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Innate Immunity
William H. Chambers, PhD
University of Pittsburgh Cancer Institute and
Department of Immunology
University of Pittsburgh School of Medicine
G.17e Hillman Cancer Center
412-623-3218
[email protected]
Recognition of Infectious Agents by Receptors in the
Non-adaptive Immune System
• Non-adaptive effectors recognize microbes via “pattern
recognition” receptors
• Pattern recognition receptors bind components of
microbes that are fundamentally different from those on
host cells, e.g. LPS, peptidoglycan
• Oligosaccharide ligands have been identified for pattern
recognition receptors
• Ligands are often called pathogen associated molecular
patterns (PAMPs)
Examples of Pattern Recognition Molecules
• fMLP receptor – N-formylated peptides produced by
bacteria, serves as a chemoattractant for neutrophils
• Macrophage Mannose Receptor/CD206 – “collectin”
family proteins that bind mannose residues on bacteria
and viruses, e.g. HIV
• Macrophage Scavenger Receptors 1-6/CD204 = MSR1 –
bind anionic polymers and acetylated LDLs, and some
structures which have lost normal expression of terminal
sialic acid “capping” residues on oligosaccharides
• Mannose Binding Lectins – serum “collectins” which
recognize a particular orientation of sugar residues and
their spacing
Toll-like Receptors
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Toll was defined as a signaling molecule in Drosophila sp. Responsible for
dorso-ventral morphogenesis via induction of apoptosis [Nusslein-Volhard, et
al. 1985]
Toll shares homology with the IL1r cytoplasmic domain which raised the
question of whether TLRs are important in immune responses
Toll was found to be important in activating Drosophila sp. non-adaptive
immunity, i.e. production of anti-fungal and anti-bacterial peptides
At least 10 TLRs in man [estimated to be between 10 and 15 in most mammals,
TLRs TLRs 11-13 defined in mice]; some can dimerize and form homo- or
heterodimers
Data suggest a role for TLR in non-adaptive responses, i.e. TLR activation
results in NFkB translocation and production of IFN, TNF and ROI
Activation via Toll/TLRs induces production of IL12 and expression of costimulatory molecules by DCs
One of the most ancient and conserved set of proteins in the immune
system…even found in plants, and have antimicrobial function
TIR Domains
• TLR and IL1rs form a superfamily that has a common
Toll-IL1r (TIR) domain
• 3 subgroups of TIRs
• Group 1 = receptors for interleukins that are produced by
macrophages, monocytes, and dendritic cells
• Group 2 = classical TLRs that bind directly or indirectly to
molecules of microbial origin
• Group 3 = adaptor proteins that are exclusively cytosolic
and mediate signaling from proteins of Groups 1 and 2
TLR Recognition
TLRs 1, 2, 4, 5 and 6 specialize in the recognition of mainly
bacterial products that are unique to bacteria and not
made by the host. Their detection therefore affords a
straightforward self:non-self discrimination.
TLRs 3, 7, 8 and 9, in contrast, specialize in viral detection
and recognize nucleic acids, which are not unique to the
microbial world. In this case, self:non-self discrimination is
mediated not so much by the molecular nature of the
ligands as by their accessibility to the TLRs. These TLRs
are localized to intracellular compartments and detect
viral nucleic acids in late endosomes-lysosomes.
TLR Expression
Receptor
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
Cell types
mf, MDCs, iDCs+, mDCs+/mf, MDCs, iDCs+, mDCs +/-, mast cells, renal
epithelial cells
mDCs
mf, MDCs, iDCs+, mDCs+/-, mast cells,
intestinal epithelial cells [low], renal epithelial
cells, pulmonary epithelial cells, corneal
epithelial cells, dermal endothelial cells
mf, MDCs, iDCs+, mDCs+/-, intestinal
epithelial cells
mf, mast cells
mf, PDCs
mf, MDCs, mast cells
mf, pDCs, B cells
Toll-like Receptors and Their Ligands
TLR family Exp.
TLR1
surface
TLR2
surface
TLR3
TLR4
endosome
surface
TLR5
TLR6
TLR7
surface
surface
endosome
TLR8
TLR9
TLR10
TLR11
TLR12
TLR13
endosome
endosome
surface
surface
?
?
Ligands (origin)
Tri-acyl lipopeptides (bacteria, mycobacteria), Soluble factors (Neisseria meningitides)
Lipoprotein/lipopeptides (a variety of pathogens), Peptidoglycan (Gram-positive bacteria),
Lipoteichoic acid (Gram-positive bacteria), Lipoarabinomannan (mycobacteria), A phenolsoluble modulin (Staphylococcus epidermidis), Glycoinositolphospholipids (Trypanosoma
cruzi), Glycolipids (Treponema maltophilum), Porins (Neisseria), Zymosan (fungi), Atypical
LPS (Leptospira interrogans), Atypical LPS (Porphyromonas gingivalis), HSP70 (host)
Double-stranded RNA (virus), poly I:C
LPS (Gram-negative bacteria), Taxol (plant), Fusion protein (RSV), Envelope proteins
(MMTV), HSP60 (Chlamydia pneumoniae), HSP60 (host), HSP70 (host), Type III repeat
extra domain A of fibronectin (host), Oligosaccharides of hyaluronic acid (host),
Polysaccharide fragments of heparan sulfate (host), Fibrinogen (host)
Flagellin (bacteria)
Di-acyl lipopeptides (mycoplasma)
Single stranded RNA, Imidazoquinoline (synthetic compounds), Loxoribine (synthetic
compounds), Bropirimine (synthetic compounds)
single stranded RNA, small synthetic compounds, (Imidazoquinoline)
Unmethylated CpG DNA (bacteria)
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Profilin
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LPS Binds TLR-4/MD-2/RP105 Complex Following
CD14 Association
MD-2
MD-2
RP105
RP105
MD-2
RP105
MD-2
RP105
TLR Signal Transduction Pathway
Toll-like receptor (TLR) signaling pathway. TLRs recognize specific patterns of microbial
components. MyD88 is an essential adaptor for all TLRs and is critical to the inflammatory response.
Lipopolysaccharide (LPS)-induced activation of signaling molecules such as IRF-3, PKR, MAP
kinase, and NF-kB has been reported, indicating the presence of the MyD88-independent pathway.
TIRAP/Mal was identified as a component specifically involved in TLR4-mediated signaling.
Inflammation
Macrophages are key players in inflammation
Neutrophil trafficking – no activation
Selectins = CD62P, CD62E
Neutrophil trafficking – with activation
TNF and IL1
Phagocytosis by
Neutrophils
Pyogenic
Local effects of TNF-a
Macrophage derived cytokines also act
systemically
The Acute Phase Response
Macrophage-derived Cytokines
Too much of a good thing: Systemic
effects of TNF-a can be lethal
Actions of TNFa
1. local containment of infection
– Increase blood flow, increase endothelial adhesion for
wbc’s & platelets
– Lack of TNFa spread of infection to blood (sepsis)
2. Systemic TNFa: septic shock
– Vasodilation, blood pressure down, shock
– Intravascular coagulation, depletion of
clotting factor & bleeding death
3. Stimulates migration of DC migration to lymph nodes &
initiation of adaptive immunity
Cytokines with chemoattractant
properties induce directed cell
migration and are called chemokines
Properties of Chemokines
Properties of Chemokines
Non-adaptive, cellular mechanisms
include cytolytic cells
Natural Killer (NK) Cells
• Lymphoid cells with an LGL morphology
• No evidence for clonality in recognition of targets
• Innate function; i.e. no clonal expansion, no
memory
• Cytolytic function vs virus infected cells and some
tumor cells
NK-mediated Response to Virus Infection
III. NK Cell Recognition Receptors
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“Missing Self” Hypothesis
Activation and Inhibition via Receptors
Recognition of “Self”
Recognition of Tumor Cells
Recognition of Virus-infected Cells
“Missing Self” Hypothesis
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NK cells do not require expression of MHC Class I determinants
for recognition of target cells.
There is, in fact, an inverse relationship between expression of
MHC Class I and susceptibility to lysis by NK cells, i.e. less
Class I equals more lysis.
Led to the hypothesis* that NK cells surveyed the surface of
target cells for “self”. If it was present, the cell was presumed to
be normal and not lysed. If self was absent, as is often the case
in tumor cells and virus-infected cells, NK cells could be
activated to lyse the “abnormal” cell.
*Ljunggren, H.G. and K. Karre, 1990. Immunology Today
11:237-244.
Recognition – NK cells
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There is no evidence supporting clonally restricted recognition
molecules expressed by NK cells, nor for recombinatorial events
being important for development of an NK cell repertoire
NK cells recognize MHC determinants, but these structures, nor
peptides expressed by MHC, are target antigens for activation of
NK lytic function
Some NK cells express CD8 homodimers, but it is unclear
whether binding to MHC Class I affects activation
NK cell recognition of targets involves a balance between
inhibitory signals and activation signals
Receptor:ligand pairs providing inhibitory signals are fairly well
defined
Receptor:ligand pairs providing activation signals are rapidly
being defined
NK Cell Gene Complex (NKC)
• The NKC is a genomic region, first described on NK cells,
encoding structurally related receptors
• NKC maps to Chromosome 12p13, 6 and 4 in man, mouse and
rat, respectively
• Type II integral membrane proteins with external domain similar
to C-type (Ca++-dependent) lectins. However, they lack amino
acid residues that coordinate binding of Ca++, and do not bind
carbohydrates in the same manner as conventional C-type
lectins. Can be expressed homo- or heterodimers.
• Highly conserved evolutionarily – found in sea squirt and several
poxviruses
• Activating and inhibitory receptors for immune cells; can be
either primary or co-stimulatory receptors.
Leukocyte Receptor Cluster (LRC)
LRC is a ~1 mb region located on chromosome 19q13.42
NK Cell Gene Complex (NKC)
- Contains genes encoding C type lectin related receptors
- Disease resistance elements mapped to this locus, e.g. Cmv1
- Conserved across species
Human – Chromosome 12
Mouse – Chromosome 6
Rat – Chromosome 4
NK Cell Inhibitory Receptors: CLRR and KIR
Name
p58.1
p58.2
p70
p140
p49
LIR1
LIR2
CD94*
NKG2A
NKR-P1B, D
p40
IRC1
p75AIRM1
Alternative Name[s]
KIR2DL1
KIR2DL2
KIR3DL1
KIR3DL3
KIR2DL4
ILT2/LILRB1
ILT4/LILRB2
KLRD1
KLRC1/CD159A
CD161B, D
LAIR1
IRp60/CMRF35H
Siglec-7
Cellular Ligand
HLA-Cw2,4,5,6
HLA-Cw1,3,7,8
HLA-Bw4
HLA-A3, -A11
HLA-G
HLA-G
HLA-F
HLA-E**
HLA-E
Clrb
?
?
Sialylated sugars
*CD94 forms heterodimers with NKG2A, -C and –E
**CD94/CD159A heterodimer is specific for HLA-E
Viral Ligand
HCMV-UL18
Target Cell membrane
NH3
IgV
IRp60
NK Cell membrane
Cytoplasm
SHP-1
COOH
I/VxYxxL
Inhibition of lytic function
ITIM
• Immunoreceptor tyrosine-based inhibitory
motif
• Based upon the amino acid motif: I/VxYxxL
• Commonly expressed in signaling receptors
in lymphocytes
• Recruits SHP-1/SHP-2 phosphatases
• Linked to inhibition of function in lymphocytes
NK Cell Activating Receptors
Name
NKp46
NKp30
NKp44
2B4
NTB-A
NKp80
CD16
CD2
DNAM-1
NKG2D
NKR-P1A
NKR-P1C
NKR-P1F
P40
IRC1
p75AIRM1
Alternative Name[s]
Ly94/NCR1
IC7/NCR3
Ly95/NCR2
CD244
KALI
KLRF1
FcgRIII
LFA-2
CD226
D12S2489E/CD159D
CD161A
CD161C
CD161F
LAIR1
IRp60/CMRF35H
Siglec-7
Cellular Ligand
Viral Ligand
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SV-HA, IV-HA
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SV-HA, IV-HA
CD48
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IgG
CD58, LFA-3
PVR/CD155, Nectin-2/CD112
MICA, MICB, MULT1
ULBP1-4
[IC-21]*
?
Clrg
?
?
Sialylated sugars
*Rat NKR-P1A binds an undefined determinant on IC-21 tumor cells
NKG2D
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Single gene
Distantly related to other NKG2 family members
Alternatively spliced isoforms (short and long) in mice
NKG2D-s and NKG2D-l, short from binds both DAP10
and DAP12
• Expressed in NK cells, CD8+ cells and macrophages
Ligands for NK Cell Activating Receptors
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Chr. 10
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MICA, MICB: Stress-inducible molecules encoded within the human
MHC, also can be induced by some infections. Normally expressed by
gastrointestinal epithelium, but also by some epithelial, lung, breast,
kidney, ovary, prostate and colon tumors, and by some melanomas.
Transmembrane with a1, a2, and a3 domains; but do not associate
with b2m and do not bind peptides.
ULBP1-4: 1-3 are GPI-linked, cell surface molecules which bind
human cytomegalovirus UL-16; 4 is a cell surface molecule with
transmembrane and cytoplasmic domains. ULBPs have a1 and a2
MHC Class I-like domains.
Rae1b: Retinoic acid inducible protein, in mice, that shares sequence
homology with ULBPs. Expressed in early embryogenesis and in some
tumors, but generally absent in normal tissues.
H60: Minor histocompatibility antigen expressed by Balb/c mice, target
for alloreactivity responses by C57Bl/6 mice.
DCs: Known that NKp30 is required for recognition of immature DCs
by activated NK cells.
IC-21: Known that rat CD161A is required for recognition of IC-21
tumor cells to mediate their lysis.
NH3
NKp46:SV-HA or IV-HA
IgC2
IgC2
FceR1g
CD3z
R
NK Cell membrane
*D
COOH
I
T
A
M
I
T
A
M
I
T
A
M
Cytoplasm
ZAP70
SYK
I
T
A
M
Activation
Function of CD150 Family Members: ITSM
(a)
CD150 structure compared with other ITSM-containing receptors that also
contain ITIMs. (b) SH2D1A-independent and SH2D1A-dependent pathways
initiated via CD150. If SH2D1A is not binding CD150, then SHP-2 binds and
promotes activation of only the Erk1 and Erk2 pathways. When SH2D1A binds
CD150, then SHIP rather tha SHP-2 binds and promotes the activation of both
the Erk and Akt kinases.
Recognition of Infected Cells by NK Cells
Neutrophils, eosinophils, basophils
• Basophils produce TNFa and mediate anti-tumor function
against TNF-sensitive tumor cell lines
• In preclinical models, eosinophils are a prominent feature
of tumor infiltrating cells in some IL4-transfected tumors,
and mediate anti-tumor lytic activity
• In a preclinical setting, neutrophils mediated ADCC
against lymphomas via bispecific mAbs (a-CD89 x aCD30)