1. Functional Anatomy of the Adaptive Immune Response
Jason Cyster
Describe the compartments within lymphoid organs 
Understand the multistep model of leukocyte transmigration
Understand the process of lymphocyte recirculation
Understand the concept of chemoattraction
Explain the process of dendritic cell (DC) maturation and antigen transport
Explain how B cells encounter antigen
Explain how lymphocytes exit from lymphoid organs
Describe the changes that promote T cell ‘homing’ to sites of inflammation
Additional comment: Lymph nodes are also sites of lymphomagenesis and sites of solid tumor metastasis and imaging for LN metastasis by MRI etc is increasingly important for staging tumor progression

2. Comment: have just learnt about how we generate large and diverse repertoires of B and T lymphocytes, so have enormous diversity but only a small number (perhaps 1 per 100,000) are specific for any given pathogen. So, if the cells specific for flu ended up in a LN in your leg, this would not be much help as flu usually infects through the lung. But lymphocytes don’t know there exact specificity as this is generated randomly. Thus the way to ensure that cells of any given specificity get the chance to see their specific pathogen, where ever it may enter the body is to have the lymphocytes continually surveying all the lymphoid tissues in the body.

3. Spleen - A filter of the blood
Two main functions of the spleen 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
Aim of this slide is to briefly discuss unique properties of red-pulp.Skip over the text to discuss while showing next slide.
Lymphoid regions (white-pulp) will be discussed in more detail in the next slides.
Try to not spend as long on the RBC point.

4. Anatomy of the Spleen Follicle Red Pulp (B zone) cord Splenic (venous)
sinus
White-Pulp cord
PALS (periarteriolar lymphoid sheath) or T zone
Pulp
vein
Careful not to spend too long on this slide
Capsule
Trabecular vein
Trabecular artery

5. Lymph contains T and B cells and dendritic cells
Lymphatics
Lymph is filtered by Lymph Nodes before returning to circulation (liters per day)
Lymph contains T and B cells and dendritic cells
(thin walled)
One-way valves
Originate in connective tissue spaces as extensively branched thin walled vessels lined by flattened endothelial cells
Fluid escapes capillaries, some is recovered by venous capillaries; the excess passes into lymphatics
Lymph flows from lymphatic capillaries to larger vessels (one-way valves) which eventually empty via the thoracic duct into veins in the neck (liters per day)
Interposed along lymphatic vessels are lymph nodes which filter the lymph
Lymph contains T cells, B cells and dendritic cells

6. Anatomy of a Lymph Node Filter antigens from the lymph
capsule
medullary sinus
subcapsular sinus
radial sinus
germinal center
(secondary follicle)
Primary follicles
(B zones)
Paracortex (T zone)
mainly T-cells
Cortex (T+B zone)
medulla
Introduce in context of being situated in lymphatic flow, filtering lymph, that there are upstream afferent lymphatics and downstream efferent lymphatics; that medullary region is full of highly phagocytic macrophages that help remove particulate material and prevent from accessing circulation.
Filter antigens from the lymph
for recognition by T and B cells
for destruction by macrophages to prevent systemic spread

7. SECONDARY LYMPHOID ORGANS
Mucosal lymphoid tissue (e.g. Tonsil, Appendix)
Splenic White-Pulp Cord
Lymph Node
lymph fluid
blood
intestinal contents ca
hev
B cell
follicle
T cell
zone
Each type of lymphoid tissue filters a different type of body fluid
Black objects are antigen-presenting cells (will come back to the properties of these cells)
From Goodnow & Cyster, Current Biology 7,R219 (1997)
red-pulp
- filter antigens from body fluids
- bring together antigen presenting cells and lymphocytes
- help bring together antigen reactive B and T cells

8. Lymphocytes traverse HEVs to enter lymph nodes and then compartmentalize in B cell follicles and T cell zones
Follicle or B zone
B cells
FDCs (follicular dendritic cells)
HEV
(High
Endothelial
Venule)
T cell zone (paracortex)
T cells
DCs (dendritic cells)
Another important use of monoclonal antibodies is to stain tissue sections
LN section stained with:
B cell marker
L-selectin ligand

9. The multistep model of leukocyte extravasation

10. Selectins
Selectins are a specialized type of lectin (sugar binding protein) that bind appropriately glycosylated membrane proteins
Three types:
L-selectin: restricted to lymphocytes
ligands on lymphoid tissue HEVs
P-selectin: made by platelets and activated (inflammed) endothelium
E-selectin: made by activated (inflammed) endothelium
E- and P-selectin ligands expressed on neutrophils, monocytes, activated T lymphocytes
L-sel (also known as CD62L) is on the lymphocyte and its ligand is on the endothelium (go back to LN section slide and see red staining for Lsel ligand); it is the opposite arrangement for P and E selectin - the selectin is on the endothelium and the ligands are on the inflammatory cells. However, the mechanism of action is the same (I.e. allow cells to attach to endothelium in a rolling interaction)
Could be good to add a diagram of a leukocyte selectin/ligand interaction

11. Chemoattractant-Cytokines or “Chemokines”
>40, small secreted proteins
Four families: C, CC, CXC, CX3C
chemokine
outside
chemokine receptor
cytoplasm
Cells with the appropriate receptor migrate (chemotax) up chemokine gradient
Chemokines also promote cell adhesion to endothelium
Lymphoid chemokines – help direct the homeostatic trafficking of cells through
lymphoid tissues (e.g. CCR7 / CCL21; CXCR5 / CXCL13)
Inflammatory chemokines – induced at sites of inflammation; can be expressed by many cell types; help recruit cells to these sites (e.g. CXCR2 / IL-8; CCR2 / MCP1)
Avoid any detail on receptor
Plerixafor (trade name Mozobil) is an immunostimulant used to mobilize hematopoietic stem cells in cancer patients. The stem cells are subsequently transplanted back to the patient. Peripheral blood stem cell mobilization, which is important as a source of hematopoietic stem cells for transplantation, is generally performed using granulocyte colony-stimulating factor (G-CSF), but is ineffective in around 15 to 20% of patients. Combination of G-CSF with plerixafor increases the percentage of persons that respond to the therapy and produce enough stem cells for transplantation.[3] The drug is approved for patients with lymphoma and multiple myeloma.[4]

12. Integrins heterodimers of  and  polypeptide chains
can be in inactive (low affinity for ligand) and active states
intracellular signals can cause ‘inside-out’ signaling in the integrin, converting it from an inactive to an active state
chemokine and antigen receptor signaling can activate integrins
bind extracellular matrix proteins (e.g.  binds fibronectin) or transmembrane proteins
(e.g. integrin LFA1 binds ICAM1;
integrin  binds VCAM1)
Subsecond time frame; cell is moving fast in the blood and has only a short time to decide ‘should I stick or should I go’
Switch-blade (mime) activation allows the rapid transition from non-binding to binding
Integrins ‘integrate signals from the outside into cytoskeletal changes’ ‘it sticks things together’  

13. Multistep Model of Lymphocyte Transmigration into Lymph Nodes
Comment that chemokines are secreted proteins but they are basic in charge and they attach to positively charged molecules (proteoglycans) displayed on the endothelium

14. same logic, different ‘area code’
Multistep cascade of lymphocyte migration to site of infection/inflammation:
same logic, different ‘area code’
Important: the chemokine receptor is on the lymphocyte and the chemokine is made by the tissue cells; chemokines are sticky and attach to proteoglycans (sugar-rich protens) on the endothelium
In the healthy tissue, none of these molecules are expressed. In inflammation they are all rapidly induced.
Note: chemokine antagonists are in trial as a method of suppressing inflammatory cell homing
Leukocyte Adhesion Deficiency - affects neutrophil homing to sites of inflammation
…Leukocyte adhesion deficiency (LAD) type I:
defects in b2 integrin -> defective neutrophil migration to inflammed skin, peritoneum; lymphocytes less affected due to continued use of a4b1, a4b7
LAD patients have recurrent bacterial infections
Other types of LAD involve defects in expression of glycosyltransferases needed to make selectin ligands and defects in intracellular signaling molecules needed for chemokine-mediated integrin activation
Natalizumab is administered by intravenous infusion every 28 days. The drug is believed to work by reducing the ability of inflammatory immune cells to attach to and pass through the cell layers lining the intestines and blood–brain barrier. Approved in Associated with 31 cases of the rare neurological condition progressive multifocal leukoencephalopathy (PML).
In Crohn’s Natalizumab may be appropriate in patients who do not respond to medications that block tumor necrosis factor-alpha such as infliximab.
CCR9 antagonist in phase III trial for treatment of Crohn’s
Prostaglandins & leukotrienes can function as chemoattractants
Leukocyte Adhesion Deficiency (LAD) – mutations affecting integrin mediated adhesion
Inhibitor of  integrin (Tysabri) in clinical use for treatment of MS & Crohn’s

15. Schematic view of a lymph node
CXCL13
Follicle
Paracortex
CCL21
Medulla
B zone (follicular) chemokine (CXCL13) attracts B cells to follicles, T zone (paracortical) chemokine (CCL21) attracts T cells to the T zone.
Activated DCs migrate from peripheral sites and enter T zone in response to CCL21
B zones produce a B cell attracting chemokine
T zone produces T cell and DC attracting chemokines
CXCL13 -> CXCR5
CCL21 -> CCR7

16. Schematic view of a lymph node
CXCL13
Follicle
Paracortex
CCL21
Medulla
B zone (follicular) chemokine (CXCL13) attracts B cells to follicles, T zone (paracortical) chemokine (CCL21) attracts T cells to the T zone.
Activated DCs migrate from peripheral sites and enter T zone in response to CCL21
B zones produce a B cell attracting chemokine
T zone produces T cell and DC attracting chemokines
CXCL13 -> CXCR5
CCL21 -> CCR7

17. DCs migrate from periphery to lymphoid organ T zone bearing Ag
immature ‘sentinel’ DCs are present in most tissues, continually sampling for antigen
by pinocytosis, phagocytosis, engulfment of dying cells
detection of pathogen-derived molecules (e.g. LPS, dsRNA, bacterial DNA) or stressed cells (necrotic cells, TNF, IL-1) causes the DCs 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 T zones
upregulate MHC and costimulatory molecules
migrate into lymphoid T zone
present antigen to T cells
Abul covers most of this so is recap; save time and just go on to show the images

18. Immature (sentinel) DCs in tracheal epithelium
longitudinal section
Sections stained in black to detect MHC class II, which is highly expressed on dendritic cells (DCs) in the epithelium as well as by macrophages in deeper layers.
From Schon-Hegrad et al., (1991) J. Exp. Med. 173, 1345
tangential section

19. B cell antigen encounter
lymph fluid
Sinus Macrophage
FDC
hev
FDCs are distinct from T zone dendritic cells (DCs) in multiple ways: FDCs are not bone marrow derived, they do not express MHC class II and they do not internalize or degrade antigen from presentation to T cells
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 (FDCs – tissue resident cells related to fibroblasts) can display antigen on their surface in an intact form for long periods

20. Lymphocytes express a receptor (S1PR1) for S1P
Lymph node egress occurs in response to a circulatory lipid (sphingosine-1-phosphate, S1P)
Lymphocytes express a receptor (S1PR1) for S1P
Egress involves migrating to S1P that is high in blood/lymph and low in the tissue
MEDULLARY SINUSES
S1P
Diagram courtesy of Ted Yednock

21. Lymphocytes express S1PR1 and exit lymphoid organs in response to S1P
thymus, spleen
lymph node, Peyer’s patch
S1PR1
S1PR1
S1P lyase
S1P lyase
FTY720
Immunosuppressant
RBC
S1P
S1P
blood
efferent lymph
Once in the blood, S1P1 is down-modulated and the cell has to enter a lymphoid tissue to regain expression
S1PR1 is required for T cell egress from thymus and for T and B cell egress from spleen, lymph nodes, tonsil
Activated lymphocytes transiently down-regulate S1PR1 and are retained in the responding lymphoid tissue until they become effector cells
FTY720 (Fingolimod) inhibits egress and was FDA approved in 2010 for treatment of relapsing-remitting Multiple Sclerosis

22. Effector T cell Trafficking
Activated T cells exit lymphoid tissue -> circulation
upregulate S1PR1 to exit lymph nodes
S1PR1 inhibitor (Fingolimod) in use for treatment of Multiple Sclerosis
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) and lipids (prostaglandin and leukotriene receptors)
Inhibitors of prostaglandin and leukotriene synthesis have mutliple anti-inflammatory effects, including antagonizing cell recruitment
adhesion molecules (e.g. integrin 1)
blocking antibody (Natalizumab/Tysabri®) in use for treatment of Multiple Sclerosis and Crohn’s disease
Segways into Abul’s talk on CMI
CCR9 antagonist in phase III trial for treatment of Crohn’s (Chemocentryx and GSK)
NSAIDs inhibit cyclooxygenase and reduce prostaglandin synthesis. Corticosteroids inhibit phospholipase A2 production by boosting production of lipocortin, an inhibitor protein.
Zileuton (ZYFLO) is an orally active inhibitor of 5-lipoxygenase, and thus inhibits leukotrienes (LTB4, LTC4, LTD4, and LTE4) formation. Used for the maintenance treatment of asthma