Immune Cells Paul Zhou Institute Pasteur of Shanghai, CAS

1. Immune CellsPaul ZhouInstitute Pasteur of Shanghai, CAS

2. Overview of lymphatic system

3. Lymphatic system1. Consist of organs, nodes and vessels2. Distribution throughout of body 3. Interface with environment4. Circulating throughout body (uni-directional, regional, connected to blood circulation system)5. Immune privilege sites

4. Organ and tissues in immune system1. primary lymphoid organs: bone marrow and thymus- ontogeny and development of lymphocytes and some non-lymphocyte cells (acquisition of capability of antigen recognition, becoming effector/memory cells, and homing)2. secondary lymphoid organs: spleen, lymph nodes, tonsils and Peyer’s patches- antigen encounter and immune activation (and effector response)3. tertiary lymphoid tissues: skin and mucosa- sites of antigen acquisition and processing and effector response

5. Cells in immune system1. Lymphoctyes: T, B and NK cells- determine the immune specificity and orchestrate the effector response (T and B cells); both innate and adaptive immunity (NK cells)2. Non-lymphocytes: monocytes/macrophages, dendritic cells and neutrophils, basophils, eosinophils and mast cells- interact with lymphocytes and play a critical role in the antigen presentation and the mediation of immunologic functions 3. Specialized epithelial and stromal cells- provide the anatomic environment in which immunity occurs

6. Important features in immune cells1. Developed from hematopoietic stem cells- ontogeny and lineage differentiation2. Replenished throughout life- proliferation, differentiation and maturation, circulation and migration3. Have great potential in response to various insults in antigen specific or non-specific manner- activation, proliferation, maturation, circulation and migration4. Activate locally and then migrate (or circulate)5. Expand and contract during immune response6. Cross talk among immune cells and between immune and non-immune cells

7. Leukocytes in blood Cell number/μl Range _______________________________________ Whole blood cells 7,400 4,500 - 11,000 (leukocytes) Neutrophils 4,000 1,800 - 7,000 Eosinophils 200 0 - 450 Basophils 40 0 - 200 Lymphocytes 2,500 1,000 - 4,800 Monocytes 300 0 - 800

8. Topics to be covered in this lecture • Primary lymphoid organs: ontogeny of immune cells • Secondary lymphoid organs: sites of immune activation • Tertiary lymphoid tissues: the sites of antigen acquisition and effector immune response • Migration and homing of immune cells

9. Primary lymphoid organs: ontogeny of immune cells

15. Bone marrow1. Cellular content of bone cavity2. A reservoir for HSCs3. A site of B cell lymphopoiesis throughout life4. sIgM expressing B cells generated in bone marrow migrate to spleen for further maturation

16. B cell differentiation in bone marrow

17. B-2 cells B-1 cells_________________________________________________Adult vs. fetal adult fetalPercentages 95 5Surface markers CD19/CD45RA CD19/CD45RA CD11b+sLgMhighsLgDlowIg repertoire highly diverse limited diverseAntigens T dependent T independent protein in nature variety including carbohydrateRequirement of Th cells yes noIg class switching yes noAffinity maturation yes no

18. Thymic architecture in human and mouse

19. Trafficking of thymocytes in T cell development and selection

20. Maturation of T lymphocytes in thymus

21. Positive and negative selection

23. Secondary lymphoid organs: sites of immune activation

24. Lymphocyte distribution (% of total) __________________________________ Blood Lymph nodes Spleen B cells 10 - 15 20 - 25 40 - 45 Th cells 50 - 60 50 - 60 40 - 50 Tc cells 20 - 25 15 - 20 10 - 15 NK cells about 10 rare about 10

25. Structure and histology of spleen

26. Follicle B cellsMarginal zone B cells

28. Histology of lymph node1. capsule2. HEV3. cortex – para-cortex (T zone) and 4. germinal center 5. sinus6. in and out lymph node

29. Lymph node structure with or without immunization

30. B cell activation and humoral immune response

32. T cell activation and cellular immune response • Two major subsets: α/β TCR and γ/δ TCR • Two lineages in α/β TCR T cells: CD4 and CD8 - differ in antigen recognition as well as regulatory and effector functions • Two subtypes in CD4 T cells: Th1 and Th2 - differ in cytokine secretion and helper functions • Naïve, effector (activated), and memory T cells • Recognize processed antigen/MHC complex on the surface of APCs • T cells become activated through signals 1 and 2 • Activated (effector) T cells become proliferative through IL-2 and IL-2R autocrine and paracrine loop and migrated to infection site(s) • Activated T cell contraction • Central memory and effector memory T cells

33. The Dendritic Cells and lymphocytes on cytospin

34. T Cell Receptors • TCR consists of two subsets: α/β and γ/δ. • Through somatic gene arrangement, the TCR displays extreme sequence diversity. • TCR expresses on the surface of T cells in a clonal fashion, in which to a given T cells only one pair of α/β or γ/δ TCR is expressed. • α/β or γ/δ TCR itself does not mediate signaling. Instead it is intimately associated with CD3 complex (γδ2ε2ζ). It is the latter that mediates TCR signaling. • The signal mediated through TCR/CD3 complex is called the signal one.

35. Human TCR gene organization

36. TCR/CD3 complex

37. Co-stimulatory molecules between T cells and antigen presenting cells

39. Effector functions of T cells • Early changes • Changes in pH • Changes in membrane potential (MTT assay) • Fluxes in cyclic nucleotides and calcium (Ca influx assay) • Late changes • Cyto-skeletal changes (morphologic change) • Activation of the cytolytic mechanism (51Cr and enzyme release assays) • Gene regulation: CD25, CD69, IL-2, IL-3, IFNγ, GM-CSF, CTLA-4, MHC class II, VLA-2, 4F2, transferrin receptors, and insulin receptors, etc. (Elisa, Elispots, intracellular cytokine staining, activation markers) • T cell proliferation (3H-thymidine and CFSE assays as well tetramer staining)

40. Tertiary lymphoid tissues: the sites of antigen acquisition and effector immune response

42. Mucosal tissues

43. Mucosal sites Lymphocyte subsets Distribution (%) Possible functions CD3+ T cells 35–40 CD4+, CD8- 65 Major T-helper cells for mucosal immunity CD4-, CD8+ 30 CTL precursors; regulatory/anergy CD4-, CD8- 2–4 Express gd TCRs Inductive tissues Peyer’s patches Naive 30–40 Circulate within the mucosal system Effector (activated) 30–35 Stimulated through M-cell pathways Memory 30–40 Homing to effector sites B220+ B cells 45–47 Include germinal center where >60% are sIgA+B cells sIgA+ ~8–10 Committed to IgA CD3+ T cells 40–50 CD4+ CD8- ~60–65 Difficult to activate via TCR

44. Mucosal sites Lymphocyte subsets Distribution (%) Possible functions Lamina propria CD4-, CD8+ ~30–35 Mature CTLs; other subset functions? CD4-, CD8- ~2–5 Express gd TCRs Memory >90 sIgA+ B cells 30–50 IgA plasma cells 10–15 Highest numbers of plasma cells in the mammalian immune system Effector tissues CD3+ T cells 85–95 CD4+, CD8- ~5–8 All express ab TCRs Intraepithelial lymphocytes CD4-, CD8+ ~75–80 2/3 are CD8 aa; 80% gd+; 50% ab+ CD4+, CD8+ ~7–10 All express of ab TCRs CD4-, CD8- ~5–8 All express gd TCRs No B cells/plasma cells

47. Skin tissue

48. Primary immune site in skin tissue

49. Background information on DCs • Initial discovery of Langerhans cells in skin by Paul Langerhans in 1868 • Dendritic cells (DCs) first described by Steinman and Cohn in mouse spleen in 1973 • Special properties of DCs in initiating immunity (i.e. antigen and pathogen recognition, uptake, process and presentation as well as pathogen dissemination) were discovered after depletion of monocytes, macrophages and B cells • Reside in most peripheral tissues, especially at sites interface with environment; but migrate to draining lymphoid nodes to activate T cells • Can be generated from their precursor cells ex vivo