IMMUNOLOGICAL TOLERANCE AND IMMUNOREGULATION

1. IMMUNOLOGICAL TOLERANCE AND IMMUNOREGULATION Yufang Shi shiyu@umdnj.edu

3. Basic Concepts • Self: • Recognized but not responded to. • Immunological tolerance: • Lack of response to a given antigen, i.e., specific immunological unresponsiveness • Self tolerance: • Individuals normally do not respond to their "self" antigens, i.e., they are "tolerant" to self. This is the basis for self - non self discrimination by the immune system. Loss of self tolerance canlead to autoimmunity. • Immunodeficiency: • Inability of the immune system to respond to a broad range of antigens. • Immunosuppression: • A disorder or condition where the immune response is reduced or absent. • Goal: • Sufficient for protection; prompt downregulation; does not persist after antigenic challenge resolved. Keep memory, eliminate effectors. • A healthy immune system is a balanced immune system, not a strong one!!

4. History • Paul Ehrlich (horror autotoxicus) • The fear of self-poisoning • First postulated the existence of autoimmune diseases in 1901. • 80 autoimmune diseases are known (multiple sclerosis, • type-I diabetes, rheumatoid arthritis, inflammatory • bowel disease, and lupus) • 2. Owen (dizygotic cattle twins, 1945) • Sir Frank Macfarlane Burnet (The Function of Antibodies, 1949) • Embryonic Ag exposure suppress future response • 4. Peter Medawar • (experimentally acquired tolerance, 1953)

5. Timing Cattle Placenta Freemartins Sterile XX calves whose external genitalia appear to be masculinized due to the hormones from the XY twin Found when the affected cow is one of a pair of twins, the other twin being XY. Lillie, F. R. 1917. J. Exp. Zool. 23: 371 - 452 Institut national de recherche pédagogique

6. Ray Owen, 1945 Dizygotic cattle twins that shared the same placenta were blood cell chimaeras, ie contained blood cells of two different haplotypes in adults and, - these two haplotypes co-existed in the adults without mutual rejection. Such chimaeric cattle will exchange skin grafts without rejection. Normally, if cells of one of the two haplotypes were transferred to an adult cattle of the other haplotype, these were promptly rejected. Thus, tolerance in the dizygotic twins resulted from the sharing of the placenta in fetal life, ie from exposure to the antigen during the development of the immune system.

7. Peter Medawar (shared a Nobel Prize with Burnet. 1960): • Autografts succeed, but allografts failed after an initial take. • “Second-set” grafts underwent accelerated rejection. • The destruction of the foreign epidermis was brought about by a mechanism of active immunization Newborn mice of CBA strain received splenocytes from A strain. Once immunological mature, they were unable to reject a graft from the A strain but remained perfectly able to reject an equivalent graft from a third strain.

8. X “Medawar’s” experiment demonstrating neonatal tolerance induction (Nobel Prize)

9. Brent, Human Immunology Volume 52, Issue 2

10. Self tolerance- “A Learning Experience” How does the immune system learn to discriminate between self and non-self during development ? The primary repertoire of T (and B) cells is enormous as a result of combinatorial diversity and imprecisional joining of VDJ or VJ segments. This repertoire contains self-reactive BCRs and TCRs and yet a normal immune system does not exhibit self-reactivity. In the lecture on T cell development mechanisms, we have been explained T cell tolerance for self antigen by negative selection. Through positive and negative selection, the resulting repertoire is therefore one of T cells that react with non-self antigens in association with self-MHC (slightly react to self-antigen plus self-MHC). However, negative selection is not complete and other mechanisms are needed in the peripheral.

11. Types of Tolerance Only antigen receptor-bearing cells can be tolerized • Central Tolerance–happens duringlymphocyte development. • Negative selection or clonal deletion of T and B cells that have receptors for self-antigens. 90% of immature lymphocyte die in thymus/bone marrow • a. TCR/BCR gene rearrangement fails • b. Not selected on thymic epithelial MHC • c. Self antigen reactive: • *Autoimmune regulator (AIRE) gene in thymic epithelial cells • Peripheral Tolerance - occurs in the periphery after lymphocyte development. • Ignorance: low dose or immunologically privileged sites; Self B cells w/o T cells: no response • Clonal contraction and AICD:LPR Fas deficient, Gld FasL deficient. Both have autoimmune phenotypes. Cannot regulate duration of response • Suppression: specific immune regulation through regulatory T cells, TGFb, or IL-10 • Anergy: Lack of co-stimulatory molecules during T cell activation; solubable or low dose antigen • Consequences:apoptosis, anergy, inactivation

12. Tolerance to Self Antigen • Antigen sequestration (lens of eye, spermatozoa). Immunologically privileged sites • Low MHC expression (i.e. hepatocytes) Immune Response to Foreign Antigens • Influence of Antigen Dose (low zone, high zone) timing/duration of exposure routes, nature of antigen, protein > CHO >> lipids, presence of adjuvants • Influence of Antibody feedback inhibition (IgG inhibits IgM), differential antigen binding affinity • Factors favoring tolerance • Age, neurological and endocrine factors, Nutritional status, MHC • Haplotypes

13. Antigen Properties • Molecular weight • smaller favors tolerance • adjuvant, aggregation favors immunogenicity • Dosage • small, large favors tolerance • intermediate favors immunogenicity • Routes of Exposure • IV exposure tolerizes naïve but not memory T cells • Rapid transit to spleen • Binds to resting B cells • Bypasses MHC and costimulatory molecule • Result is anergy • Oral, intratracheal, obital exposure • Can activate mucosal T cells to secrete TGF. special DC • Cryptic epitopes

14. Regulation by antibody (end-product inhibition). • Passively administered antibody can prevent an antibody response (von Behring). This principle is utilized in the prevention of erythroblastosisfetalis by RHOGAM (Many women are given RhoGAM around the 28th week of pregnancy) • 1. Passive antibody inhibits primary response > secondary response. • 2. High affinity antibody of IgG subclass is best. Subunits (Fab) are far less effective. The effect is specific and is primarily on B cells; helper T cells are relatively resistant. • 3. Antibody made in response to an antigen may homeostatically regulate its own level. • -Antibody may act by limiting the amount of antigen (afferent masking), • - by inactivating specific lymphocytes (as central action immune complexes) or • -by blocking immune effector function (efferent inhibition; e.g. in human neoplasia or enhancement of transplanted allografts). • Antibodies directed at idiotypes and anti-idiotypic suppressor cells also play a role in regulating responsiveness. • Epitope on Ig tip

15. Mechanisms of Intravenous immunoglobulin (IVIg) • Modulation of complement activation products • Suppressing idiotypic antibody • Saturating Fc receptors on macrophages • Suppressing various inflammatory mediators including • cytokines, chemokines, and metalloproteinases.

16. Acquired Tolerance • In many cases, experimental unresponsiveness may be mediated by suppressor (T) cells, which actively prevent an immune response. • Original experiments of Gershon and Kondo (1970) showed that T cells were required for tolerance induction. Moreover, T cells from tolerant mice suppressed B cells from normal mice. • Active suppression by T cells also seen in some responses under Ir gene control and in the regulation of IgE responses (Tada). • Suppression commonly induced by systemic administration of antigen-coupled to self cells; route of injection is important (i.v. favors; intradermal gives contact sensitization). • Suppressor T cells produce factors. These factors may act directly on T/B targets. However, until recently, there has been some evidence that these factors are encoded by TCRa and b chain loci. • Can be adaptively transferred.

17. Cells Currently Known to Bear the Capacity to Suppress Immune Responses T Suppressor cells (CD8+), Discovered by Richard Gershon CD4+CD25+ Treg Veto cells CD4-CD8- Th3

18. Five No’s of Suppressor T Cells • No identifiable cell surface marker • No clone or cell lines with suppressor activity • T hybridomas producing antigen specific suppressor factors • has no TCR rearrangement • No I-J gene exist • No antigen specific suppressor factor gene identified

19. Ts Cells are Back Nature Immunology5, 469 - 471 (2004)

20. Regulatory T Cells (CD4+CD25+) • Naïve T cells to syngeneic lymphocyte deficient mice develop multiorgan autoimmunity. Plus activated T cells, prevent • CD4+CD25+GITR+CD45RBlow FOXP3 • Produce TGF, IL-10. • Active in vitro and in vivo • Suppress proliferation of responding CD4+ and CD8+ T cells by inhibiting the production of IL-2, as well as inhibiting the upregulation of IL-2 receptors • Suppress immune response to tumor antigens • Depletion of CD25+ cells, enhances tumor rejection.

22. Veto Cells • The veto cell can be a CD8+ CTL. • For the veto function, the TCR is not required. • The veto function is a backward action of CTL. • Only self-reactive CTL are suppressed by veto cells.

23. Upon recognition of class I on the veto cell by TCR on the effector T cell, Fas is up-regulated on effector cells, allowing for FasL on the veto CTL to induce apoptosis Reich-Zeliger et al. 2004 J. Immunol. 173:6660

24. Th3 From Weiner, Microbes and Infection , 3:947

25. Idiotypic Regulation The idiotypes expressed by T and B cells can be recognized by the immune system. 1. Anti-idiotypic responses may serve to down-regulate an ongoing immune response. 2. Since some anti-idiotypes (Ab2) may look like antigen, they can be used as "vaccines” 3. V region genes code for idiotype 4. Anti-idiotype response can initiate control loops How important is the idiotypic network?

26. IL-4 Morphine Stress Th0 FasL Suicide Th2 Fratricide Fas Th1 TRAIL DR4 Pro-Inflammatory Cytokines Anti-Inflammatory Cytokines IL-4 IL-10 IL-13 IL-5 IL-6 IFN-g IL-2 LT Th1 response Cellular Immunity Th2 response Humoral Immunity T Helper Cell Differentiation and Function Antigen APC IL-12

27. A Model Depicting the Role of Apoptosis in Th1 and Th2 Balance DC1 DC2 TCR ? MHC+Ag MHC+Ag IL-12 Thp IL-4 Stat 4 ERM T-bet Stat 6 GATA-3 c-Maf IRS Th1 Th2 FLIP Suicide Fratricide TRAIL Fratricide CD95L

28. Tolerance can be broken • New clones of T and B cells appear in the absence of antigen • Viral infection can create a cytokine milieu to turn on quiescent (anergic) cells • New epitopes are introduced either by viral modification or iatrogenically

29. Summary • DEFINITION: Tolerance may be defined as a specific unresponsive state induced by prior exposure to an antigen. Therefore, tolerance is a learned process and is specific. • 1. Tolerance is manifested in both T and B cells. • 2. The kinetics of tolerance differ in B vs T cells. • a. T cells exhibit more rapid tolerance induction than B cells. • b. Tolerance is lost in B cells more rapidly than in T cells. • c. T cells are tolerized by lower doses of antigen (tolerogen) than B cells. • Waning reflects the differentiation of new T and B cells in the absence of • sufficient tolerogen, and can be prevented with more antigen. • 4. Tolerance can be broken • a. New clones of T and B cells appear in the absence of antigen • b. Viral infection can create a cytokine milieu to turn on quiescent (anergic) cells • c. New epitopes are introduced either by viral modification or iatrogenically • d. Cross-reactive or modified antigens. New epitopes are introduced either by viral • modification or iatrogenically • 5. Autoimmunity results when tolerance is lost or broken. Often the response is of low avidity to self.

30. Factors Affecting Tolerance Induction A. Age: Young immunologically immature animals show tolerance antigen exposure. 1. Immature B cells lack surface IgD and fail to resynthesize IgM receptors after capping. 2. Antigen is poorly localized and presented in immature animals B. Route of exposure: i.v. or oral exposure favors tolerance. S.c. or intradermal favors immunity. Intramuscular favors Th2. C. Dose of antigen: High doses favor tolerance; however, repeated low doses can also cause tolerance D. Associated antigens: Coupling of antigens to self Ig or self cells enhances tolerogenicity. 1. Coupling nucleosides to self carriers can prevent anti-DNA in genetically autoimmune mice.

31. Immunosuppressive cytokines, factors and drugs TGF, IL-10 Corticosteroids, prostaglandins, Neuropeptides, FasL, TRAIL, TNF CTLA4 Cyclosporin, rapamycin, FK506 Apoptotic Cells???

32. Cell Death and Immune Responses