Functional Anatomy of the Lymph Node and other Secondary Lymphoid Organs

Jan 27, 2015

Elizabeth Repasky, Ph.D. Dept of Immunology- 5-321 CGP

The Immune System scattered organs cells that “wander”

Innate Immunity

• Cells constantly patrol body • Only a few types of cells – Macrophages, DCs, NK cells, neutrophils – Recognize pathogens by the pattern of their microbial surface components rather than by a specific antigenic sequence • Copy number is high • Guaranteed supply • Rapidly recruited to site of infection • Immediate response Adaptive Immunity

• Number of specialized cells (different specificities) ranges in the millions • Copy numbers for each are very low= just a few hundred • Relevant clones must be activated and numbers greatly expanded • Encounters with DC presenting cognate Ag is required and occurs by chance Challenges to the Immune System

• High diversity of B/T–cell repertoire • 20-2,000 cells for each specificity, therefore rather rare • Encounters between APCs and T-cells are “stochastic” • Need to facilitate the encounters with pathogens/ antigen presenting cells • How to solve the “needle in a haystack”?? Adaptive Immunity Depends on Secondary Lymphoid Tissue (SLO) • Secondary (peripheral) lymphoid tissue is specialized to: – Trap antigen/ capture pathogens – Facilitate interactions between cells to initiate an immune response – Provide factors to support survival and differentiation of lymphocytes • Particular organs developed for high throughput: – Lymph node …… filters lymph – Spleen………….. filters blood – Peyer’s patches/tonsils… digestive tract “Lymph nodes are compact immunological projections of the patch of peripheral tissue that they drain.” -Lammerham and Sixt Immuno Rev 2008

• DCs collect information/ antigens in the periphery • migrate by afferent lymphatics to draining LN • “The big advantage of this system is that instead of scanning the whole periphery, naïve T cells just visit lymph nodes…” Lymph node is the best understood SLO

Questions?? 1. How do “the players” enter the lymph node? • Pathogens/ Antigens • APCs (DCs) • Lymphocytes 2. Where do they meet 3. How does the structure facilitate productive interactions 4. How do cells leave Understanding the functional anatomy of the lymph node… “The anatomy of the LNs is complex, extremely dynamic and until 2001, it was largely ignored by immunologists.” -Lammerham and Sixt Immuno Rev 2008

- changed by development of intravital microscopy - 2 photon confocal microscopy allows visualization of living cells in tissues to a depth of several hundred microns

Lymphatics begin in the periphery

System draws pathogens into lymph to filter and engender an immune response; prevents them from entering blood and becoming systemic infection http://www.lymphnotes.com/article.php/id/151/ Lymphatic vessels begin as blind ends in the periphery

Randolph et al, Nat Rev Immunol, April 2005 Lymphatic vessels begin as blind ends in the periphery

polygonal plexus beneath the epidermis

Randolph et al, Nat Rev Immunol, April 2005 Lymphatic Drainage in Skin

Mouse tail

Randolph et al, Nat Rev Immunol, April 2005 Langerhans cells in the skin bacteria

Alitalo et al, Nature 438, 2005 Lymph nodes filter lymph

Lymphatic vessels

Lymph node Lymph node histology

Stroma (fixed cells)- the internal framework and supporting tissue of an organ capsule and trabeculae Parenchyma (migrating cells- the essential, functional cells unique to that organ Cell types in Lymph Node • Langerhan cell- epidermis • Dermal DC • Fibroblastic reticular cell- stromal cell • Follicular dendritic cell (non- hematopoetic) • Macrophage • T cells • B cells • Plasma cells

Lymph node “skeleton”

• Stroma (reticular tissue) is a form of fibrous connective tissue consisting of a 3-d meshwork of reticular cells and reticular fibers. – Fibroblastic reticular cells: star-shaped cells with a central nucleus and many long thin cytoplasmic processes by which the individual cells connect. – Reticular fibers run along the processes and in the space between the cells- are wrapped up by the cells that make them. Parenchyma: predominantly lymphocytes and some macrophages and dendritic cells.

Compartments • Cortex – Follicles – B-cells • Paracortex – T-cells, DCs – HEVs • Medulla – Plasma cells – medullary cords & sinuses Crivatello Trends in Immunology April 2004 Cell Trafficking into and out of lymph nodes: Lymphocytes and dendritic cells enter lymph nodes by different routes. Blood

Lymph

FRC= fibroblastic reticular cells form channels to T-cell zone- guide DC’s to vicinity of HEVs Miyasaka and Tanaka, 2004 Nat Rev Immunol Conduits connect subcapsular sinus and perivenular channel surrounding HEVs

Von Andrian Nat Rev Immunol Nov 2003 “Remote control of monocyte (lymphocyte) recruitment”

Von Andrian Nat Rev Immunol Nov 2003 The conduit system: FIBERS= FRC- fibroblastic reticular cells + enclosed reticular fibers

= reticulum

Lymphocytes & myeloid cells are in spaces

Roozendaal et al. Int Immunol 20:1483-1487 (2008) Fibroblastic reticular cells produce fibers and surround them forming conduits

Bajenoff et al, Immunity 25:889 (2006) SEM picture of lymphocytes associated with FRC fibers in the T cell zone. The arrowheads indicate lymphocyte microvilli extending from the T cell to the FRC fibers.

Bajenoff et al, Immunity 25:889 (2006) DCs sample fluid in conduits

Batista & Harwood. Nature Rev Immunol Jan 2009: 15 Sixt et al Immunity 22: 19 (2005) The Fibroblastic Reticular Cell Conduit

Anderson, AO, and ND Anderson. 1975. Studies on the structure and permeability of the microvasculature in normal rat lymph nodes. Amer J Path 80: 387 (first evidence that FRC Conduit conducted 40KDa Horse Radish Peroxidase tracer from LN Subcapsular Sinus to HEV wall and lumen within a minute after intralymphatic inoculation) Function of conduit system: lymph

• Connects subcapsular sinus to outside of HEVs • Transports small molecules (<70kDa) rapidly to HEVs • Could include chemokines from sites of inflammation= “remote control system” • Larger particulates= Pathogens (bacteria and viruses) rapidly caught by macrophages in subcapsular and medullary sinuses Functions of conduit system: Ag

• Resident DCs interspersed with FRCs sample the fluid inside conduits • Explore lumen of conduits by cellular projections • Pick up antigens and show them to T-cells that pass by to initiate response • Maintenance depends on DCs entering from the site of antigen (emigrated= second wave of antigen presentation) FRCs secrete protein recognized by mAB ERTR-7

• used to identify and visualize FRCs by intravital 2-photon microscopy An ERTR-7-stained thick section (30 μm) presented in 3D, showing a fibrous and complex network of interconnected strands of FRCs. Bajenoff et al, Immunity 25:889 (2006) uropod

A T cell (blue) is shown along with the associated FRC fibers stained with ERTR-7 (green) and desmin (red) mAb ER TR7 reacts with intracellular component of fibroblasts Bajenoff et al, Immunity 25:889 (2006) desmin- intermediate filament found in muscle cells T-cell moving through lymph node stroma

Movie S5. T Cells Migrate along the FRC Network (AVI 18793 kb) Dynamic image of T cell (red) migration along the FRC network (green). The trails of three of the T cells are highlighted in the second part of the movie with colored dots to help visualize the path taken along the fibers by a given T cell (z stack = 12 μM). The playback speed is 300× in the first part of the movie and 150× in the second part when the tracks are highlighted.

Bajenoff et al, Immunity 25:889 (2006) How do T-cells leave the parenchyma and get back into the lymph and get out of the lymph node?

Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells

Grigorova, Schwab, Phan1, Pham, Okada & Cyster

Nature Immunol Jan 2009

Cortical sinus LN

Wheater’s Histology http://www.nature.com/ni/journal/v10/n1/suppinfo/ni.1682_S1.html

T-cell egress to cortical sinuses (Grigorova et al Nature Immun Jan 2009) Supplemental info & links to movies

See movie #2 Cortical sinus entry of T cells is dependent on S1P1 (30 minutes) http://www.nature.com/ni/journal/v10/n1/extref/ni.1682-S3.mov

See movie #3 LYVE-1+ cortical sinus probing by Edg1+/+ and Edg1-/- T cells during entry decision-making (30 minutes)

See movie #9- Flow of cells beneath the capsule at the medullary side (20 minutes)

Proposed Model: 1) cortical sinus probing

2) S1P1 mediated entry 3) capture in area of flow 4) passage to medullary sinuses 5) flushing into efferent lymph What about B cells? Where does Ag recognition occur: B cells recognize whole, unprocessed antigen

1. SCS mΦ 2. Paracortical DC’s

1

2

Batista & Harwood. Nature Rev Immunol Jan 2009: 15 Immune Complexes

• Opsinized antigens= antigens with antibodies bound to them= antigen/antibody complexes • Can “fix” complement-

1. Subcapsular sinus mΦ bind Immune Complexes Complement receptors recognizes C3 fragments of complement FCγRIIB recognizes Ab FC region

1

Batista & Harwood. Nature Rev Immunol Jan 2009: 15

2. B cells enter follicle via HEV and encounter DCs in paracortex

2

Batista & Harwood. Nature Rev Immunol Jan 2009: 15 Activated B cells move to follicles and form germinal centers Two types of lymphoid follicle Mantle Zone (cap) of resting B cells

Germinal Center Light zone: more mature, smaller centrocytes contact follicular dendritic cells Dark zone: closely packed, Primary Secondary rapidly dividing centroblasts

Germinal Centers •Are formed when activated B cells enter lymphoid follicles and proliferate •Somatic hypermutation •Affinity maturation •Isotype switching •Selected B cells will mature to plasma cells or become memory cells Follicular dendritic cell: APC • Present in germinal centers • Specialized non-hematopoetic stromal cell • Displays whole Ag on surface; Ag “depot” prolongs immune response – Have CR1, CR2 complement receptors – Have FC receptors – B-cells can carry Ag to the FDC • Critical for selection of B-cells that will produce antibodies that will recognize the antigen • Since affinity maturation occurs here, play role in selecting high affinity B-cells blue= CD4 T cells

Red= follicular dendritic cells Green= centrocytes (B cells) green= Ki67 proliferating centroblasts, somatic hypermutation Contemporary multiphoton microscopy has revived an interest in the functional histology of the lymphoid organs. The superficial location of some lymph nodes has facilitated visualization of the behavior of these cells and this is a very active research area. Functional Histology of the Spleen

• Technical limitations in Intravital microscopy have prevented visualization of cell interactions and movements in the spleen (depth and anatomical location) • However, can postulate that they are similar to that described for lymph node Spleen

capsule

trabeculae

Red pulp

Central artery

White pulp ~ paracortex Localization of cells in the white pulp Peripheral white pulp (nodules) B-cells S S Red pulp

∗ S

Artery

PALS T-cells Depicted with closed circulation (∗)

Functional histology • T-cell zone- T cells interact with DCs and B cells • B- cell follicles- clonal expansion and activated B cells, isotype switching, somatic hypermutation • Marginal zone- analogous to LN subcapsular sinus – Blood enters MZ and all cells enter the white pulp through the marginal zone – recognition of blood borne pathogens – innate immunity.. macrophages – adaptive immunity..APCs to T cell zone

In gut: Peyer’s patches Each patch is a collection of many individual lymphoid follicles (pink) scattered between the microvilli each may have 70 follicles 20 yr old, colonoscopy- Peyer’s patches form a ring in the distal ileum

Jung et al. Int J Inflam 2010 SEM of M cells

rikenresearch.riken.jp M-cells (microfold cells) at the surface of Peyer’s patches are important for antigen uptake from gut

Dome: has dendritic cells

see Fig 1.20 p22 of Janeway

SED- subepith dome TDA- thymus dependent area Read:

Peyer’s Patch Dendritic Cells Sample antigens by Extending Dendrites through M Cell-Specific Transcellular Pores

Lelouard, Fallet, Bovis, Meresse and Gorvel Gastroenterology 2011 PP DC’s extend processes through M cells to explore lumen

Lelouard et al, Gastroenterology (2012) DC’s & M cells in Peyer’s patches PP DC’s extended processes take up microspheres

M-cell= red DC= green Microsphere= yellow

~20 min

Lelouard et al, Gastroenterology (2012) DC’s & M cells in Peyer’s patches Image/movie websites-

http://www.nature.com/ni/journal/v10/n1/suppinfo/ni.1682_S1.html movies: #2- entry is S1P1 dependent #3- probing #9- exit and flow efferent