1. (from Cyster, Immunol. Rev., 2003. 195:5-14)

2. (from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399-433) White pulp nodule within spleen

3. Follicular dendritic cells (FDC) - abundant complement receptors and Fc receptors - focus immune complexes within the B cell follicle - crucial for the development of effective isotype-switched and memory B cell responses. FDC precursors are radiation-resistant Dendritic cells and lymphocytes are from radiation-sensitive bone marrow-derived precursors Development of GC structures (PNA + ) depends on intercellular signaling via CD40 and CD40L, CD19, CD28, and B7-2, among others. In the absence of T cells, spleens have no GC’s, but do have white pulp nodules containing NK, B and DC, and FDC clusters Mesenteric LN are different - can have ‘GC’s’ without FDC clusters

4. (from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399-433) TNF, LT (or LT  ) and LTß (LT1  ß2) are structurally related and encoded within same 25 kb portion of MHC III TNFRI and TNFRII broadly-expressed LTßR expressed on stromal cells in various tissues LTß-mediated responses involve cell-cell contact

5. LT-/- mice: Loss of most lymph nodes, and Peyer’s Patches. Single mesenteric LN’s in 2-4% of mice. Spleens show loss of discrete T and B cell zones, loss of marginal sinus MAdCAM-1–staining, loss of discrete B cell follicles and FDC networks, and loss of (PNA+) GC B cell clusters Impaired specific Ig responses and affinity maturation TNFRI-/- and TNF-/- mice: No loss of lymph nodes Spleens show absence of marginal sinus MAdCAM-1 staining, lack of discrete B cell follicles and FDC networks, and lack of splenic GC. But - segregation of T and B cell zones retained

6. LTßR +/+ India ink draining from footpad identifies popliteal lymph node LTßR -/- No popliteal lymph node (from Fütterer et al, Immunity1998;9:59-70)

7. Bone marrow chimera experiments (eg. LT  -/- BM -> irradiated WT recipient, generates mice with segregated T/B zones, but no FDC’s) show that: The ability to form discrete white pulp B cell and T cell zones is a fixed feature of the microenvironment, imprinted by the time mice reach maturity. Whereas - B cell follicle structure (FDC clusters) is dependent on continued presence of LT  - or LTß-expressing cells TNF- or TNFRI-deficient mice also lack FDC clusters Indirect evidence that FDC response to LTß and TNF is required: Radiation-resistant LTßR- and TNFRI-expressing cells are required to generate FDC clusters in bone marrow chimeras

8. Irradiation chimeras (previous slide) also showed that: LT-expressing cells that are required for formation and maintenance of FDC network are bone marrow-derived. B cell MiceLymphocytesFolliclesFDCMadCAM-1 WT++++ RAG-/-NK only--+/- BCR-/-NK, T--+/- TCR-/-B, NK+++ CD3  B+++ transgenic Conclusion: LT- and TNF-expressing cells that are required for formation and maintenance of FDC network in spleen are B cells (from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399-433)

9. Mice without secreted TNF but with functional normally- regulated and expressed membrane-bound TNF (Mem-TNF∆/∆ mice) were created by knocking-in the uncleavable ∆1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF-/-), mem-TNF supported many features of lymphoid structure, except generation of primary B cell follicles. Splenic chemokine production was nearly normal in Mem-TNF mice Mem-TNF was suboptimal for development of inflammation. Ruuls et al, 2001, Immunity 15:533 Mem-TNF Tg mice have normal LN development

10. Site-directed mutagenesis of the TACE cleavage site in exons II and III - removal of amino acids 1-9 and K 11 ->E 11 mutation. Homologous recombination to ES cells, injection into blastocysts, breeding chimeric animals -> +/- X +/- -> ∆/∆ Mem-TNF mice A: LPS-induced TNF in cell supernatants of peritoneal-exudate cells (PEC’s) B: FACS-staining for surface TNF on LPS-stimulated PEC’s C: TNF RNA in LPS-stimulated PEC’s Not shown - expression of LT  and LTß was unaffected Ruuls et al, 2001, Immunity 15:533

11. SPLEEN No primary B cell follicles T:B segregation less than in WT, but improved over TNF-/- Marginal zone metallophilic macrophages restored MadCAM restored PNA + Germinal centres CR1 + FDC networks

12. Peyer’s Patch organization restored Lymph Nodes - similar to spleen Ruuls et al, 2001, Immunity 15:533

13. Rescue of chemokine expression in spleen of mem-TNF ∆/∆ mice