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Home , Follicular B cell, High endothelial venules, Histology, Red pulp, Spleen, Trabecular arteries

Review of basic features of the /Histology of The - Anatomy and Integration in the regulation of immune responses. Advanced E. Repasky 1/28/14 Lymphoid organs are classified as:

Primary lymphoid organs • • marrow • Lymphatic nodules of the distal intestinal tract (e.g. ileum and appendix)

Secondary (effector) lymphoid organs/ • Spleen & nodes (organs) • Mucosal associated lymphoid tissue (MALT), e.g. and lymphatic nodules in the lamina propria

Ross, Fig. 14.1 Primary Lymphoid Organs:

The and the thymus and the Gut-Associated Lymphoid Tissue (e.g. appendix, terminal ileum) are the initial “education centers” of the immune system

In these organs, lymphocytes (T cells in the thymus, B cells in bone marrow and gut) differentiate into immunocompetent cells (i.e. they can recognize “self” vs. “nonself”)

This differentiation is said to be -independent

The lymphocytes then enter the and lymph to populate: • and mucosae • • secondary lymphoid organs

Secondary Lymphoid Organs:

The lymph nodes, lymphatic nodules, , spleen are the secondary “education centers” of the immune system

In these organs, immunocompetent lymphocytes differentiate into immune effector and memory cells that undergo antigen- dependent activation and proliferation in these organs.

These lymphocytes then carry out their functions in the: • connective tissue • secondary lymphoid organs • mucosal surfaces lining epithelia They participate in: • mediated immunity (mostly “cytotoxic” T cells) • Humoral responses (production of ) (B cells, also requires “helper” T cells. Lymphocytes in peripheral blood smear

Mizobuti histology slide set These are B and T-cells that have undergone antigen-INDEPENDENT differentiation and are trafficking through the bloodstream on their way to lymphoid organs/tissue. Cytokines and (along with selectins and integrins) mediate EXTRAvasation of lymphocytes into tissues. Tether Roll Arrest Migrate

blood flow

cytokines chemokines

L. Stoolman APCs and other cells MALT: intraepithelial lymphocytes: γδT-cells (neither helper nor cytotoxic): first to see

U-M Histology Collection Intraepithelial lymphocytes

Shown here in resp. epith.

Homing mediated by “” (a sort of lymphocyte “GPS”)

U-M Histology Collection LYMPHOCYTES IN CONNECTIVE TISSUE: MALT = mucosa-associated lymphoid tissue

LN

Ross and Pawlina, Histology: A Text and Atlas U-M Histology Collection Diffuse lymphoid tissue Primary lymphatic nodule/follicle (LN) Lamina propria (LP) of gut shown here, but can be Aggregation of lymphocytes in lamina propria or found associated with mucosae anywhere in the gut, respiratory, and genitourinary tracts. Secondary follicles/nodules

• Contain germinal centers

• Arise when B-lymphocytes are presented with appropriate antigen, receive T-cell help, and then begin proliferating as

• Lymphoblasts differentiate into plasma cells or memory cells; aberrant lymphoblasts undergo .

Ross and Pawlina, Histology: A Text and Atlas Microfold, or “M” CELLS Modified intestinal epithelial cells that assist in by conveying from the intestinal lumen to underlying compartments housing lymphocytes and .

Source Undetermined M cells: TEM

Source Undetermined After antigen presentation and T-cell help, activated B-cells set up germinal centers in secondary follicles

Secondary follicle germinal centers

• Arise when B-lymphocytes are presented with appropriate antigen, receive T-cell help, and then begin proliferating as lymphoblasts

• Lymphoblasts differentiate into plasma cells or memory cells; aberrant lymphoblasts undergo apoptosis.

Ross and Pawlina, Histology: A Text and Atlas : high magnification

U-M Histology Collection. Slide 175. Source Undetermined Plasma Cells are mature B lymphocytes

U-M Histology Collection Junquiera and Carneiro. Basic Histology. Tenth Ed. 2003 Black arrows indicate several plasma cells White arrows = Golgi regions EM of Plasma Cells

Source Undetermined Source Undetermined So, associated with just about any mucosa (GI, respiratory, genitourinary), you may see:

• Intraepithelial lymphocytes (T-cells) • Diffuse lymphoid tissue: – B-cells – T-cells – APCs • Primary nodules • Secondary nodules – Germinal center with lymphoblasts and mphages

Source Undetermined Regions of extensive lymphoid infiltration: Peyer’s patches

Aggregates of lymphoid follicles in the ileum.

Source Undetermined Appendix

Blind sac extending from the caecum

• primary and secondary follicles in lamina propria and submucosa

• So, clearly a secondary lymphoid …

• However, also a site of antigen-INDEPENDENT differentiation (similar to Bursa of Fabriscus is birds)

• So, also a primary lymphoid organ

Sorry about the various “primary” and “secondary” nomenclature; that’s just the way it is… Ross and Pawlina, Histology: A Text and Atlas Tonsils: MALT of the oropharynx

United States Federal Government S

Ross and Pawlina, Histology: A Text and Atlas The palatine tonsils are paired structures made of dense accumulations of lymphatic tissue located in the mucous membrane of the junction of the oropharynx and oral cavity. The tonsils dip down into the underlying CT, forming crypts. There are also and pharyngeal tonsils (under the roof of the nasopharynx and around the opening of the Eustachian tubes). Key features: crypts, abundant nodules, stratified squamous Wanderlust: lymphocytes don’t just stay in one place

From the MALT, lymphocytes can squeeze into lymph vessels…

S.K. Kim. U-M Histology Collection ..go through larger lymphatic channels in the …

U-M Histology Collection ..and end up at a .

U-M Histology Collection Lymph Nodes

Main functions:

1. Filter lymph, thereby promoting lymphocyte contact with antigen

2. Provides necessary microenvironment for antigen-dependent differentiation

Ross, Fig. 14.1 Lymphoid circulation in the takes place in both the blood stream and the lymphatic vessels, a separate vessel system that carries cells of the lymphoid system and their products (cytokines, , etc.).

United States Federal Government Lymph node structure

Original Image: http://academic.kellogg.cc.mi.us/herbrandsonc/bio201_McKinl ey/f24-10a_lymph_node_and__c.jpg

Ross Textbook of Histology Lymphatic Circulation Through a Lymph Node

Lymph nodes filter lymph

1. Afferent lymphatic vessels drain lymph into the Subcapsular Sinus

2. Lymph then passes to the Trabecular sinuses

3. From there, the lymph goes to the Medullary sinuses.

4. Lymphocytes and macrophages pass easily between these sinuses and the tissue of the lymph node.

5. Macrophages in sinuses Original Image: http://human.freescience.org/images/Illu_lymph_node_structure.png monitor the fluids. Macs phagocytose the antigenic material and present it to T- and B-cells Lymph Node Structure

- Capsule & subcapsular sinus - Trabeculae & trabecular sinuses sinuses contain lymph, macrophages, and reticular cells

- Cortex: • superficial cortex (B-cells) -primary follicles/nodules -secondary follicles/nodules (i.e. with germinal centers)

• “deep” cortex (T-cells, dendritic cells)

- Medulla: • medullary cords (B-cells, plasma cells) • medullary sinuses (lymph, more macrophages, plasma cells, and reticular cells)

U-M Histology Collection High magnification view of a sinus (subcapsular sinus shown here)

U-M Histology Collection M=, Ly=lymphocytes, RF/RC= (and associated ) From the sub-capsular sinus, lymph percolates through trabecular sinuses, and finally into medullary sinuses

U-M Histology Collection Reticular (Reticulin) Fibers

• Form a delicate supporting framework for highly cellular tissues (endocrine glands, lymph nodes, , bone marrow, spleen, ). • Composed mainly of Type III , with a moiety that reduces Ag+ to metallic sliver = argyrophilic. • Special stain: silver impregnation to visualize. • Thinner than type I collagen (Type III fibrils are 30-40 nm diameter; type I fibrils are ~200 nm diameter)

Source Undetermined Reticular Fibers (type III collagen) made by reticular cells (specialized )

Top left: Ross and Pawlina, Histology: A Text and Atlas. Others: Sources Undetermined Medullary sinuses drain into EFFERENT lymphatics that exit from the of the lymph node

U-M Histology Collection Blood Circulation Through a Lymph Node

1. Blood enters through an at the hilus

2. branch from hilar artery to feed into beds

3. Capillary beds are drained by high endothelial *

4. HEVs drain into hilar

*HEVs are sites where lymphocytes can leave blood stream to enter the lymph node tissue bed. U-M Histology Collection U-M Histology Collection

Site of:

• Fluid absorption (via aquaporin-1 channels), which causes lymph flow

• EXIT of lymphocytes from bloodstream via diapedesis

Source Undetermined Source Undetermined Source Undetermined Source Undetermined The Thymus

T-cell education

Self vs. nonself distinctions

Cell-mediated immune functions

Populates effector organs

Lymph nodes Lymphatic nodules Spleen Tonsils Ross, Fig. 14.1 The Thymus is a Primary Lymphoid (Immune) Organ Responsible For the Education of T-Cells

Located over the great vessels of the in the area of the body called the mediastinum

Develops from an invagination of EPITHELIUM of the 3rd pharyngeal pouch, so an endodermal organ.

Specialized epithelial cells (called epithioreticular cells) that are joined to one another by long processes with desmosomes on the extremities of the cells (like starfish joined together at the tips) make up the bag-like support for:

Lymphocytes that, when the organ is young, fill this “bag”.

NOTE: There are generally no B cells in the Thymus.

The Young Thymus Surrounded by a CT capsule; cortex has a lot of lymphocytes, fewer in the medulla THERE ARE NO GERMINAL CENTERS IN THE THYMUS!

Gray’s Anatomy

Ross and Pawlina, Histology: A Text and Atlas Source Undetermined The Thymus undergoes a process called THYMIC INVOLUTION, as T cells leave the thymus to populate other lymphoid effector organs, the organ shrinks, leaving only the epithelioretucular cells

U-M Histology Collection The young thymus

Thymus at puberty

U-M Histology Collection Overview of T-cell “education”

1. Naïve T-cells enter medulla via diapedesis across venules 2. Pass into cortex to undergo POSITIVE selection: • Presented with MHC and self or non-self antigens by ERCs • T-cells that recognize MHCs and self/non-self antigens “pass” this selection process and survive (those that don’t undergo apoptosis) 3.Move into medulla to undergo NEGATIVE selection: • T-cells that recognize SELF antigens displayed by self MHCs (i.e. are :autoreactive”) are eliminated 4.Differentiate into helper (CD4+) or cytotoxic (CD8+) T-cells and leave medulla via diapedesis across venules Blood-Thymus Barrier Education of T-cells must occur in a very controlled environment such that antigens are ONLY presented by epithelial reticular cells.

To ensure that no other cells or free antigens are present, there is a very tight BLOOD-THYMUS BARRIER consisting of: 1. The blood capillary wall • endothelial cells • endothelial cell basal laminae •

2. Perivascular connective tissue • type III collagen • macrophages

3. Epithelioreticular cell layer • basal lamina of the epithelial reticular cells (type I ERCs) • epithelial reticular cells

(NOTE: T-cells can enter thymus ONLY via bloodstream – NO AFFERENT LYMPH VESSELS!) Macrophage

Source Undetermined Source Undetermined Source Undetermined Source Undetermined High mag view of medulla

Source Undetermined T-cells that survive selection process allowed to cross (INTRAvasation) to enter circulation. Hassall’s corpuscles Type VI ERCs; not very well known, but produce interleukins (such as IL-4 and IL-7) and so likely influence T-cell differentiation

Source Undetermined The Spleen

Filters the blood

Destroys old red blood cells

Serves as an immune organ

Divided into (RBC/ recycling) (responsible for immune functions)

Ross, Fig. 14.1 Spleen

• On August 13th, 2012 Jason Witten suffered a lacerated spleen when he was tackled by Oakland Raiders’ linebacker Rolando McClain during the first quarter of the Cowboys’ first preseason game. “To make things even more complicated, the spleen receives 10-15% of the body’s blood supply all on its own. That means that if you bruise it, lacerate it, or, in the worst case scenario, rupture it, the amount of internal bleeding that may result can immediately turn life-threatening. That is not all. Even though it was initially confirmed that Jason Witten had only a laceration, it has been well documented that spleen injuries can worsen, and possibly progress to outright rupture, 10 days or more after the initial even when appropriate precautions are taken. In other words, the Cowboy doctors had to make absolutely sure Witten’s spleen laceration had completely healed before letting him do what tight ends do. A re-injury or rupture of a still injured spleen would have been catastrophic; it is fair to say that the Cowboys were not going to put Witten on the field without medical clearance, signed medical waiver or not. Does any of this sound familiar? It might. If you or any of your friends ever suffered from the dreaded kissing disease , or “mono,” your doctor probably told you that you had to avoid contact sports. The reason is that your spleen helps your body fight certain kinds of , such as ones caused by the Epstein-Barr Virus, the virus responsible for mono. To do so, it goes into overdrive and swells. Just as a balloon gets more and more fragile as it grows, so does the spleen, along with its massive blood supply.”

“If Witten’s spleen did rupture with his original injury or thereafter, surgery would have been necessary to repair the spleen or remove it altogether, as while the spleen is helpful in fighting certain infections, it is not an essential organ. That was not the case, and Witten is off to the races for the Cowboys and fantasy football players everywhere. Unlike a dreaded hamstring strain that can persist all season long, this was a finite injury that has completely healed. Witten has seen limited catches tonight thus far, but it is safe to say that he can be expected to resume his role as one of Tony Romo’s favorite targets very soon.” Physiologic Functions

A. Filtering – splenic blood flow 1. removal of abnormal red blood cells – approximately 20 ml of aged RBC are removed daily

2. removal of abnormal WBC, Howell Jolly bodies

Howell-Jolly bodies are round, purple nuclear fragments of DNA in the red Function

• The spleen is a sophisticated filter that monitors and manages blood cells and immune functions • During fetal development the spleen produces red and white blood cells • By the fifth month of gestation the spleen no longer has hematopoietic function but retains the capacity throughout life • Red cells that pass through the spleen undergo a “cleaning” or repair • Abnormal and old cells are destroyed Function cont.

• lose their nuclear remnants and excess membrane before entering the circulation • RBC’s coated with IgG and IgM are removed and destroyed – The spleen is the site of destruction in autoimmune disease states (ITTP and hemolytic ) – Parasites such as can be removed as well • The spleen is involved in specific and nonspecific immune responses (promotes phagocytosis and destruction of bacteria) Anatomical Organization

MacNeal: 1929 The spleen is organized into regions called the red pulp and white pulp, which are separated by an interface called the (MZ).

Circulation: An “open” circulation: Afferent arterial blood ends in sinusoids at the MZ that surrounds the white pulp. Blood flows through the sinusoid spaces and red pulp into venous sinuses, which collect into efferent .

Splenic red pulp: serves mostly to filter blood and recycle iron from aging red blood cells.

Monitoring of most of the blood occurs in the red pulp and MZ.

Spleen- not only trap and remove blood borne antigens but also initiate innate and adaptive immune responses.

Spleen: anatomy

Cancer.gov, Wikipedia, http://commons.wikimedia.org/wiki/File:Illu_spleen.jpg White Pulp

Spleen Structure

The white pulp is circular in structure and is made up mainly of lymphocytes. It functions in a manner similar to the nodules of the lymph node.

The red pulp surrounds the white pulp and contains mainly red blood cells and macrophages. The main function of the red pulp is to phagocytize old red blood cells. Red Pulp

Localization of macrophage subtypes in the spleen

Section of a mouse spleen showing 1. marginal metallophilic macrophages (CD169+, green, or CD169+ and Treml4+, yellow) 2. marginal zone macrophages (SIGNR1+, blue, or + + SIGNR1 and Treml4v , light blue) By Juliana Idoyaga, Ph.D. Ralph M. + 3. red pulp macrophages (Treml4 , red) Steinman. Rockefeller University

ORGANIZATION OF THE SPLEEN

Ross, 14.29 Immune Functions • Monitoring antigens in blood • Proliferation of lymphocytes Of the Spleen • Production of humoral antibodies

Hematopoietic • Formation of blood cells in fetal life Functions • Removal and destruction of RBCs & platelets • Retrieval of iron from RBC hemoglobin Of the Spleen • Storage of RBCs and platelets (more so in non-human species) Splenic Circulation

1. Blood enters via splenic artery at hilus 2. Splenic artery branches into trabecular (which travel within connective tissue trabeculae). 3. give off branches known as central arteries which leave the and enter the substance of the spleen (covered by a peri-arterial lymphatic sheath). 4. Central arteries branch into penicillar arterioles that piece through the lymphatic sheath and spill into splenic cords. 5. Blood percolates through splenic cords and across walls of splenic sinuses. 6. Splenic sinuses drain into pulp veins. 7. Pulp veins drain into . 8. Trabecular veins drain into splenic vein at the hilus. Wheater’s, Functional Histology, Fifth Edition, 2006 Organization of the spleen: white pulp and red pulp White pulp: lymphatic aggregations around “central” arteries: periarterial lymphatic sheath (PALS): T-cells lymph nodules: B-cells Red pulp: cords and sinuses

U-M Histology Collection White pulp function Blood and antigens pour into red pulp (more on that later) Antigen presentation takes place in MARGINAL ZONE T-cells (from PALS) provide “help” to activate mphages and B-cells • activated mphages stimulated to destroy ingested material (e.g. bacteria) • activated B-cells set up proliferative germinal centers

U-M Histology Collection As the body is exposed to antigens and the immune system mounts an immune response in the form of antibody production, lymph nodules (w/ germinal centers) appear in the white pulp of the spleen.

U-M Histology Collection PALS w/ secondary follicle

Shown here with “central” artery cut in cross section –note that the CA has been pushed off to the side by the rapid expansion of cells in the germinal center (GC)

RP= red pulp MZ= marginal zone (antigen presentation) dashed circle = T-cell rich zone

Ross, plate 35-3 Scanning EM of a Splenic Sinus (SS) and Cord of Billroth

The cords contain, RBCs, (N), macrophages (M), blood platelets (P)

A reticular cell framework (RC) supports the cord. The sinus is bounded by the epithelial cells that form the basket-like structure of the sinus (VS)

Ross 14.30a Spleen (red pulp) at high power (40x)

sinus

cord

cord sinus

U-M Histology Collection Percolation of blood into splenic sinuses

Here, you are inside the sinus looking through to the cord, where both a macrophage (M) and a (N) are outside the sinus. Note that the endothelial cells have a rodlike appearance.

Ross and Pawlina, Histology: A Text and Atlas; Source Undetermined A B Splenic sinuses and cords A. red pulp B. higher mag of venous sinus and C. silver-stained section

C

Original Image: http://immuneweb.xxmu.edu.cn/Lymphoid%20Syste m.files/UntiHE20.jpeg

Ross and Pawlina. Histology: A Text and Atlas, Plate 36. Figure 1, 2, 3. SPLEEN: venous sinus showing rodlike endothelial cells

Source Undetermined SPLENIC CIRCULATION Sinuses drain into splenic pulp veins, which, in turn, drain into trabecular veins. Trabecular veins travel within trabeculae and drain into splenic vein at the hilus. red pulp white pulp

U-M Histology Collection Source Undetermined Sites of Haemopoiesis

• Yolk sac

• Liver and spleen

• Bone marrow – Gradual replacement of active (red) marrow by tissue inactive (fatty) – Expansion can occur during increased need for cell production

Leukocytes in the spleen- See Text in Review by: Bronte and Pittet, 2013 Various subsets of T and B cells Dendritic cells Macrophages that exert discrete functions

Red pulp macrophages are specialized to phagocytose aging red blood cells and regulate iron recycling and release, whereas MZ macrophages and metallophillic macrophages express a unique set of pattern-recognition receptors and removes at least certain types of blood born bacteria and viruses in the MZ

MZ also also contains MZ B cells and DCs which take up passing antigens and migrate to the white pulp to promote antigen presentation to lymphocytes.

Access to white pulp is largely restricted to B cells, CD4+ and CD8+ T cells and DC.

Exit of leukpcytes from spleen occurs motely through the splenic veins in the red pulp, although some cells in the white pulp can exit the organ locally via a network of efferent lymphatic vessels. Control of immune cell migration and functionally occurs also through several types of splenic stromal cells. Immune cell in the spleen PRIOR TO immune activation

Circulating T and B cells- usually gain access to secondary lymphoid organs in search of their cognate antigens.

Trafficking and position of lymphocytes within defined splenic microenvironments enables scanning of APCs and is guided by stromal cell networks, integrins, chemokines, and other factors. Distinct chemokines attract and maintain B and T cells to their respective zones:

CXCL13 attracts B cells expressing the receptor CXCR5 to follicular zones, CCL19 and CCL21 attract CCR7+ T cells and antigen presenting DCs in zones.

IVM studies show that CCR7 ligand interactions not only guide T cell homing, but also stimulate basal T cell motility inside lymphoid organs. Both processes facilitate T cell-DC interactions and thus antigen screening by T cells.

Resident B cell lineages

B cell lineages: Follicular and MZ B cells

Follicular B cells recirculate and participate mainly in T cell –dependent immune responses,

MZ B cells reside between the MZ and red pulp, capture antigens carried in the blood via complement receptors, and promote both T cell independent and dependent immune responses

Follicular and MZ-B cells express CXCR5 and thus can be expected to respond to follicular attracting activity mediated by its ligand CXCL13.

However, MZ B cells can overcome such activity by expressing high amount of the sphingosine-1 phosphate -1 (S1PR1) and S1PR3 receptors. Binding of the lysophopholipid S1P to these receptors triggers a chemotactic response and promotes MZ B cell accumulation in the MZ and red pulp where S1P is found in higher concentrations.

MZ B cells

Were initially viewed as sessile immune cells: they express high amounts of cell surface such as integrins (VLA-4 and LFA-1) which allow them to bind to the stromal cells, and confer resistance to local shear forces of blood flow that would otherwise direct the cells to the circulation.

However, IVM show that MZ B cells shuttle continually between the MZ and follicles/ The mechanisms that control this oscillation involves transient S1PR1 desensitization (for migration from the marginal zone into the follicle) followed by S1PR1 resensitization (for migration back to the MZ).

This provides a mechanism by which these cells can deliver opsonized antigens from the open blood circulation to the follicles. Subsequent antigen presentation to follicular BG cells, most notably by follicular DCs, establishes the humoral immune response.

Unlike MZ B cells, follicular B cells fail to express sufficient amounts of integrins and, for this reason, cannot adhere to the MZ stroma. Together: these findings suggest that distinct integrin-dependent adhesion capabilities of B cells subets force follicular B cells to recirculate to distant follicles in search for antigens but enrich the MZ with a of B cells that is equipped with specialized antigen-sensing functions. NK T cells

The spleen also has a sizable population of NKT cells, which sense antigens and are involved in a broad range of immune responses by secreting cytokines and inducing downstream activation of adaptive immune cell types.

Lipid antigen presentation is facilitated by CD1d, which is expressed at elevated amounts by MZ B cells.

IVM shows that that splenic NK T cells locate mostly in the MZ and red pulp. These cells respond to lipid antigens specifically in these regions but only in presence of MZ B cells. Presumably, MZ B cells undergo physical interactions with NKT cells to facilitate sensing of blood-borne antigens and NKT cell stimulation.

** Activation of both T and NKT cells likely depends on the positioning of B cell lineages in distinct splenic compartments…manipulating B cell compartmentalization should also considerably alter antigen presentation and the outcome of adaptive immune responses. Resident

The spleen hosts all major types of mononuclear phagocytes, including macrophages, DCs and - key protectors of the because they identify pathogens and cellular , remove dying cells and foreign material, regulate tissue and inflammatory responses and shape adaptive immunity. Additionally, they can contribute to many diseases.

Metschnikoff- 1884— Recent research continues to reveal surprising aspects of these cells. Resident phagocytes- cont.

Until recently, tissue resident macrophages were viewed a mostly descendants of circulating monocytes; however, genetic and cell fate mapping studies suggest that in the steady state, most macrophages and monocytes represent distinct lineages.

Circulating monocytes derived from hematopoietic stem cells (HSCs) and discrete intermediary progenitors, which occupy specialized niches of the bone marrow , many tissue resident macrophage populations, including , liver, , peritoneal, bone marrow and red pulp splenic macrophages, are established prior to birth either from elements present in the yolk-sac or from embryonic fetal liver precursors.

Thus, under steady state, most splenic and other macrophages can generated independently of adult HSCs and, by extension, of HSC-derived circulating monocytes. And thus have the capacity to self-maintain throughout adult life. Macrophage maintenance is Not restricted to the steady state, but can be preserved after enhanced cell turnover triggered by exogenous challenges. Discrete Macrophage subsets

MZ macrophages may have a distinct monocytic origin, but the reason remains unclear.

Monocyte precursors may confer unique attributes to these cells. e.g., -derived macrophages appear better equipped than their yolk- sac derived counterparts to trigger and shape immune responses, a feature that might be required for efficient MZ macrophage activity.

MZ macrophages –like gut macrophages,--might never face a truly resting environment but instead be repeatedly exposed to exogenous antigens, which trigger monocytes accumulation and local differentiation into ‘activated” macrophages . Endogenous circulating ligands, such as apoptotic cells, might also promote the “tonic” maintenance of tolerogenic macrophages.

A longstanding paradigm?

…states that monocytes are cells that either circulate freely in blood (CX3CR1 int Ly6C hi cells) or patrol blood vessels (CX3CR1 hi Ly6Clo cells) but irreversibly differentiate into DCs or macrophages upon recruitment to either lymphoid or nonlymphoid tissue.

Departing from this paradigm, bona fide undifferentiated monocytes, including CX3CR1 int Ly6Chi and CX3CR1hi Ly6C lo subsets, accumulate in the spleen under steady state and outnumber circulating monocytes.

IVM indicates that monocytes assemble in cluster of 20-50 cells in the subcapsular red pulp, near venous sinuses and collecting veins, along the perimeter of the organ. The cells closely resemble their blood monocyte counterparts and are morphologically, phenotypically and functionally distinct from splenic macrophages and DCs. How splenic monocytes are maintained and whether they contribute to the replenishment of MZ macrophage is unknown; however, inflammatory signals can mobilize these cells en mass to distant organs. Splenic DCs

Originate from bone marrow HSCs, specialize in antigen processing and presentation, and are key initiators and controllers of adaptive immunity.

Besides IFN producing plamacytoid cells, splenic DCs contain at least two classical subsets:

CD8α+ CD11b- cells present in T cell zones, and responsible for the uptake of dying cells and cross presentation of antigens to CD8+ T cells, and CD8α- CDllb+ cells, preferentially found in the red pulp and MZ, expressing MHC class II peptide complexes and presenting antigens to CD4+ T cells. Cells RECRUITED to the spleen

The spleen has no afferent lymph vessels and collects its leukocytes directly from the blood.

Besides circulating immune cells that continuously migrate into and out of the resting spleen, diseases can recruit additional cells to the organ.

Listeria monocytogenes, a Gram+ bacterium that infects the spleen and liver, involve a sequence of actions that includes CCR2-dependent mobilization of CX3CR1 int Ly6CHi monocytes from the bone marrow to the blood stream, followed by CX3CR1-dependent accumulation in the spleen-- MZ and T cells zones and differentiation in DC-like cells that produce TNF-a and inducible NO synthase …these events are important for early control of the . Cells AMPLIFIED or PRODUCED in the spleen 5 decades of research: at least a fraction of bone marrow HSPC can enter the circulation in the steady state …these circulating HSPCs which resists cell death by upregulating the “don’t eat me” signal CD47, traffic through peripheral tissues and lymphs and can re-engraft bone marrow niches, Extramedullary hematopoiesis: when circulating HSPCs produce lineage-descendant cells outside the bone marrow. - occurs predominantly in the liver in the developing embyo but also in adult tissues including the spleen. Under specific disease conditions, splenic HSPCs profoundly expand and produce progeny locally. Splenic hematopoiesis has been reported in several animal models of disease including , atherosclerosis, myocardial , and .

When it occurs, they are more skewed to myelopoiesis at the expense of and . An active area of investigation involves whether splenic and marrow cells are similar… But, many studies are now showing the accumulation of immature myeloid derived suppressor cells- MDSCs in tumor bearing animal, in the spleen. And also in the tumor stroma. These cells, as well as -macrophage progenitor derived splenic monocytes and neutrophils are CD11b+Gr-1+ in mice and likely overlap with each other. Cells mobilized FROM the spleen

CD4+ and CD8+ T cell redistribution from the spleen to non lymphoid tissues following recognition of cognate antigens in the splenic white pulp.

In addition to lymphocytes, the spleen contributes mononuclear phagocytes in various disease settings.

Ischemic myocardial injury increase the motility of reservoir monocytes and induces a massive exit of this population to the circulation. A fraction of the deployed splenic monocytes accumulates in the ischemic myocardium were they contribute to .

Mechanisms of monocyte release are distinct in bone marrow and spleen: emigrationof bone marrow monocytes depends on CCR2 signaling whereas in the spleen it is controlled by angiotensin II. This hormone, whose concentration increases in serum after , directly contributes to monocyte expulsion by signaling through the angiotensin type 1 receptor expression on the surface of reservoir monocytes. Tumor associated macrophages

In addition to bona-fide , reservoir monocytes, CX3CR1 int Ly6C hi monocytic cells newly produced in the spleen also enter the circulation and relocate to distant tissues. In the context of cancer, these cells can migrate to the tumor stroma and contribute new tumor associate macrophages (TAMS) throughout cancer progression.

This activity might be bad for the host because TAMS can stimulate tumor growth and their density predicts patient survival in several cancer types.

Newly made CX3CR1 int Ly6Chi splenic monocytes contribute macrophages in other diseases, including atherosclerosis, and myocardial infarction.

Spleen and antigen tolerance

Splenectomy can selectively abrogate peripheral tolerance indicating that are involved in this process.

Peripheral tolerance is characterized by systemic immune unresponsiveness to repeatedly applied, or large amount of, antigen (high antigen zone tolerance), or to mucosal contact with the antigen (oral tolerance).

Spleen in mice and humans

Mostly similar anatomically, although the human MZ is more organized, with clearly identifiable outer and inner layers surrounded by a large peri follicular zone.

A big difference is that hypersplenism occurs in mice in response to tumors; this main sign of hypersplenism is not commonly associated with the development of human solid tumors.

Interestingly, spleen enlargement is commonly seen in tumor cell transplantable models, but to lesser extent or not at all in genetically engineered models of cancer in which, nonetheless, splenic HDPC and myeloid activity significantly increase. So, and extramedullary hematopoiesis are not necessary connected.

On the other hand, several examples of increased presence and activity of splenic HSPCs in human suggest that the embryonic role of the spleen in blood formation can be observed in adult life.

Splenic hematopoiesis is observed in metastatic of difference origins, including lung, and …

• http://www.nature.com/nature/journal/v493/n7 434/fig_tab/nature11738_SV2.html • • Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress • · Tal I. Arnon, • · Robert M. Horton, • · Irina L. Grigorova • · & Jason G. Cyster • Nature 493,684–688 (31 January 2013)