(from Cyster, Immunol. Rev., 2003. 195:5-14). (from Fu and Chaplin, Annu. Rev. Immunol. 1999....

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

(from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399-433)

White pulp nodule within spleen

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-resistantDendritic 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


•TNF, LT (or LT) and LTß (LT1ß2) 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

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 clustersImpaired specific Ig responses and affinity maturation

TNFRI-/- and TNF-/- mice: No loss of lymph nodesSpleens 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

LTßR +/+India ink draining from footpad identifies popliteal lymph node

LTßR -/-No popliteal lymph node

(from Fütterer et al, Immunity1998;9:59-70)

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

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 cellMice Lymphocytes Follicles FDC MadCAM-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


• 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

Site-directed mutagenesis of the TACE cleavage site in exons II and III - removal of amino acids 1-9 and K11->E11 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’sC: TNF RNA in LPS-stimulated PEC’s

Not shown - expression of LT and LTß was unaffected



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

Peyer’s Patch organization restoredLymph Nodes - similar to spleen



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

(from Cyster, Immunol. Rev., 2003. 195:5-14). (from Fu and Chaplin, Annu. Rev. Immunol. 1999....

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