K. Speirs, L. Lieberman, J. Caamano, C. Hunter, P. Scott
Journal of Immunology 172, 752 (2004)
Cutting Edge:
NF-κB2 is a Negative Regulator of
Dendritic Cell Function
(2004)
Introduction
Introduction
Dendritic Cells (DCs)
the most important antigen-presenting cells (APC)
after activation DCs can activate CD4+ T-Cells (helper)
and CD8+ T-Cells (killer)
mature DCs also produce immune-stimulatory cytokines
(IL-12, IL-6, TNF-α)
Introduction
Introduction
T-Cell Activation by DCs
2 signals required:
presentation of the antigen on MHC molecules
co-stimulatory signals
(CD80, CD86)
Introduction
Introduction
Maturation of Dendritic Cells
after ingestion of pathogens DCs are activated through
innate immune receptors (eg. LPS triggers TLR-4)
mature DCs increase their expression of MHC class II and
co-stimulatory molecules
NF-κB family member RelB is known to play an important
role in DC maturation
Introduction
Introduction
NF-κB
5 known members of the NF-κB family:
NF-κB1, NF-κB2, RelA, RelB, c-Rel
regulated by inhibitory proteins (IκBs) and the precursors of
NF-κB1 and NF-κB2 (p105 and p100)
absence of one or more NF-κB components leads to
dramatic changes in the immune system
(e.g. chronic inflammation)
NF-κB signaling
NF-κB signaling
NF-κB activation in principle
NF-κB signaling
NF-κB signaling
NF-κB activation pathways
differences:
activating receptors
involved kinases
inhibtory proteins
active complexes
kinetics
NF-κB signaling
NF-κB signaling
Alternative NF-κB pathway
Variation:
active dimer
NF-κB signaling
NF-κB signaling
Regulation of RelB
in HeLa cells RelB has been shown to be regulated only by
NF-κB2 / p100
Is RelB also exclusively inhibited by p100 in dendritic cells?
Æ Testing RelB activity in DCs from NF-κB2 knock-out mice
NF-κB signaling
NF-κB signaling
Summary
stimulation of a dendritic cell (by e.g. LPS)
p100
?
activation of RelB
upregulation of MHC and co-stimulatory
molecules
activation of T-Cells
Results
Results
RelB in Cytoplasm and Nucleus
NF-κB2 KO DCs show much higher levels of RelB in the nucleus
- even without stimulation!
Results
Results
DNA-binding ability of RelB
Æ EMSA and supershift
EMSA: Electrophoretic mobility shift assay
oligo nucleotides with specific protein binding site
nuclear extracts (with DNA binding proteins)
specific antibodies
probe-protein-antibody
complex
probe-protein complex
free probe
direction of
migration
Results
Results
DNA-binding ability of RelB
WT and KO DCs show no differences in quantity of the
p50/p50 complex (EMSA)
KO DCs show an additional p50/RelB complex
(confirmed by supershift, no differences for other NF-κBs)
Results
Results
NF-κB2 represses RelB activation in WT DCs
Æ Differences in the phenotype?
KO DCs show normal development and
morphology
Results
Results
Quantification of surface molecules
KO DCs show higher baseline expression of MHC II and CD86
KO DCs are more responsive to stimulation
same effects were shown in vivo with splenic DCs!
Results
Results
Cytokines in the supernatants
KO DCs produce normal levels of IL-12, TNFα, IL-6 and IL-1β
Æ Cytokine production does not depend on NF-κB2
Æ Expression of surface molecules and cytokine production are
differentially controlled
Results
Results
Activation of AG-specific CD4+ T-Cell response
measurement of proliferation (with CFSE) and IFN-γ production
(in presence of LPS)
Proliferation and IFN-γ production are
increased by KO DCs
Æ KO DCs induce a stronger AG-specific
CD4+ T-Cell response
Results
Results
Activation of AG-specific CD4+ T-Cell response
KO DCs can present antigens
even without microbial stimulation
Æ correlation with higher baseline expression of co-stimulatory
molecules
Results
Results
CD4+ T-Cell response in vivo
injection of
activated WT DCs
24 h
unactivated WT DCs
activated KO DCs
labled OT-II T-Cells
into Thy1.1 mice
unactivated KO DCs
72 h
recovery of
spleenocytes
Results
Results
CD4+ T-Cell response in vivo
spleenocytes were stained for Thy1.2 and CD4
Æ KO DCs induce T-Cell proliferation much better than WT DCs
Discussion
Discussion
Summary
NF-κB2 KO DCs show an enhanced maturation status:
- elevated nuclear levels of RelB
- increased expression of MHC Class II, CD80, CD86
- increased ability to induce AG-specific CD4+ T-Cell
responses
NF-κB2 has a crucial role in regulation of RelB
Æ required to prevent DC hyperactivation and inadequate
adaptive immune responses
Discussion
Discussion
Summary
RelB is regulated by the alternative NF-κB pathway
Open Questions:
phenotype of NF- κB2 KO DCs might be influenced by p52
deficiency
direct influence of p100 on RelB
influence of other IκBs
Discussion
Discussion
Clinical Implications
mutations in NF-κB2 result in cutanous T-Cell Lymphomas
Æ contribution of hyperactive DCs?
Inhibitors of NF-κB2 could boost T-Cell responses in
vaccination therapies
Inhibitors of p100 degradation could prevent hyperactive DC
phenotypes in certain autoimmune diseases
Frank Schmitges – Molecular Mechanisms in Health and Disease
2005
Thank You for Your
Attention