Anatomy of Immune Responses! Micro 204: Molecular and Cellular Immunology!! Lecturer: Jason Cyster!!

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1 Anatomy of Immune Responses! Micro 204: Molecular and Cellular Immunology!! Lecturer: Jason Cyster!!

2 Lecture Outline 1. What are Secondary Lymphoid Organs and how do they function? 2. Why are Dendritic Cells so effective at initiating adaptive immune responses? 3. Where do B cells come in contact with antigen? 4. How do B cells find helper cells specific for the same antigen?

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4 Lymphatics ne-way valves (thin walled) Lymph is filtered by Lymph Nodes before returning to circulation (liters per day) Lymph contains and B cells and dendritic cells

5 SECONDARY LYMPHOID ORGANS Splenic White-Pulp Cord! Lymph Node! Mucosal lymphoid tissue (e.g. onsil, Appendix)! B cell follicle red-pulp! cell zone - filter antigens from body fluids! - bring together antigen presenting cells and lymphocytes! - help bring together antigen reactive B and cells!

6 ymphocytes traverse HEVs to enter lymph nodes and then compartmentalize in B cell follicles and cell zones Follicle or B zone! - B cells! - FDCs HEV! cell area (paracortex)! - cells! - DCs! LN section stained with:! B220! PNAd!

7 Lymphoid organ chemokine expression in lymph node! CXCL13 (BLC)! CCL21 (SLC)! CCL19 (ELC)! -> CXCR5! -> CCR7! -> CCR7! from Cyster, 1999! Science 286, 2098!

8 CXCR5 is required for B cell migration into follicles W B cells -> W! CXCR5-/- B cells -> W! red = transferred B cells! brown = endogenous B cells!

9 Lymphocyte migration within lymphoid tissue wo-photon microscopy of intact lymph node! High speed imaging at depths up to 500 µm! Demonstrate that naïve B and lymphocytes undergo extensive random migration behavior! 5-6 µm / min for B cells! µm / min for cells!

10 he infrastructure of the lymph node! Follicle! zone! Scanning EM of collagen fiber network in rat LN after removal of cells! from Gretz et al., 1997, Imm. Rev. 156, 11!

11 Schematic view of a lymph node Afferent Lymphatic B CXCL13! dendritic cell B zone HEV zone B CCL21! * * * * Efferent Lymphatic Vein Artery In mice lacking CXCL13 or CXCR5, B cells fail to home to B zones (follicles)! In mice lacking CCL21 and CCL19 or CCR7, cells and DCs fail to home to zones!

12 Summary 1! Secondary lymphoid organs:! lymph nodes, spleen, Peyer s patches! function to filter antigen from body fluids! bring together antigen, antigen-presenting cells and antigenspecific lymphocytes! support lymphocyte activation and differentiation events!

13 2. Why are Dendritic Cells (DC) so effective at initiating adaptive immune responses? immature sentinel DCs are present in most tissues, continually sampling their microenvironment for antigen! by pinocytosis, phagocytosis and engulfment of dying cells! detection of pathogen-derived or damage associated signals (e.g. LPS, dsrna, bacterial DNA, necrotic cells, NF, IL-1, CD40L) causes the cells to mature! decrease adhesion to local tissue cells (e.g. keratinocytes)! increase expression of receptors (CCR7) for chemokines made by lymphatic endothelial cells and lymphoid organ zones! process internalized Ag, upregulate MHC and costimulatory molecules! migrate into lymphoid zone! present antigen to cells! Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med :1142.!

14 Immature (sentinel) DCs in peripheral tissue! rat tracheal epithelium! longitudinal section tangential section Schon-Hegrad et al., (1991) J. Exp. Med. 173, 1345

15 Chemokine CCL21 (SLC) expression by lymphatic endothelium! in situ hybridization to detect CCL21 mrna expression! Liver! Small Intestine! gut lumen! bright field! dark field! under bright field illumination, deposited silver grains appear black;! under dark field (Nomarski) optics they appear silver! from Gunn et al.,! 1999, PNAS 95, 258!

16 DCs migrate from periphery to lymphoid organ zone! bearing antigen! contact! sensitizer! (FIC)! Skin draining Lymph Node (day 1)! B zone zone DC ote: immature DC of skin are known as Langerhan s Cells

17 Summary 2! DC are effective at initiating immune responses because:! he immature cells are located in sentinel positions! hey are highly efficient at processing and presenting antigen! hey migrate rapidly to lymphoid zones! hey express high levels of costimulatory molecules for provoking activation of cells! DC influence the differentiation pathway of the cell in terms of cytokine induction and homing receptor profile!

18 3. Where do B cells come in contact with antigen? Sinus Macrophage FDC antigen lymph fluid! hev! B cells bind intact antigen through their surface Ig / B cell receptor (BCR) Antigen that enters via blood or lymph reaches the follicle and can be captured directly by B cells Follicular dendritic cells (FDC) can display antigen on their surface in an intact form for long periods

19 Deposition of Immune Complexes occurs in distinct phases What is an immune complex?! 15 min 2h 8h Capsule Follicle FDC PE Immune Complex Complement Receptor-1 (CD35) B cells (B220) Immune Complexes are made up of Antigen, Antibody (IgM or IgG) and (typically) Complement (C3b). hey are a form of opsonized antigen. hey will usually be multivalent (contain multiple units of the antigen). Antigens coated by C3b alone are also termed opsonized and are handled in a similar way

20 PE-IC association with SCS macrophages and B cells! CD169! PE-IC! B220! 2 h after PE injection!

21 Follicular Dendritic Cells (FDCs) Resident in lymphoid follicles! highly extended processes, can contact many migrating B cells! not of hematopoietic origin and thus not related to DCs of zone (possibly of mesenchymal fibroblastic origin)! produce CXCL13! Development dependent on Lα1β2 and NF! Express receptors that bind antigen coated in complement C3d (CRs) and antibody (FcRs)! Play a role in the Germinal Center reaction! Scanning EM of isolated FDC! Skazal et al JI 134, 1349

22 Multiple modes of follicular B cell encounter with antigen

23 4. How do B cells find helper cells specific for the same antigen?!

24 Changes in lymphocyte homing during! -dependent antibody responses! D C B F B F B G C B B F M P! P! P! P! P! Antigen encounter /B collaboration near the follicle/ zone boundary Plasma Cell and Germinal Center formation B Antigen-specific B cell Antigen-specific cell P! Antigen-specific Plasma cell

25 B cell antigen receptor engagement induces B cell movement to outer zone i.v. antigen 6-8 hr Spleen! Spleen! brown = all endogenous B cells! red = antigen specific B cells!

26 BCR engagement increases CCR7 surface levels! HEL-specific Ig-transgenic! Non-transgenic! CCR7! CXCR5!

27 B cells deficient in zone chemokine receptor fail to migrate to follicle / zone boundary! Wildtype Ig-tg B cells! CCR7 deficient Ig-tg B cells! F F

28 Activated B-cell localization in outer zone determined by balanced responsiveness to and B zone chemokines! zone! B zone (follicle)! vessel! zone! stromal cell! CCR7! +Ag! B! CXCR5! B zone! stromal cell!

29 Migration of activated cells to B cell area is CXCR5 dependent! - some activated CD4 cells become Follicular Helper cells ( FH )! - -upregulate CXCR5 and downregulate CCR7 and S1PR1 (causing them to stay in the responding lymphoid organ and move into follicle)! - upregulate costimulatory molecules (e.g. ICOS) and cytokines (e.g. IL21) that facilitate B cell responses! - -undergo prolonged (SAP-dependent) adhesive interactions with B cells!

30 Summary 4! Antigen specific B cell - CD4 encounter:! Cells move to a common location in lymphoid tissue! B- conjugate pairs are highly motile! Antigen specific conjugates persist for >10 min, some for more than 1 hr! Antigen non-specific conjugates persist <10 min!

31 Effector cell rafficking! Activated cells exit lymphoid tissue -> circulation! ability to re-enter lymphoid tissue is reduced (decrease in CCR7, L- selectin)! Increased ability to enter inflammed tissue due to increased expression of:! ligands for E- and P- selectins! receptors for inflammatory chemokines (e.g. CXCR3)! adhesion molecules (e.g. integrin a4b7)!

32 Effector cells in non-lymphoid tissue! Effector cells attracted to site in response to chemokines! produced by tissue cells exposed to microbial products (e.g. epithelial cells, keratinocytes, mast cells, macrophages)! Macrophages and DCs in tissue present Ag to CD4 cells! CD4 cells release cytokines that activate macrophages to promote killing of ingested organisms! All cells (except RBC) express MHC class I and can be recognized (and killed) by effector CD8 cells!

33 ranslational impact of understanding anatomy of immune responses Augmenting effects of pre-existing antibody on antibody response (vaccination) Use of DCs as anti-cancer adjuvants Use of chemokines to agument vaccine response Shin & Iwasaki 2012 Nature Efforts ongoing to build artificial LNs

34 Vaccines Most vaccines work by inducing neutralizing Abs! attenuated forms of microbes (treated to abolish infectivity and pathogenicity, but retain antigenicity) are most effective! route of administration important e.g. oral administration of Polio vaccine ensures generation of neutralizing IgA! immunization with inactivated microbial toxins generates toxin neutralizing antibodies! Led to world-wide eradication of Small Pox! May soon (?) achieve eradication of Polio! Many vaccines still needed - HIV, Malaria, Schistosomes, Mtb etc!

35 Vaccination and long-lasting immunity Bordetella pertussis, whooping cough and the DP vaccine dilemma B. pertussis lodges into cilia in the respiratory tract U.S. pertussis cases have steadily climbed since switching from inactivated whole cell vaccine (DP) to an acellular vaccine (DaP) containing 1-4 B. pertussis proteins (free of endotoxin)

36 Classification of Licensed Vaccines

37 Recommended Reading! Shin H, Iwasaki A. A vaccine strategy that protects against genital herpes by establishing local memory cells Nature 491(7424):463-7!! Braun A, Worbs, Moschovakis GL, Halle S, Hoffmann K, Bölter J, Münk A, Förster R. Afferent lymph-derived cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration. Nat Immunol Aug 14;12(9):879-87!! Schumann K, Lämmermann, Bruckner M, Legler DF, Polleux J, Spatz JP, Schuler G, Förster R, Lutz MB, Sorokin L, Sixt M. (2010) Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells. Immunity 32, !! Phan G, Grigorova I, Okada, Cyster JG Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat Immunol. 8: !! M. Bajénoff, J. Egen, L. Koo, J. Laugier, F. Brau, N. Glaichenhaus, R. Germain Stromal Cell Networks Regulate Lymphocyte Entry, Migration, and erritoriality in Lymph Nodes. Immunity25, !! Miller MJ, Safrina O, Parker I, Cahalan MD. (2004) Imaging the Single Cell Dynamics of CD4+ Cell Activation by Dendritic Cells in Lymph Nodes. J Exp Med. 200:847-56!! Mempel R, Henrickson SE, Von Andrian UH. (2004) -cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:154-9.!! Okada,., Miller, M.J., Parker, I., Krummel, M.F., Neighbors, M., Hartley, S.B., O Garra, A., Cahalan, M.D. and Cyster, J.G Antigenengaged B cells undergo chemotaxis toward the zone and form motile conjugates with helper cells. PLoS Biology 3: e150.!! Useful Reviews:! Cyster, J.G. (2010) B cell follicles and antigen encounters of the third kind. Nat. Immunol. 11, 989! Lammermann and Sixt M. (2008) he microanatomy of cell responses. Imm. Rev. 221, 26-43! Alvarez D, Vollmann EH and von Andrian UH. (2008) Mechanisms and consequences of dendritic cell migration. Immunity 29, 325! Cyster JG. (2005) Chemokines, Sphingosine-1-Phosphate, and Cell Migration in Secondary Lymphoid Organs.! Annu Rev Immunol ! Itano AA, Jenkins MK. (2003) Antigen presentation to naive CD4 cells in the lymph node. Nat Immunol. 4:733-9

38 Spleen - A filter of the blood wo functions carried out in separate regions! 1) White-pulp is where immune responses against blood-borne antigens occur! 2) Red-pulp is responsible for monitoring and removing old or damaged RBCs!! Red-pulp consists of thin walled venous sinuses and dense collections of blood cells (including numerous macrophages) that form red-pulp cords (or cords of Billroth)! Blood supply: branches of central arteries open directly into red-pulp cords, adjacent to the splenic sinuses (open circulation)! Released RBC must cross the sinus walls; interendothelial slits are a major mechanical barrier and only the most supple, mechanically resilient RBC survive; old and damaged cells are removed by macrophages!

39 Anatomy of! the Spleen! Follicle! (B zone)! Splenic (red pulp)! cord! Splenic (venous)! sinus! PALS or zone! (periarterial lymphoid sheath)! Pulp! vein! Capsule! rabecular artery! rabecular vein!

40 Lymphocyte homing and recirculation (PP)! Adapted from Anderson, artnscience.us