Innate Immunity

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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
• chamberswh_at_msx.upmc.edu

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Phagocytic Cell Function Macrophages and
Neutrophils
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Macrophages in Tissues Regulate Migration of
Leukocytes Into Sites of Inflammation and of
Pathogens Out of Sites of Inflammation
Macrophages are in normal tissues, neutrophils
are not!
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Bactericidal Agents in Phagocytic Cells
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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)

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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

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Toll-like Receptors

• Toll was defined as a signaling molecule in
  Drosophila sp. Responsible for dorso-ventral
  morphogenesis via induction of apoptosis
• Toll shares homology with 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 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 co-stimulatory molecules
  by DCs

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Toll-Like Receptors

• TLRs 1, 2, 4, 5 and 6 seem to 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.

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TLR Expression

• Receptor Cell types
• TLR1 dimer mf, MDCs, iDCs, mDCs/-
• TLR2 dimer mf, MDCs, iDCs, mDCs /-, mast
  cells, renal epithelial cells
• TLR3 mDCs
• TLR4 mf, MDCs, iDCs, mDCs/-, mast cells,
  intestinal epithelial cells low, renal
  epithelial cells, pulmonary epithelial cells,
  corneal epithelial cells, dermal endothelial
  cells
• TLR5 mf, MDCs, iDCs, mDCs/-, intestinal
  epithelial cells
• TLR6 dimer mf, mast cells
• TLR7 mf, PDCs
• TLR8 mf, MDCs, mast cells
• TLR9 mf, pDCs, B cells

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Toll-like Receptors and Their Ligands

• TLR family Ligands (origin)
• TLR1 Tri-acyl lipopeptides (bacteria,
  mycobacteria), Soluble factors (Neisseria
  meningitides)
• TLR2 Lipoprotein/lipopeptides (a variety of
  pathogens), Peptidoglycan (Gram-positive
  bacteria),
• Lipoteichoic acid (Gram-positive bacteria),
  Lipoarabinomannan (mycobacteria), A
  phenol- soluble modulin (Staphylococcus
  epidermidis), Glycoinositolphospholipids
  (Trypanosoma cruzi), Glycolipids (Treponema
  maltophilum), Porins (Neisseria), Zymosan
  (fungi), Atypical LPS (Leptospira interrogans),
  Atypical LPS (Porphyromonas gingivalis), HSP70
  (host)
• TLR3 Double-stranded RNA (virus)
• TLR4 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)
• TLR5 Flagellin (bacteria)
• TLR6 Di-acyl lipopeptides (mycoplasma)
• TLR7 Imidazoquinoline (synthetic compounds),
  Loxoribine (synthetic compounds), Bropirimine
  (synthetic compounds)
• TLR8 ?

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LPS Binds TLR-4/MD-2/RP105 Complex Following CD14
Association
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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.
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Nod-like Receptors NLRs

• A new family of intracellular receptors including
  at least 5 types of molecules NOD1-5, NALP1-14,
  CIITA, IPAF and NAIP have recently been defined
• These molecules contain an leucine rich repeat
  domain LRR, a domain for oligomerization
  NACHT, one or more of three signaling domains
  PYD-NALPs CARD-NODs/CIITA/IPAF BIRs-NAIP
• Stimulate cytokine release IL1b/IL18 or
  caspases apoptosis

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Macrophages are key players in inflammation
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Macrophage-derived Cytokines
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Local effects of TNF-a
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Macrophage derived cytokines also act systemically
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The Acute Phase Response
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Too much of a good thing Systemic effects of
TNF-a can be lethal
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Cytokines with chemoattractant properties induce
directed cell migration and are called chemokines
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Properties of Chemokines
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Properties of Chemokines
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Non-adaptive, cellular mechanisms include
cytolytic cells
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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

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III. NK Cell Recognition Receptors

• Missing Self Hypothesis
• Activation and Inhibition via Receptors
• Recognition of Self
• Recognition of Tumor Cells
• Recognition of Virus-infected Cells

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Missing Self Hypothesis

• 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 11237-244.

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Recognition NK cells

• 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
• Receptorligand pairs providing inhibitory
  signals are fairly well defined
• Receptorligand pairs providing activation
  signals are rapidly being defined

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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.

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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
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Leukocyte Receptor Cluster (LRC)
LRC is a 1 mb region located on chromosome
19q13.42
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NK Cell Inhibitory Receptors CLRR and KIR

• Name Alternative Names Cellular
  Ligand Viral Ligand
• p58.1 KIR2DL1 HLA-Cw2,4,5,6
• p58.2 KIR2DL2 HLA-Cw1,3,7,8
• p70 KIR3DL1 HLA-Bw4
• p140 KIR3DL3 HLA-A3, -A11
• p49 KIR2DL4 HLA-G
• LIR1 ILT2/LILRB1 HLA-G HCMV-UL18
• LIR2 ILT4/LILRB2 HLA-F
• CD94 KLRD1 HLA-E
• NKG2A KLRC1/CD159A HLA-E
• NKR-P1B, D CD161B, D Clrb
• p40 LAIR1 ?
• IRC1 IRp60/CMRF35H ?
• p75AIRM1 Siglec-7 Sialylated sugars
• CD94 forms heterodimers with NKG2A, -C and E
• CD94/CD159A heterodimer is specific for HLA-E

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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

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NK Cell Activating Receptors

• Name Alternative Names Cellular Ligand
  Viral Ligand
• NKp46 Ly94/NCR1 ? SV-HA, IV-HA
• NKp30 IC7/NCR3 ?
• NKp44 Ly95/NCR2 ? SV-HA, IV-HA
• 2B4 CD244 CD48
• NTB-A KALI ?
• NKp80 KLRF1 ?
• CD16 FcgRIII IgG
• CD2 LFA-2 CD58, LFA-3
• DNAM-1 CD226 PVR/CD155, Nectin-2/CD112
  
• NKG2D D12S2489E/CD159D MICA, MICB, MULT1
  ULBP1-4
• NKR-P1A CD161A IC-21
• NKR-P1C CD161C ?
• NKR-P1F CD161F Clrg
• P40 LAIR1 ?
• IRC1 IRp60/CMRF35H ?
• p75AIRM1 Siglec-7 Sialylated sugars

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ITAM

• Immunoreceptor tyrosine-based activating motif
• Based upon the amino acid motif
  YxxL/Ix6-8YxxL/I
• Serves as a signaling partner to transmembrane
  receptors with a charged residue in the
  transmembrane region which allows docking of
  signal transducers such as DAP12, CD3z-CD3z
  homodimers, CD3z-Fcer1g heterodimers
• Activation of cells either via PI3 kinase, or
  ZAP70 or Syk tyrosine kinases

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NKG2D

• 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

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Ligands for NK Cell Activating Receptors

• 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.

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Recognition of Infected Cells by NK Cells
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Pre-adaptive immune mechanisms or innate-like
lymphocytes
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gd-T cells

• Clonal receptor expression
• Two major subsets
• -Diversity in repertoire in gd-T cells in
  lymphoid tissues
• -Limited repertoire in IEL gd-T cells
• Do not need to recognize antigen in the context
  of MHC
• Recognize antigens directly, e.g. HSP or MHC
  Class IB
• May have important regulatory effects
• Localized at internal epithelial surfaces
• No memory

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NK/T Cells

• Potent producers of cytokines
• Ligands are glycolipids bound to the Class I-like
  CD1d molecule
• Highly skewed in TCR gene utilization one alpha
  chain vs three beta chains
• No boost/memory
• Important as anti-tumor effectors for liver
  metastases

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B-1 CD5 Cells

• Limited Clonal receptor expression
• Produce IgM antibody, often to oligos
• No memory
• Primarily found in the peritoneal cavity

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B-1 Cell Function