1. Immunology: Immune Tolerance Peter A. Savage, Ph.D. Assistant Professor Department of Pathology psavage@bsd.uchicago.edu PATH 30100 01 Cell Pathology / Immunology (Spring 2016) Tuesday, April 19, 2016

2. Mechanisms of T cell tolerance Immune tolerance: a state in which the immune system is unresponsive to substances or tissues that have the capacity to elicit an immune response

3. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

4. Immune Tolerance Immune tolerance: a state in which the immune system is unresponsive to substances or tissues that have the capacity to elicit an immune response Immune tolerance restricts immune responses to: self tissues tumor-expressed antigens chronic viruses commensal microorganisms Classifications: recessive tolerance: autoreactive lymphocytes are deleted or functionally inactivated dominant tolerance: autoreactive lymphocytes are suppressed in trans central tolerance: deletion or inactivation of T/B cells in the thymus or bone marrow peripheral tolerance: deletion, inactivation, or suppression of T/B cells at peripheral sites

5. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

6. The breakdown of immune tolerance to self tissues can lead to autoimmunity

7. Many autoimmune diseases are associated with distinct HLA allotypes, implying T cell-dependent disease pathogenesis

8. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

9. The development of antibody responses requires collaboration of antigen-specific B and T cells

10. Optimal differentiation of CD8 + T cells requires CD4 + T cell help CD4 + helper T cells license DCs via CD40L and secrete paracrine cytokines for CD8 + T cells

11. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

12. Dominant and recessive tolerance collaborate to prevent autoimmunity “recessive tolerance” “dominant tolerance” “recessive tolerance”

13.  T cell development in the thymus Central tolerance: The recognition of self antigen by CD4 + thymocytes can lead to either clonal deletion or differentiation into the Foxp3 + Treg lineage self-reactive “recessive tolerance” “dominant tolerance”

14. Dominant and recessive tolerance collaborate to prevent autoimmunity Recessive tolerance: Antigen-specific lymphocytes reactive to self antigens must be purged or inactivated throughout life. Analogy: constantly plugging a leaky dam Dominant tolerance: Foxp3 + regulatory T cells selected early in life prevent autoimmune reactions in the periphery throughout life. Analogy: training a police force Advantages of dominant tolerance: One Treg cell specific for a self antigen can suppress autoreactive T cells recognizing ANY self antigen presented by the same DC.

15. Regulatory T cells suppress conventional T cells in trans by sequestering B7 costimulatory molecules expressed by antigen presenting cells

16.  T cell development in the thymus The recognition of self antigen by CD4 + thymocytes can lead to either clonal deletion or differentiation into the Foxp3 + Treg lineage self-reactive “recessive tolerance” “dominant tolerance”

17. TCR affinity model of CD4 + T cell development in the thymus high TCR affinity promotes clonal deletion intermediate TCR affinity promotes Treg development

18. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

19. T cells that recognize self antigen with high avidity are selectively purged from the repertoire by clonal deletion Zehn and Bevan. T cells with low avidity for a tissue-restricted antigen routinely evade central and peripheral tolerance and cause autoimmunity. Immunity (2006) vol. 25 (2) pp. 261-70 “V  5”: transgenic TCR  chain reactive to Ova 257-264 peptide presented by K b (CD8 + T cells) “Rip-mOva”: transgene in which membrane-bound Ova is expressed under control of rat insulin promoter (Rip) “K b -Ova 257-264 ”: peptide/MHC tetramer staining reagent used for identification of K b -Ova-specific T cells high-avidity OVA-specific T cells are deleted, but low- avidity cells are spared

20. Low-avidity self-reactive T cells that are spared from clonal deletion can cause autoimmunity when reactivated via bacterial infection “Lm-Ova”: Listeria monocytogenes bacterium engineered to express Ova protein Zehn and Bevan. T cells with low avidity for a tissue-restricted antigen routinely evade central and peripheral tolerance and cause autoimmunity. Immunity (2006) vol. 25 (2) pp. 261-70 V  5xRip-mOva mice develop high blood glucose (a hallmark of diabetes) following infection with Lm-Ova due to autoimmune attack of the pancreas

21. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

22. T cell activation in the absence of costimulatory signals induces anergy B7 CD28

23. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

24. Sakaguchi et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. JI (1995) vol. 155 (3) pp. 1151-64 Identification of CD4 + CD25 + regulatory T cells (CD25 was used a marker of Tregs before the discovery of Foxp3) Transfer of CD4 + CD25 - T cells into immune-deficient (nude) mice induces autoimmunity Co-transfer of CD4 + CD25 + T cells prevents autoimmunity transfer of CD4 + CD25 - conventional T cells induces auto-antibodies co-transfer of CD4 + CD25 + (regulatory) T cells suppresses autoimmunity

25. Brunkow et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. (2001) vol. 27 (1) pp. 68-73 Autoimmunity in scurfy mice is due to a mutation in Foxp3 Scurfy mutant mice develop autoimmunity that can be transferred to new hosts via T cell transfer The phenotype is X-linked, and is caused by mutation of the Foxp3 gene

26. Bennett et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. (2001) vol. 27 (1) pp. 20-1 Human IPEX autoimmune syndrome is associated with mutations in FOXP3 IPEX = Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked

27. Regulatory T cells are required throughout life for the prevention of catastrophic autoimmunity Kim et al. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol (2007) vol. 8 (2) pp. 191-7 Foxp3 DTR mice express the human diphtheria toxin receptor (DTR) in Foxp3-expressing cells Injection of diphtheria toxin (DT) leads to ablation of Foxp3 + Tregs systemic ablation of regulatory T cells leads to fatal autoimmunity within two weeks

28. Dominant and recessive tolerance collaborate to prevent autoimmunity Recessive tolerance: Antigen-specific lymphocytes reactive to all self antigens must be purged or inactivated throughout life. Analogy: constantly plugging a leaky dam Dominant tolerance: Foxp3 + regulatory T cells selected early in life prevent autoimmune reactions in the periphery throughout life. Analogy: training a police force Advantages of dominant tolerance: One Treg cell specific for a self antigen can suppress autoreactive T cells recognizing ANY self antigen presented by the same DC.

29. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

30. Peripherally induced Tregs (pTreg) specific for environmental antigens can develop extrathymically taken from Bilate and Lafaille, Annual Review of Immunology, 2012 pTreg (iTreg) reactive to food or commensal-derived antigens can develop from conventional T cells in the gut

31. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

32. Logistical problem: how does the immune system establish tolerance in the thymus to tissue-restricted antigens that are expressed in the periphery? Patients with the autoimmune syndrome APECED harbor loss-of- function mutations in AIRE

33. Aire-expressing medullary thymic epithelial cells (mTECs) promiscuously express a diverse array of peripheral tissue-restricted antigens Kyewski and Derbinski. Self-representation in the thymus: an extended view. Nat Rev Immunol (2004) vol. 4 (9) pp. 688-98 Aire is a transcriptional regulator that acts on repressed chromatin to promote the promiscuous expression of transcripts in thymic mTECs

34. A dual role for Aire in the enforcement of immunological tolerance? recessive vs. dominant tolerance TRAs medullary thymic epithelial cell (mTEC) Autoimmune regulator (Aire) tissue restricted antigens (TRAs)

35. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

36. Mechanisms of B cell tolerance Also: Without help from CD4 + T cells, T-dependent B cell responses will stall and will not proceed through isotype switching, affinity maturation, etc. Therefore, CD4 + T cell tolerance impinges upon B cell tolerance

37. Autoreactive B cells undergo receptor editing to generate new specificities Pelanda and Torres. Central B-cell tolerance: where selection begins. Cold Spring Harbor Perspectives in Biology (2012) vol. 4 (4) pp. a007146

38. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

40. Dendritic cells continuously present self antigen to T cells under non-inflammatory conditions Scheinecker et al. Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. JEM (2002) vol. 196 (8) pp. 1079-90 dendritic cells (DCs) purified from gastric-draining lymph nodes or peripheral lymph nodes “TXA-23”: CD4 + T cell clone specific for a peptide from the gastric antigen H + /K + -ATPase “D0.11.10”: CD4 + T cell clone specific for Ova peptide low-level stimulation of T cells specific for gastric antigen by DCs isolated from draining lymph nodes

41. Delivery of antigen to dendritic cells under non-inflammatory conditions induces T cell tolerance Bonifaz et al. JEM (2002) vol. 196 (12) pp. 1627-38 “  DEC-205:OVA”: an antibody-Ova fusion protein that targets Ova to DEC-205 + DCs in vivo “isotype:OVA”: an antibody-Ova fusion protein for which the antibody has irrelevant specificity “  CD40”: anti-CD40 antibody, which delivers a strong activation signal to DCs delivery of OVA antigen to DCs via  DEC-205:OVA fusion leads to deletion of OVA-specific T cells

42. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

43. PD-L1 expression on virally-infected cells restricts T cell effector function via the PD-1 T cell inhibitory receptor the inhibitory ligands PD-L1 and PD-L2 can be upregulated by APCs, stromal cells, and tumor cells

44. Immune Tolerance - Outline 1. Breakdown of immune tolerance can lead to autoimmunity 2. A successful immune response requires a number of carefully coordinated collaborative events 3. T cell tolerance a. Recessive T Cell Tolerance Clonal deletion (negative selection) Functional inactivation (anergy) b. Dominant T Cell Tolerance Thymic-derived Foxp3 + regulatory T cells (tTreg) Peripherally induced Tregs specific for environmental antigens (pTreg) c. Aire and promiscuous gene expression in the thymus 4. B cell tolerance receptor editing, clonal deletion, anergy 5. Dendritic cells coordinate tolerance and immunity 6. Inhibitory signals restrict T cell responses CTLA-4, PD-1, TGF-  receptor 7. Immune privileged sites (sequestration from immune recognition)

45. Several facets of the immune system promote selective responses to immunogenic foreign antigens

46. Factors promoting susceptibility to autoimmunity

47. Mechanisms of T cell tolerance Immune tolerance: a state in which the immune system is unresponsive to substances or tissues that have the capacity to elicit an immune response