Transcript (from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399

(from Cyster, Immunol. Rev., 2003. 195:5-14)
White pulp nodule within spleen
(from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399-433)
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
•TNF, LT (or LTa) and LTß (LT1aß2) 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
(from Fu and Chaplin, Annu. Rev. Immunol. 1999. 17:399-433)
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
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. LTa-/- 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 LTa- 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.
Mice
WT
RAG-/BCR-/TCR-/CD3e
transgenic
B cell
Lymphocytes Follicles
FDC
MadCAM-1
+
NK only
NK, T
B, NK
B
+
+
+
+
+/+/+
+
+
+
+
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)
Mem-TNF Tg mice have normal LN development
• Mice without secreted TNF but with functional normallyregulated 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
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’s
C: TNF RNA in LPS-stimulated PEC’s
Not shown - expression of LTa and LTß was unaffected
Ruuls et al, 2001, Immunity 15:533
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
Ruuls et al, 2001, Immunity 15:533
Peyer’s Patch organization restored
Lymph Nodes - similar to spleen
Ruuls et al, 2001, Immunity 15:533
Rescue of chemokine expression in spleen of mem-TNF ∆/∆ mice
Ruuls et al, 2001, Immunity 15:533