Organogenesis of secondary lymphoid tissues (SLT).

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Organogenesis of secondary lymphoid tissues (SLT).

• Cytokines, chemokines and cell adhesion molecules.

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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.

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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.

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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 !
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Neonate lymph node structure.
B
B
T
T
T
T
Plasma cells Macrophages
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Neonate Peyers Patch Structure.
Gut lumen
Follicle
Villus
HEV
B
B
T
T
Dendritic cells.
NALT has a related structure.
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Spleen
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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.
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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 IL7Ra with an
antibody, blocks PP formation. But if PP has
started, it finishes.
Stops LN/PP development
.
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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 IL7Ra
with an antibody, blocks PP formation. But if PP
has started, it finishes.
LT-R
No LNs or PPs
Prevents PP formation
The time at which these inhibitors or activators
are added determines which LNs and PPs are
affected.
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Mutant mice with defective lymphoid organogenesis
Essential secondary lymphoid tissue (SLT) genes.
Gene deletion (knock-outs) has revealed major
molecular players.
Cytokines and receptors. LTa, LTb, LTbR, TNFR,
TranceR, Trance, IL7, IL7R Common cytokine
receptor g-chain (gc). Signal transduction
molecules and transcription factors. Jak3, Nik,
IKKa, rel-a, rel-b, traf6, Id2, Ror-g, Ikaros,
NFkb2. Chemokines and receptors. CCR7, CXCR5,
CXCL13.
Other essential genes may be missed if they give
a lethal embryonic phenotype (CXCR4).
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HOW DO THESE MOLECULES CO-ORDINATE LYMPHOID
TISSUE DEVELOPMENT? Peyers patches as a model.
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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 IL7Ra-expressing
cells.
Distribution? Anti-mesenteric side.
How do they know where to start?
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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 IL7Ra cells. Further
clustering with stromal cells.
Accompanied by new blood vessel supply
angiogenesis. ECs express markers that allow for
blood cell homing.
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Are some of the essential SLT genes involved in
making the inducer cells?
LN and PP recovered by injecting normal LTIC
inducer cells into mice lacking genes
involved in generating the
LTICs.
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What do the inducers bring with them?
Members of the TNF superfamily central role of
Lymphotoxin.
So, if a receptor is required for LN and PP
formation, maybe molecules involved in signalling
from that receptor are also required
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Signalling through LTbR on organiser cells.
IKKa IKKb IKKg
Cytoplasmic retention proteins.
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Removing the LTbR signal transduction pathway
reduces chemokine expression by organiser cells,
so fewer LTIC inducers are attracted.



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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
Tissue
.
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Chemokine receptors and integrins on inducer
cells.
INDUCERCELL
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
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CXCR5/CCR7 co-operation in LN/PP development.
IL7R-/- Most LN present. MLN present but
smaller. No PP. CXCR5-/- or CXCL13-/- Some LN
present. MLN present but badly organised. Few
PP CCR7-/- Most LN and PP present, but badly
organised. CXCR5-/-CCR7-/- No PPs or LNs except
MLN, which is badly organised. CXCL13-/-IL7Ra-/- N
o PPs or LNs, including MLN.
Some LN/PP use CXCL13 only. Some use CXCL13 and
CCL19/21. MLN use CXCL13 and IL7Ra ligand.
MLN Mesenteric LN.
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Stimulation through CXCR5 and CCR7 not just
migration.
CXCL13 through CXCR5, CCL19/21 through CCR7
causes the LTICs to - up-regulate
Lymphotoxin LTa1b2 expression. - adhere to
VCAM-1 by activating a4b1 integrin.
Outcome - Adhesion of inducer cells to
stromal organiser cells. - Stimulation of
LTbR. AND LTbR stimulation causes the
up-regulation of the CXCL13 and
CCL19/21 chemokines, and IL7.
POSITIVE FEEDBACK LOOP.
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Model for early SLT organogenesis.
LN
Inducer
IL7R ligands
PP
Organiser
Cytokines, chemokines, and cell adhesion
molecules.
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What next in Peyers Patch development, once cell
clusters form?
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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.
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Induction of changes in the local epithelium.
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-3a,
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.
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Ectopic lymphoid tissue formation tertiary
lymphoid tissue.

• Inappropriate formation of lymphoid tissue can
  occur in many chronic inflammatory diseases.
• 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 LTa/b 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
  LTa1b2 and LTbR.

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Transgenic expression of BLC/CXCL13 in the
pancreas using the Rat insulin promoter.
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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., 11183.
• Owen (1999) Sem. Immunol., 11157.
• Pictures from
• Honda (2001) J. Exp. Med., 193 621.
• Adachi (1997) Int. Immunol., 9507.
• 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.