functional anatomy of the lymph node and other secondary ... · 1/27/2015 · functional anatomy...
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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
http://www.lymphnotes.com/article.php/id/151/
System draws pathogens into lymph to filter and engender an immune response; prevents them from entering blood and becoming systemic infection
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
Randolph et al, Nat Rev Immunol, April 2005
polygonal plexus beneath the epidermis
Lymphatic Drainage in Skin
Randolph et al, Nat Rev Immunol, April 2005
Mouse tail
Langerhans cells in the skin
Alitalo et al, Nature 438, 2005
bacteria
Lymph nodes filter lymph
Lymph node
Lymphatic vessels
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.
Crivatello Trends in Immunology April 2004
Compartments • Cortex
– Follicles – B-cells
• Paracortex – T-cells, DCs – HEVs
• Medulla – Plasma cells – medullary
cords & sinuses
Parenchyma: predominantly lymphocytes and some macrophages and dendritic cells.
Cell Trafficking into and out of lymph nodes: Lymphocytes and dendritic cells enter lymph nodes by different routes.
FRC= fibroblastic reticular cells form channels to T-cell zone- guide DC’s to vicinity of HEVs
Miyasaka and Tanaka, 2004 Nat Rev Immunol
Lymph
Blood
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
Roozendaal et al. Int Immunol 20:1483-1487 (2008)
The conduit system: FIBERS= FRC- fibroblastic reticular cells + enclosed reticular fibers
Lymphocytes & myeloid cells are in spaces
= reticulum
Fibroblastic reticular cells produce fibers and surround them forming conduits
Bajenoff et al, Immunity 25:889 (2006)
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.
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)
A T cell (blue) is shown along with the associated FRC fibers stained with ERTR-7 (green) and desmin (red)
Bajenoff et al, Immunity 25:889 (2006)
uropod
mAb ER TR7 reacts with intracellular component of fibroblasts 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
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) 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)
http://www.nature.com/ni/journal/v10/n1/extref/ni.1682-S3.mov
http://www.nature.com/ni/journal/v10/n1/suppinfo/ni.1682_S1.html
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
Batista & Harwood. Nature Rev Immunol Jan 2009: 15
What about B cells? Where does Ag recognition occur: B cells recognize whole, unprocessed antigen
1. SCS mΦ 2. Paracortical DC’s
1
2
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
Batista & Harwood. Nature Rev Immunol Jan 2009: 15
1
2. B cells enter follicle via HEV and encounter DCs in paracortex
Batista & Harwood. Nature Rev Immunol Jan 2009: 15
2
Activated B cells move to follicles and form germinal centers
Two types of lymphoid follicle
Primary Secondary
Mantle Zone (cap) of resting B cells
Germinal Center Light zone: more mature, smaller centrocytes contact follicular dendritic cells Dark zone: closely packed, 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
Red= follicular dendritic cells Green= centrocytes (B cells)
blue= CD4 T 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
Red pulp
White pulp ~ paracortex
trabeculae
Central artery
Localization of cells in the white pulp
S
S
S
PALS T-cells
Peripheral white pulp (nodules) B-cells
Red pulp
Artery
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
Jung et al. Int J Inflam 2010
20 yr old, colonoscopy- Peyer’s patches form a ring in the distal ileum
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
see Fig 1.20 p22 of Janeway
Dome: has dendritic cells
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
Lelouard et al, Gastroenterology (2012) DC’s & M cells in Peyer’s patches
PP DC’s extend processes through M cells to explore lumen
Lelouard et al, Gastroenterology (2012) DC’s & M cells in Peyer’s patches
M-cell= red DC= green Microsphere= yellow ~20 min
PP DC’s extended processes take up microspheres
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
Image/movie websites-