Mucosal Immunity-I

1. Mucosal Immunity-I Sarah Gaffen, Ph.D. Division of Rheumatology & Clinical Immunology Spring 2009 sig65@pitt.edu

2. Overview • Organization- what is a mucosal surface? • Development, NALT, GALT etc • Effectors • IELs • M cells • IgA • Commensal organisms • Mucosal tolerance & homeostasis • Mucosal diseases: lung, gut, mouth

3. Mucosal Immunity • Comprises the “surface exposed” parts of the body (gut, mouth, genital tract, lung, eye, etc) • Contrasts with “systemic immune system” (spleen, LN) • Most infections start at mucosal surfaces • Probably the most evolutionarily ancient

4. Anatomical/Physical Barriers • Skin - acidic, anti-microbial peptides • Mouth - enzymes, anti-microbial peptides, directional flow of fluid to stomach • Stomach- low pH, digestive enzymes, fluid flow to intestine • Large intestine -normal flora compete for niches • Airways and lungs- cilia, mucus, coughing/ sneezing, macrophages in lung alveoli

7. Unique features of Mucosal I.S. • Close association of mucosal epithelial layers and lymphoid structures • Diffuse or organized • Specialized Ag-uptake mechanisms • Example: Microfold (M) cells in the gut • Effectors • IgA • Specialized T’s, activated even w/o infection • Strong immunosuppressive environment • Suppressive responses to commensals

8. Mucosal-Associated Lymphoid Tissue (MALT) • Each mucosal surface has immune tissue, more or less organized • GALT (gut): Peyer’s Patches, appendix • NALT (nasopharynx): Adenoids, Tonsils • BALT (bronchial): iBALT • Genital tract? Not as clear, no lymphoid follicles.

10. Antimicrobial Defenses of the Lung • Structural Defenses • Glottis • Cough reflex • Airway secretions • Mucociliary transport • Innate Defenses • Resident alveolar macrophages • Recruited polymorphonuclear leukocytes • Recruited monocytes

11. Prototypical mucosal surface: Gut • Largest mucosal surface • Specialized Ag-uptake • Large # of specialized lymphocytes • Villi- large surface area for nutrient exchange • Peyer’s Patch • secondary lymphoid organs within wall of intestine • Rich in B cell follicles, smaller T cell areas • Surrounded by epithelium interspersed with “microfold” (M) cells

14. M (microfold) Cells • Transport antigens across the mucosal surface • Transport achieved via vesicles; “transcytosis” • Extensively folded – large surface area • Creates a “pocket” for DCs, lymphocytes • DC’s migrate to: • T cell areas in PP • mesenteric LNs • Can be exploited by pathogens to gain entry into

16. Blue=epithelial cells Red = T cells Green = B cells

17. Luminal antigens are taken up by M cells and presented to T cells by macrophages EMBO reports 7, 7, 688–693 (2006)

19. Lymphocyte circulation in Mucosa • Naïve lymphocytes that arrive at PP or mLNs enter via HEVs, just like other 2° organs • If they are activated, they lose expression of CCR7 and L-selectin (which normally directs cells to peripheral LNs) • They gain expression of the a4:b7 integrin, which binds to “MAdCAM-1” on gut (and other mucosal) endothelial cells • They also express CCR6, CCR9 and CCR10, which directs them to gut

20. Adhesion molecules in the Integrin family are heterodimers sharing similar a and b chains b3-7 Integrins b1 Integrins b2 Integrins MAC-1 CD11b, CD18 aM, b2 a4, b7 VLA-4 a4, b1 LFA-1 aL, b2

21. Initial steps in leukocyte migration are mediated by interactions with endothelium Selectin dep. Integrin dep

23. CCL20 CCR6

24. “Common Mucosal Immune System” • Lymphocytes primed at one mucosal area can recirculate to other mucosal surfaces • This is because MAdCAM-1, mucosal chemokines are expressed on vasculature of other mucosal sites • Therefore, priming at one site can provide protection at another • e.g. Immunization in the nose can protect against infection in lung (flu)

25. The “Common” Mucosal Immune System

26. T Mucosal effector site Mucosal inductive site Antigen Antigen SIgA SIgM (IgG) M a/b g/d M M B T CD8 APC APC T IgG(J) B DC T FDC B T a/b pIgR B B Lymphatic vessel T T (SC) a/b IgA+J T B B CD4 IgA+J IgM+J HEV Naive cells B B T Organized mucosa-associated lymphoid tissue (MALT) B Lymph node B Peyer's patches Isolated lymphoid follicles (ILFs) Appendix Waldeyer's ring (NALT) M cell Endothelial gatekeeper function Peripheral blood

27. IgA • The dominant class of Ab in the mucosal immune system • Can exist in multiple forms • Serum- IgA is mostly monomer • Mucosa • IgA is dimer linked by J chain • After transcytosis, associated with secretory component

28. IgA is the most heterogeneous of Ig isotypes: 3 molecular forms

29. 1% 13% 6% 80% Ig-producing cells in human tissues Mucosal Tissues Systemic Tissues Gastrointestinal, respiratory, Bone marrow, genito-urinary tracts; eyes; lymph nodes, spleen Salivary, lacrimal, mammary glands 1% 17% 30% 52% 10 ~2.5 x 10 cells IgM IgG IgA IgD 10 >6 x 10 cells

30. Daily production rates (mg) of IgA and IgG in humans (70kg adult)

31. IgA • Up to 5 GRAMS of IgA produced daily! • Certain pathogens can cleave IgA as a defense mechanism • How does IgA get across? “Poly-Ig receptor” • Remains tethered to “secretory component” =sIgA • Class switching mediated by TGFb • IgA-secreting B cells express a4b7, which binds to MAdCAM-1 and directs them to mucosal tissues

32. Biological activities of IgA antibodies

33. IgA • Up to 5 GRAMS of IgA produced daily! • Certain pathogens can cleave IgA as a defense mechanism • How does IgA get across? “Poly-Ig receptor” • Remains tethered to “secretory component” =sIgA • Class switching mediated by TGFb • IgA-secreting B cells express a4b7 and CCR6, which directs them to mucosal tissues

34. Figure 9-26 IgA can prevent pathogen adherence to epithelium

35. O O O O O | | | | | P V P S T P P T P S P S T P P T P S P S Cl. ramosum H. influenzae 1 H. aegyptius N. gonorrhoeae 1 N. meningitidis 1 Str. pneumoniae Str. sanguis Str. oralis Str. mitis Gemella haemolysans Prevotella Capnocytophaga H. influenzae 2 H. parahaemolyticus N. gonorrhoeae 2 N. meningitidis 2 Bacterial IgA Proteases-IgA Hinge Region

36. IgA • Up to 5 GRAMS of IgA produced daily! • Certain pathogens can cleave IgA as a defense mechanism • How does IgA get across? “Poly-Ig receptor” • Remains tethered to “secretory component” =sIgA • Class switching mediated by TGFb • IgA-secreting B cells express a4b7 and CCR6, which directs them to mucosal tissues

38. IgA • Up to 5 GRAMS of IgA produced daily! • Certain pathogens can cleave IgA as a defense mechanism • How does IgA get across? “Poly-Ig receptor” • Remains tethered to “secretory component” =sIgA • Class switching mediated by TGFb • IgA-secreting B cells express a4b7 and CCR6, which directs them to mucosal tissues

39. TGF-b is chiefly responsible for directing the IgA ‘switch’

40. IgA • Up to 5 GRAMS of IgA produced daily! • Certain pathogens can cleave IgA as a defense mechanism • How does IgA get across? “Poly-Ig receptor” • Remains tethered to “secretory component” =sIgA • Class switching mediated by TGFb • IgA-secreting B cells express a4b7 and CCR6, which directs them to mucosal tissues

41. Differential expression of chemokines in tissues explains distribution of T and B cells between mucosal effector sites

42. Ontogenic development of mucosal immunity • Newborn infants have virtually no S-IgA in secretionsbut all the cellular machinery is present: • SC in intestine by gestational day ~40 • J chain+ B cells by day ~110 • Mucosal T cells by day ~100-120 • sIgA+ B cells in intestinal aggregates by day ~120 • Peyer’s patches by day ~200 • Ag stimulation required to activate CMIS • colonization by commensal microbiota NB: germ-free animals remain with poorly developed immune systems

43. Development of Igs in Infancy 100 Placental transfer of IgG  % relative to adult levels 0

44. Mucosal T or T-like cells • Located in MALT, also scattered throughout • In the gut, found in: • LAMINA PROPRIA • EPITHELIUM (intraepithelial lymphocytes, IELs) • LP T cells: cause inflammation in disease • IBD, celiac disease (gluten allergy) • May be involved in tolerance • IELs: 90% T cells, 80% are CD8+ • High proportion of gd-T cells, IL-17+ cells

46. Mucosal T cells/IELs • High proportion have memory phenotype • CD45RO (hu) • Gut homing markers (CCR6, CCR9, a4:b7 integrin) • Constitutively produce IFNg, IL-10 • IELs • 80% are CD8+, 50% are a:a form of CD8 (do not see conventional Ags+MHC, but Class Ib MHC) • Many gd+ T cells, high levels of NKG2D (induced in epithelial cells under stress) – repair? Danger? • Intracellular granules with perforin, granzymes • Restricted VDJ usage

48. T cells in PP and lamina propria respond differently to antigens Naive CD4 T cell Intestinal lamina propria Differentiated CD4 T cell pIgA Cytokines B cell plasma cell

49. Characteristics of Intraepithelial lymphocytes (IELs) • Large granular lymphocyte morphology • CD3+, CD8+ • aE, b7 integrin expression • gd TCR+ more common • Alternative pathways of activation • Produce IL-2, IFN-g • Function: cytotoxic, immunoregulatory

50. IL-4 IL-5 IL-10 TH2 IL-2 IFN-g TNF IL-17A IL-6 TNF IL-22 TH1 TH17 CD4+ T-cells IL-4 x x IFN-g x IL-12 TGF-beta, IL-6, IL-23