graft drove the curiosity to understand the mechanisms of tolerance. The observations a zoologist, Owens, that Dizygotic bovine twins could accept grafts from each other but their siblings from other pregnancies could not tolerate such grafts led Medawar to perform a series of experiments to induce tolerance in mice.

10. Experiment of Medawar on immunologic tolerance
He and his collaborators Brent and Billingham under the hypothesis that early exposure to foreign antigens might facilitate tolerance induction performed this experiment.

12. 2. Induction of immunologic tolerance to antigen in adult
Antigen and immunologic tolerance:
Concentration of antigens
Type of antigen: monomer, aggregates
Pathway of antigen entering body
Features of determinant: tolerogenic epitope
Variation of antigen

13. High and low dose tolerance

14. Tolerance in T and B cells
Induction of tolerance in T cells is easier and requires relatively smaller amounts of tolerogen than tolerance in B cells. Maintenance of immunological tolerance requires persistence of antigen. Tolerance can be broken naturally (as in autoimmune diseases) or artificially (as shown in experimental animals, by x‑irradiation, certain drug treatments and by exposure to cross reactive antigens).
Tolerance may be induced to all epitopes or only some epitopes on an antigen and Tolerance to a single antigen may exist at B cell level or T cells level or at both levels.

15. Factors affecting tolerance role of antigen
Factors which affect response
Favor immune response
Favor tolerance
Physical form of antigen
Route of injection
Dose of antigen
Large, aggregated, complex molecules, properly processed
Subcutaneous or intramuscular
Optimal dose
soluble, aggregate-free, simple small molecules, not processed
Oral or, sometimes, intravenous
Very large or very small dose
Tolerance to soluble antigens:
A state of tolerance to a variety of T-dependent and T-independent antigens has been achieved in various experimental models. Based on these observations it is clear that a number of factors determine whether an antigen will stimulate an immune response or tolerance (Table 1).

16. Individual and immunologic tolerance:
Heredity, Age, Gender, Health

17. Factors affecting tolerance the role of host
Factors that affect response
Favor immune response
Favor tolerance
Age of responding animal
Differentiation state of cells
Fully differentiated; memory T & B cells
Older, immuno-logically mature
Newborn (mice), immuno-logically immature
Relative undifferentiated B cell with only IgM, T cells in the thymic cortex

18. Host age and antigen dose affect tolerance
newborn
adult

19. Part Ⅲ Mechanism of Immunologic Tolerance

20. 1. Central tolerance:
Central tolerance occurs in the central lymphoid organs as a consequence of immature self-reactive lymphocytes recognizing ubiquitous self-antigen.
2. Peripheral tolerance:
tolerance was induced in peripheral organs as a result of mature self-reactive lymphocytes encountering tissue-specific self antigens under particular conditions

22. 1. Central tolerance Clonal deletion (apoptotic cell death)
During maturation of lymphocytes in the thymus for T cell or in the bone marrow for B maturation, immature lymphocytes that recognize ubiquitous self-antigen with high affinity are deleted by negative selection

23. Clonal deletion: negative selection of T cells in the thymus
Clonal deletion: Functionally immature cells of a clone encountering antigen undergo a programmed cell death. For example, auto-reactive T-cell are eliminated in the thymus following interaction with self antigen during their differentiation (negative selection). Clonal deletion has been shown to occur also in the periphery. B cells expressing only IgM (no IgD) on their surface when exposed to antigen are eliminated.

24. Central Tolerance

25. Negative selection of B cells in bone marrow
Clonal deletion: Functionally immature cells of a clone encountering antigen undergo a programmed cell death. For example, auto-reactive T-cell are eliminated in the thymus following interaction with self antigen during their differentiation (negative selection). Clonal deletion has been shown to occur also in the periphery. B cells expressing only IgM (no IgD) on their surface when exposed to antigen are eliminated.

26. 2. Peripheral tolerance ① clonal deletion and clonal ignorance:
large tissue specific antigen delete specific T cells.
self-reactive lymphocytes remain viable and functional but do not react to the self antigens in any detectable way.
② Clonal anergy and inactivation:
functional inactivation without cell death: lack co-stimulatory signal

29. Clonal anergy in T cells
Clonal anergy: Auto-reactive T cells when exposed to antigenic peptides that do not possess co-stimulatory molecules (B7-1 or B7-2) become anergic to the antigen.

30. Clonal anergy in B cells
Also, B cells when exposed to large amounts of soluble antigen down regulate their surface IgM and become anergic and short lived. These cells also up regulate Fas molecules on their surface. An interaction of these B cells with Fas-ligand bearing cells result in their death via apoptosis.

31. ③ Action of Suppressor lymphocyte (Ts)
④ Action of cytokines: TGF- , IL-10
⑤ Holdback in signal tranduction
⑥ Immunologically privileged sites
anatomic barrier: clonal ignorance

32. Pathways to Peripheral Tolerance
Activated T cells
Normal Response
CD28 B7
Proliferation & differentiation
Antigen Recognition without co-stimulation
Anergy
CTLA4 B7
Functionally Unresponsive
CTL4-B7 interaction
Fas
Activation induced cell death
Adapted From:   Van Parijs: Science, Volume 280(5361).April 10,
Fas-FasL interaction
Apoptosis
FasL
Inhibition of proliferation &
effector action
Cytokine-mediated suppression
Cytokine regulation
cytokines

33. The Two Signal Hypothesis for T-cell Activation
Mature
Dendritic
cell
APC
Activated
TH cell TH
cell
MHC II
TCR
CD28 B7
Signal 2

34. Hypothetical mechanism of tolerance in mature T cells
Signal 1
CD28
Resting
B-cell
APC
TH0 cell
Tolerant
T cell
Tolerance (anergy or apoptosis)
from lack of signal 2

35. Summary: Lack of co-stimulation can lead to tolerance (anergy)
Activated T cells
CD28 B7
Proliferation &
differentiation
Normal Response
Antigen Recognition without co-stimulation
Anergy

36. Functionally Unresponsive (Anergic) T cell
Regulation by CTLA-4
CTLA4
CTLA4-B7 interaction
Functionally Unresponsive (Anergic) T cell B7
Activated T cell

37. Functionally Unresponsive T cell
Regulatory T cells
Production of IL-10 or TGF-b
Functionally Unresponsive T cell
Regulatory T cell

38. Pathways to Peripheral Tolerance

39. Inhibition by Antibody Feedback
Passively administered antibody can prevent an antibody response
Antibody produced during an immune responses leads to elimination of antigen (stimulus)
Less antigen available to stimulate specific cells
Immune complexes can bind to inhibitory receptors
Application: RhoGam for Erythroblastosis Fetalis

40. Major Immune Inhibitory Receptors
B cells
FcgRII
T cells
CTLA4
NK cells
KIR (killer cell Ig-like receptors),

41. Anti-Idiotypes and Immune Regulation
Definition
anti-idiotype response-antibody produced against immunoglobulin or TCR idiotypes that serve to down-regulate immune response
The epitope for an responsive anti-idiotype molecule (antibody, BCR, or TCR) is the internal image formed by the CDR region of the respective epitopes antigen receptor

42. Idiotype/Anti-idiotype network

43. Part Ⅳ Immunologic Tolerance and Clinic Medicine

44. 1. To induce immunologic tolerance
Prevent the rejection of organ allografts and xenografts
Treat autoimmune diseases
Treat allergic diseases

45. 2. To terminate immunologic tolerance
To treat tumor:
enhance first signal or second signal
To treat infection diseases

46. And now for a clinical case….

47. Inhibitor Impact
Inhibitors have a major impact on treatment outcome. When inhibitors are present, hemostasis is often difficult to control. Therefore normal therapy typically fails, which leads to increased morbidity and mortality. The presence of inhibitors is also associated with the decreased ability to perform needed or elective surgery. FIX inhibitors pose specific problems in treatment. (See next slide.)

48. Patient Presentation
6 year old male, ER with unexplained bruising associated with minor trauma
Patient has minimal clotting activity
FVIII levels <1% of normal
Patient given i.v. FVIII concentrate i.v. and released but returns in two weeks with same problem
Repeated FVIII treatment
However, FVIII is ineffective.

49. Issues Coagulation factor inhibitors (anti-FVIII activity) Basis?
Lack of tolerance. Why?
Prevalence/impact
20-30% FVIII, less FIX
Treatment/problems
FVIII concentrate or rFVIII
Inhibitors develop that neutralize FVIII
Therapy?
Porcine FVIII with less cross-reactivity
Tolerance (high dose)
Gene therapy
Overview
Inhibitors are antibodies that counteract the therapeutic actions of factor concentrates currently used to treat hemophilia bleeding episodes. Inhibitor development therefore represents one of the greatest obstacles for physicians caring for persons with coagulation disorders. In the first part of today’s presentation, the prevalence and impact of coagulation factor inhibitors will be addressed. In addition, the advantages and disadvantages of current inhibitor treatment options will be presented. The second part of the presentation will discuss recombinant factor VIIa, a new therapeutic agent for managing bleeds in patients with inhibitors to FVIII or FIX. The mechanism of action and production of rFVIIa as well as preclinical and clinical rFVIIa data will also be presented. The final part of the presentation will address future challenges concerning the use of rFVIIa in the treatment and maintenance of bleeding episodes in inhibitor patients.

50. What are Inhibitors? IgG; commonly subclass 4, mixed 1 & 4 Occur in
Congenital factor deficiency = alloimmune
Previously unaffected = autoimmune
Associated with pregnancy, autoimmunity, malignancy, multi-transfusion, advanced age etc.
Inhibitors
Inhibitors in persons with hemophilia A or hemophilia B are almost always alloantibodies of the IgG immunoglobulin class and are of the IgG1 and IgG4 subtypes. Inhibitors occur either in congenital deficiencies (alloimmune states) or they are acquired in previously unaffected individuals (autoimmune states).

51. Summary Definition of immunologic tolerance
Features of immunologic tolerance
Induction of immunologic tolerance
Mechanism of immunologic tolerance
Clinical application of immunologic tolerance