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

Neonate lymph node structure.BTTTTBPlasma cellsMacrophages

Neonate Peyers Patch Structure.BTBGut lumenHEVVillusFollicleTDendritic cells.NALT has a related structure.

Spleen

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.

.Stops LN/PPdevelopmentRevealed 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.

LT-RNo LNs or PPsPrevents PP formationRevealed 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.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.LTa, LTb, LTbR, TNFR, TranceR,Trance, IL7, IL7RCommon cytokine receptor g-chain (gc).

Signal transduction moleculesand 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).

HOW DO THESE MOLECULES CO-ORDINATE 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 IL7Ra-expressing cells.How do they know where to start?Distribution?Anti-mesenteric side.

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.Step 2: Colonising the developing SLT with inducers.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?LN and PP recovered by injecting normal LTIC inducer cells into mice lacking genes involved in generating the LTICs.

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 What do the inducers bring with them?

Signalling through LTbR on organiser cells.IKKaIKKbIKKgCytoplasmicretention proteins.

***Removing the LTbR 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 flowTissue- Three different chemokines.

Chemokine receptors and integrins on inducer cells.Chemokine production by organiser cells causesActivation 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).Chemokines are not just chemoattractantsNull mice: Cooperation between CXCR5 and CCR7 for LN and PP development.INDUCER CELL

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 PPCCR7-/-Most LN and PP present, but badly organised.CXCR5-/-CCR7-/-No PPs or LNs except MLN, which is badly organised.CXCL13-/-IL7Ra-/-No PPs or LNs, including MLN.MLN Mesenteric LN.Some LN/PP use CXCL13 only.Some use CXCL13 and CCL19/21.MLN use CXCL13 and IL7Ra ligand.

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.

Model for early SLT organogenesis.Cytokines, chemokines, and cell adhesion molecules.IL7RligandsLNPPOrganiserInducer

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

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.

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.

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.

Transgenic expression of BLC/CXCL13 in the pancreasusing the Rat insulin promoter.

References.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.Some of the key papers: