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Organogenesis of secondary

lymphoid tissues (SLT).


Cytokines, chemokines and cell
adhesion molecules.

Lymphoid tissue: primary and secondary


sites.
Primary lymphoid tissue.
Bone marrow, and foetal liver.
Thymus (absent in nude mice whn transcription factor mutation).
Secondary lymphoid tissue (SLT).
Spleen
- Developmentally separate from other SLT. Distinct genes involved:
hox11, Bapx1, Wilms tumour suppressor (WT1), capsulin
- Architecture often disrupted when LN and PP lost by mutation but
the spleen is still there.
Lymph nodes (LN).
- Mucosa-associated lymphoid tissues (MALT).
- Bronchial associated lymphoid tissue (BALT).
- Nasopharyngeal-associated lymphoid tissue (NALT).
- Gut associated lymphoid tissue (GALT).
- Peyers patches (PP).
- Lymphoid clusters.

Function of secondary lymphoid tissue.


To permit efficient interactions between
antigen, antigen-presenting cells, lymphocytes
and other regulatory cells.
To provide a controlled environment for the
development of immune responses.

Lymph node location.


The lymphatic vasculature drains tissue fluid, cells and
antigens from most tissues, through LNs and back into blood
via thoracic duct.

Axillary armpit.
Brachial On bicep, underneath pectorals.
Deep cervical Behind salivary gland.
Superficial cervical In front of salivary gland.
Inguinal Adherent to skin of groin.
Lumbar Behind split of abdominal aorta.
Mediastinal Thymic region.
Mesenteric Mesentery of small intestine
and pancreas.
Popliteal Behind the knee.
Pancreatic Between pancreas and stomach.
Renal Between aorta and kidneys.
Sacral In front of the split of the abdominal aorta.
Sciatic Below sciatic nerve.
Facial draining the face.

They are always in the same place !

HEV

Neonate lymph node structure.

Blood

High endothelial venules: HEV

Afferent lymphatic: antigen, APCs, T cells and tissue fluid.


Network of channels connect
lymphatic to blood vessels.
Pathways for antigens,
other peripheral molecules,
and cells.
Paracortical cords act as T cell
channels.

B
T

Cortex

T
T
Dendritic cells.
Plasma cells
Macrophages

Paracortex

Medulla

Efferent lymphatic: cells, antibodies and tissue fluid.

Neonate Peyers Patch Structure.


Gut lumen
M cells

Follicle

Antigen delivery different


from LNs. Use M (microfold)
cells.
Follicle-associated
epithelium

Villus

HEV

T
Efferent lymphatic vessel

Dendritic cells.

To mesenteric LN.

NALT has a related structure.

Spleen
DCs in T and B cell areas.
Red Pulp

Marginal sinus
Antigen
and cell
entry

Central
arteriole

PALS
T

White Pulp

Conduits
lined with
fibroblasts

Marginal
zone (MZ)
Adherent
MZ B cells

Macrophages

Time line of the development of lymphoid organs.


Different parts of the secondary lymphoid tissue system develop at different times.
Environment induces further enlargement and development after birth.

First PP forms at border of duodenum and ileum, and they are then generated successively, one by
one towards the lower intestine at regular intervals, although the final number is variable.

Revealed by in situ analysis (limited by embryonic LN size) or by inhibiting or activating


various receptors at different points of embryogenesis
(i) blocking lymphotoxin(LT) with a LT-R-Ig fusion protein. Stops PP and LN development.
(ii) using an LT-R agonistic antibody in LT null mice. LNs and PPs are rescued.
(iii) blocking IL7R with an antibody, blocks PP formation. But if PP has started, it finishes.

LT
LT-R-Ig
LT-R
Make a LN
or PP

Stops LN/PP
development
.

Revealed by in situ analysis (limited by LN size) or by inhibiting or activating various receptors


at different points of embryogenesis
(i) blocking lymphotoxin(LT) with a LT-R-Ig fusion protein. Stops PP and LN development.
(ii) using an LT-R agonistic antibody in LT null mice. LNs and PPs are rescued.
(iii) blocking IL7R with an antibody, blocks PP formation. But if PP has started, it finishes.

LT gene
deleted

IL7?

Mimics LT
LT-R

Inhibits IL7R
IL7R
Make a LN
or PP

PPs
LNsNo
andLNs
PPsor
are
rescued

Prevents PP formation

The time at which these inhibitors or activators are added determines


which LNs and PPs are affected.

Mutant mice with defective lymphoid organogenesis


Essential secondary lymphoid tissue (SLT) genes.

Gene deletion (knock-outs) has revealed major molecular players.

Cytokines and receptors.


LT, LT, LTR, TNFR, TranceR,
Trance, IL7, IL7R
Common cytokine receptor
-chain (c).
Signal transduction molecules
and transcription factors.
Jak3, Nik, IKK, rel-a, rel-b, traf6,
Id2, Ror-, Ikaros, NFk2.
Chemokines and receptors.
CCR7, CXCR5, CXCL13.

Other essential genes may be missed if they give a lethal embryonic phenotype (CXCR4).

HOW DO THESE MOLECULES COORDINATE LYMPHOID TISSUE


DEVELOPMENT?
Peyers patches as a model.

Step 1: Formation of the early Peyers Patch

organiser.

Clustering of stromal organiser cells around a lymphatic vessel.


- visualised with antibodies to VCAM-1 or ICAM-1 (Cell Adhesion Molecules).
May be directed by IL7R-expressing cells.
Distribution?
Anti-mesenteric side.

Anti-VCAM-1 stain

Adachi Int Immunol. 9, 507.


Stromal organiser cell (VCAM-1+/ICAM-1+)
IL7R+ cell

How do they know where to start?


Lymphatic

Mesenchyme

Blood vessel

Step 2: Colonising the developing SLT with inducers.


A subset of blood cells migrate out of the blood vessel, between the cells of
the HEV, and into the developing SLT.
These are specialised cells termed Lymphoid Tissue Inducing Cells (LTICs)
or inducers. May be same as early IL7R+ cells.
Further clustering with stromal cells.

chemokines
Stromal organiser cell (VCAM-1/ICAM-1)
IL-7R+ inducer cell ?
MAdCAM-1+ HEV

LTICs (inducers)
Lymphatic

Mesenchyme

Blood vessel

Accompanied by new blood vessel supply angiogenesis.


ECs express markers that allow for blood cell homing.

Are some of the essential SLT genes involved in making the inducer cells?

INDUCER

Stem cell

Multipotent
Precursor.

Restricted blood
borne SLT
inducer.
LN and PP recovered by injecting normal LTIC inducer cells into mice lacking genes
involved in generating the LTICs.

What do the inducers bring with them?


Members of the TNF superfamily central role of Lymphotoxin.

On inducer
cells.
On organiser
cells.

TRANCE
RANKL
OPG

TRANCER
RANK
(on inducer cells)

So, if a receptor is
required for LN
and PP formation,
maybe molecules
involved in
signalling from that
receptor are also
required

Signalling through LTR on organiser cells.

IKK
IKK
IKK
Cytoplasmic
retention proteins.

Removing the LTR signal transduction pathway reduces chemokine


expression by organiser cells, so fewer LTIC inducers are attracted.

*
*
*

Getting the inducer cells to the right place.


Chemokines direct the homing and migration of LTICs during development,
and control leukocytes in the adult.

Blood flow

- Three different chemokines

Rolling

Firm Adhesion

INTEGRINS
Extravasation

SELECTINS

Tissue

CHEMOKINES

Chemokine receptors and integrins on inducer cells.


CXCL13/BLC

CXCL12/
SDF1

IL7R

CD4
CXCR5

CXCR4

INDUCER
CELL

CCL19/ELC

CCR7

CCL21/SLC

41 integrin
(VCAM-1 receptor)
47 integrin
(MAdCAM-1 receptor)

CD11c
L-selectin (PNAd receptor)

Chemokine production by organiser cells causes


Activation of integrins to adhere inducers to endothelial cells at these sites,
via MadCAM-1 (in PP and early LN), or other integrin ligands (VCAM-1).
Null mice: Cooperation between CXCR5 and CCR7 for LN and PP development.

Chemokines are not just chemoattractants

CXCR5/CCR7 co-operation in LN/PP development.


IL7R-/CXCR5-/- or CXCL13-/CCR7-/CXCR5-/-CCR7-/CXCL13-/-IL7R-/MLN Mesenteric LN.

Most LN present. MLN present but smaller. No PP.


Some LN present. MLN present but badly organised. Few PP
Most LN and PP present, but badly organised.
No PPs or LNs except MLN, which is badly organised.
No PPs or LNs, including MLN.

Some LN/PP use CXCL13 only.


Some use CXCL13 and CCL19/21.
MLN use CXCL13 and IL7R ligand.

Honda et al (2001) J. Exp. Med., 193, 621.

Stimulation through CXCR5 and CCR7: not just migration.


CXCL13 through CXCR5, CCL19/21 through CCR7 causes the LTICs to:
- up-regulate Lymphotoxin LT 1 2 expression.
- adhere to VCAM-1 by activating 41 integrin.

LT12

Outcome:
- Adhesion of inducer cells to stromal organiser cells.
- Stimulation of LTR.

AND LTR stimulation causes the up-regulation of the


CXCL13 and CCL19/21 chemokines, and IL7.

POSITIVE FEEDBACK LOOP.

Model for early SLT organogenesis.

LN
Inducer

PP

IL7R
ligands

Organiser

Cytokines, chemokines, and cell adhesion molecules.

What next in Peyers Patch development, once cell clusters form?


EPITHELIAL CHANGES
M cells

Follicle

Follicle-associated
epithelium

B
B

HEV

T
Efferent lymphatic vessel

COMPARTMENTALISATION

Compartmentalisation probably driven by CXCL13, and CCR7


ligands, controlled by TNF family members look in the spleen.
Organisation of splenic B and T cell
zones controlled by chemokines and
TNF family members, even though
the development of the spleen
framework is normal.

CXCR5 B cellls.
CCR7 T cells.
CXCR4 T and B cells.

B cells green
T cells blue

Normal

CCR7-/-

CXCR5-/-

CCR7-/-CXCR5-/-

Lymphotoxin from B cells causes CXCL13 release from splenic


stromal cells in developing follicle, attracting more B cells.

Induction of changes in the local epithelium.

Electron micrographs of
FAE (left) and M cell (right).

Signals from the PP (from B cells?),


induces formation of the Follicle
Associated Epithelium (FAE) involved in
antigen/pathogen uptake.
M cells form and invaginations fill with
lymphocytes (memory B and CD4+ T
cells).
FAE can also regulate cell influx into the PP region.
- FAE makes chemokines: MIP-3, which signals through CCR6, and CCL9, which
signals through CCR1.
- Mice without CCR6 lack DCs under FAE, and cant mount good gut immune response.
- Abs against chemokine CCL9 reported to reduce DC number.

Ectopic lymphoid tissue formation tertiary lymphoid


tissue.
Inappropriate formation of lymphoid tissue can occur in many
inflammatory diseases.

chronic

Autoimmune diabetes.
Rheumatoid arthritis.
H. pylori (stomach) B. burgdorferi (skin) infection.
Hashimotos thyroiditis.
Sjogrens syndrome.

Important for strong responses to autoantigens and loss of self-tolerance.


Therapeutic potential in disrupting these structures?
Transgenic expression of CXCL13/BLC or CCL21/SLC or LT/ in the pancreas,
induces the formation of lymphoid tissue in this tissue.
The chemokine-induced formation of lymphoid tissue in these experiments is
dependent on LT12 and LTR.

Transgenic expression of BLC/CXCL13 in the pancreas


using the Rat insulin promoter.
B cells

T cells

DCs

HEVs

SLC

Accumulate injected labelled B cells

References.
Some of the key papers:

Mebius (2003). Nat Rev Immunol., 3, 292.


Several excellent reviews in Immunological Reviews issue 195
(2003).
Ansel and Cyster (2001) Curr. Opin. Immunol. 13: 172.
Debard (1999) Sem. Immunol., 11:183.
Owen (1999) Sem. Immunol., 11:157.
Pictures from:
Honda (2001) J. Exp. Med., 193: 621.
Adachi (1997) Int. Immunol., 9:507.
Hashi (2001) J. Immunol., 166: 3702.
Neutra (2001) Nat. Immunol., 2: 1004.
Finke (2002) Immunity, 17, 363.
Luther (2003) J Exp Med 197 1191.
Ohl (2003) J Exp Med 197, 1199.