Cytolytic Cell Effector Mechanisms

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Cytolytic Cell Effector Mechanisms

• William H. Chambers, PhD
• Associate Professor of Immunology
• Department of Immunology
• University of Pittsburgh School of Medicine
• Associate Director for Basic Research
• University of Pittsburgh Cancer Institute

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NK Progenitors Adult
Periphery
Stimulus Days
Cytokines IFNg GM-CSF TNFa
IL2 IL12 IL15 IL21 IL23 IL27 IFNa, -b
Proliferation
Enhanced CTX, with broader specificity
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NK cell activating receptors

• Loss of the inhibitory signal does not, in and
  of itself, provide signals to kill target cells
• Some receptors able to activate NK cells to kill
  target cells have been defined NKG2D, Ly49D,
  Ly49H, NKp30, NKp44, NKp46, CD161A
• Some activating receptors are members of the
  C-type lectin e.g. NKG2D and IgSF NKp30
  superfamilies
• IgSF members often referred to as KARs
• Associate with an adaptor molecule e.g. DAP10
  containing an ITAM. Associate via a charged
  residue in the TM domain
• Some ligands for activating receptors have been
  defined, e.g. RAE-1 for NKG2D

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NKG2DMICA, MICB, ULBPs
COOH
COOH
CTLD
CTLD

NK Cell membrane
R
Cytoplasm
NH3
NH3
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Signaling Activation of Lytic Function Virus
Infected Cells
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Recognition T cells

• T cells recognize antigen via a heterodimeric
  receptor TCR that is clonally distributed
• Heterodimeric TCR is either ab or gd pairing of
  chains
• Antigen is recognized in the context of MHC
  molecules
• CD8 homo- or heterodimer binds MHC Class I and
  serves as a co-receptor
• Cytolytic cells are primarily CD8, but some CD4
  T cells are also cytolytic

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Activation of T cells changes the expression of
several cell surface molecules
CD8
CD8
High affinity IL2r CD122/CD132/CD25 MHC Class
II some T cells
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2. CTL differentiation

• Tc1 and Tc2 cells
• Similar to CD4 T cells, clones of CD8 T cells
  can be differentiated based upon profiles of
  cytokine production
• IL2 IL4 in primary cultures of CD8 T cells
  induces development of clones secreting IL4 and
  IL5, termed Tc2 cells
• IL2 IFNg in primary cultures of CD8 T cells
  induces development of clones secreting IFNg,
  termed Tc1 cells
• Tc1 and Tc2 retain their patterns of cytokine
  production in vivo

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B. The Process Used by Cytolytic Effector Cells

• First step is conjugation which is dependent upon
  adhesion molecules, particularly CD11a/CD18
  LFA-1 and CD2 binding CD54 and CD58,
  respectively
• Second step involves recognition and activation
  of the lytic machinery of effector cells via one
  of several signaling pathways. Many observations
  also indicate a role for LFA-1 and CD2 in
  activation. Similarly, CD244 2B4 binding of
  CD48 contributes to NK cell activation.
• Third step is the delivery of the lethal hit
  granule exocytosis, death receptor mediated
  pathways
• These three steps occur rapidly in the context of
  a short-lived immunologic synapse between CTLs or
  NK cells and their target cells although there
  are some differences in the synapses formed by
  these two cell types
• Recycling of the effector cell and delivery of
  the lethal hit to additional target cells

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Immunologic Synapse
Fig. 1. The secretory synapse. (a and b) CTL
(left) target (right) conjugate stained with
antibodies against cathepsin D (blue), LFA-1
(green) and talin (red) (c and d) CTL (left)
target (right) conjugate stained with antibodies
against granzyme A (green), Lck (red) and actin
(blue). Single confocal sections are Shown in a
and c, and reconstructions of serial sections in
the z-axis to show the interface between the two
cells are shown in b and d., (e) Diagram showing
spatial organization of proteins observed during
granule secretion. Stinchcombe, J. 2003. Sem.
Immunol. 15301.
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Localization of Lytic Granules In Cytolytic Cells
to the Immunologic Synapse
Figure 1. The secretory synapse.(a) Polarization
of secretory granules at the immunological
synapse formed by CTLs and target cells. The
granules are labeled with electron-dense
horseradish peroxidase and are dark. They cluster
in the region of the MTOC and move around the
MTOC and Golgi apparatus to dock at the plasma
membrane for secretion. (b) Confocal section of
two CTLs, both polarized towards the same target
cell. Granules are stained with antibodies to
cathepsin (green), actin (blue) and the T cell
receptor-associated protein Lck (red). (c)
Section through the interface between the upper
CTL in b and the target cell shown face on to
reveal the secretory domain in the immunological
synapse formed between CTLs and target cells.
Trambas, CM, 2003
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Steps in Granule Movement/Exocytosis
Bossi, G. et al., 2002. Immunol. Rev. 189152
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Delivery of Graules to the Immunologic Synapse

• On contact with the target cell, the MTOC
  polarizes towards the target cell
• Granules move along microtubules towards the
  polarized MTOC
• Centrosome moves to and contacts plasma membrane
  at the cSMAC cluster
• Actin and IQGAP1 are cleared from the synapse
• Granules delivered directly to the plasma
  membrane
• Novel mechanism for delivering secretory granules

Stinchcombe, J, et al. 2006. Nature 443462
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Video of Granule Exocytosis
http//www.nature.com/ni/journal/v4/n5/extref/ni05
03-399-S1.gif
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Cytolytic Effector Cells Deliver the Lethal Hit
Using Multiple Mechanisms

• Granule Exocytosis necrotic cell death/membrane
  disruption/colloidal osmotic lysis/activation of
  apoptosis
• Death Receptor Activation of apoptosis
• - secreted cytotoxins
• - cell surface expressed cytotoxins

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Components of Cytolytic Granules

• Perforin/Cytolysin/C9RP
• Granzymes
• Chondroitin Sulfate A proteoglycan
• Granulysin only defined in man
• Lysosomal enzymes aryl sulfatase
• Cathepsin B
• Granulysin
• Occasionally reported to contain TNFa

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Components of Lytic Granules Form Pores in
Lethally Hit Target Cells
15-16 nm
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Modeling of Perforin Function
Voskoboinik et al. 2006. Nature Reviews
Immunology 6940
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Structure of Perforin
Ca-dependent membrane binding
Unknown function
Unknown function
MAC-like
Lytic activity as recombinant protein
Unknown function
Voskoboinik et al. 2006. Nature Reviews
Immunology 6940
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Voskoboinik et al. 2006. Nature Reviews
Immunology 6940
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Specificity of Granzymes Grz

• Granzyme Category Specificity Chrmsml. location
• GrzA trypsin arg/lys 5q11-12 h
• GrzB aspase aspartic acid 14q11.2 h
• GrzC ? ?
• GrzD-G
• GrzH chymase phenylalanine 14q11.2 h
• GrzK trypsin arg/lys 5q11-12 h
• GrzM elastase meth/leu/isoleu 19p13.3
• Caspase-independent induction of apoptosis, slow
  via induction of single-strand nicks in DNA and
  prevention repair
• Caspase-dependent induction of apoptosis, rapid
• Mice only

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Comparison of Granzyme Grz Functions
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Given the nature of perforin-mediated
cytotoxicity, what limits autolysis of effector
cells?
Catalfamo, M, 2003, Curr. Opin. Immunol.
15522 Balaji, K, et al. 2002 J Exp Med 196493
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Given the nature of perforin-mediated
cytotoxicity, what limits autolysis of effector
cells?

• Cathepsin B is contained in granules of NK cells
  and CTLs
• Cathepsin B is translocated to the cell surface
  when granule membranes fuse with the plasma
  membrane
• Cathepsin B cleaves perforin at the killer cell
  surface limiting pore formation
• Proposed as a means of protection of the effector
  cell during target cell destruction

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Protection from Lysis Effectors and Targets

• Protease inhibitor 9 (PI-9) blocks granzyme B
  activity
• member of the serine protease inhibitor (serpine)
  family
• glutamic residue in the P1 site of its reactive
  loop
• found in cytoplasm and nucleus
• expression in lymphomas protects them from lysis
• expression in DCs protects them from lysis by
  CTLs
• homolog found in mice, SPI-6

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What is the in vivo importance of granule
exocytosis?

• Expression of immune effector function by NK
  cells and T cells against tumor and
  virus-infected cells
• Immune regulatory activity via reduction of
  expanded T cell populations following an immune
  response, or by elimination of antigen presenting
  cells controversial

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Consequences of Cytolysin Deficiency Transgenic
Mice

• Cytolytic cells from perforin-/- mice are
  deficient in lytic activity in vitro
• Perforin-/- mice have increased susceptibility to
  non-cytopathic and cytopathic viruses higher
  titers of Herpes and MCMV
• Perforin-/- mice have an increased incidence in
  lymphomas, particularly when crossed with p53-/-
  mice
• IL12 and IL21 activated anti-tumor function in
  vivo requires perforin, as cytokine boost in
  anti-tumor function lacking in perforin-/- mice

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Consequences of Genetic Defects Related to
Perforin or Granule Exocytosis

• Individuals with perforin mutations lethal,
  inherited disease, familial haemophagocytic
  lymphohistiocytosis FHL believed to be the
  consequence of a failure to down-regulate an
  immune response following a viral infection for
  which protection is strictly perforin-dependent,
  suggests perforin is important for immune
  regulation
• Deletion of Rab27a, a small GTP-binding protein,
  results in a lack of ability to exocytose
  granules Griscelli Syndrome/Ashen mouse
• Mutation in LYST/CHS1 results in defects in
  membrane fusion of lysosome/granules which
  results in the formation of a giant granule which
  is not exocytosed Chediak-Higashi Syndrome/Beige
  mouse
• Mutation in the Rab geranylgeranyl transferase
  RGGT gene results in a failure to translocate
  granule to the site of the synapse
  Hermansky-Pudlak Syndrome/Gunmetal mouse

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Steps in Granule Movement/Exocytosis
Reduced NK killing, may be due to less actin
accumulation at synapse
Granule fusion with membrane
Granule motility
Granule motility
Granule movement from microtubule to synapse
Bossi, G. et al., 2002. Immunol. Rev. 189152
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Consequences of Granzyme Deficiency Transgenic
Mice

• By comparison to perforin, relatively poorly
  characterized for in vivo significance
• Considerable functional redundency among
  granzymes
• GrzA-/- mice are basically normal in lytic
  function and resistance to virus
• GrzB-/- mice reduced capacity to induce, and
  delayed induction of, apoptosis
• GrzA/B-/- reduced lytic capacity and increased
  susceptibility to infection with ectromelia virus

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Voskoboinik et al. Nature Reviews Immunology 6,
940952 (December 2006) doi10.1038/nri1983
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Elimination of intracellular pathogens can be
mediated by a novel protein, granulysin, which is
associated with granules
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Characteristics of Granulysin

• First defined by subtractive hybridization
  looking for new genes in day 3-5 activated T
  cells 1987
• 9K polypeptide
• 5 a-helices connected by short loops similar to
  saposin, positively charged may cluster on cell
  surface to express function
• produced by T cells, NK cells, induced in
  monocytes TLR2 pathway
• Broad lytic activity against tumor cells and
  cells infected by intracellular pathogens
• Unlike perforin, granulysin does not lyse
  non-nucleated cells, intrinsic pathway for
  apoptosis via mitochondria
• Found to be a chemoattractant for monocytes,
  CD45RO memory T cells CD4 and CD8, NK cells
  and mDCs
• Activates monocytes to produce IFNg and
  chemokines,such as MCP-1 and RANTES

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Saposin like protein Saplip family members
Granulysin
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Granulysin-mediated Induction of Apoptosis
Krensky, A., et al. 2005. Am. J. Transpl. 51789
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Mechanism of Induction of Apoptosis by Granulysin

• 1) Binds to tumor cell surface on the basis of
  charge
• 2) Sphingomyelinase SM is activated, resulting
  in increased ceramide concentration fast
  process
• 3) Binding results in an increase in
  intracellular calcium and decrease in
  intracellular potassium
• 4) Mitochondria are damaged resulting in release
  of cytochrome C and apoptosis inducing factor
  AIF slow process
• 5) Activation of caspases and endonucleases
  resulting in apoptosis

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Granulysin Directly Kills a Variety of
Microorganisms
Stenger, S., et al. Science 282121, 1998
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Granulysin and Perforin Work In Concert to Lyse
Intracellular M. tuberculosis

Stenger, S., et al. Science 282121, 1998
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Biological Significance and Consequences of
Granulysin Deficiency

• Granulysin homolog/ortholog has not been found in
  mice
• Potential disease associations with polymorphisms
  or differences in granulysin as yet unidentified
• mRNA in urine may be a very good biomarker for
  acute graft rejection
• siRNA depletion of granulysin resulted in loss of
  CTL ability to lyse C. neoformans
• Required for CTL lysis of M. tuberculosis
  infected macrophages
• Tumor bearing individuals had comparable levels
  of perforin relative to normals, but remarkably
  lower levels of granulysin suggests levels of
  granulysin have some prognostic value for
  progression of cancer

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Death Receptor Pathways

• NK cells and CTLs express multiple members of the
  tumor necrosis factor super family TNFSF, e.g.
  TNFa, LTa TNFb, TNF-related apoptosis inducing
  ligand TRAIL/Apo2L, CD95L FasL/Apo1L, Apo3L,
  DR-6L
• Type II glycoproteins, expressed as homo- or
  heterotrimers on cell surface or as secreted
  proteins
• Many have corresponding receptors which recruit
  adaptor proteins with a characteristic death
  domain which is required for intracellular
  transmission of the cytotoxic signal, e.g. FADD,
  TRADD
• There are, however, some receptors signal
  apoptosis without death domain containing
  adaptor molecules, e.g. TNF-R2
• lt22 TNFSF members, lt29 TNFSFR

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TNFSF and Receptors

• TNFa TNF-R1 (CD120a/p55), TNF-R2
  (CD120b/p75)
• LTa TNF-R1 (CD120a/p55), TNF-R2 (CD120b/p75)
• TRAIL 5 receptors, TRAIL-R1 (DR4), TRAIL-R2
  (DR5), TRAIL-R3, TRAIL-R4, TRAIL-R5
  (osteoprotegerin)
• CD95L CD95 (Fas/Apo-1)
• Apo3L TRAMP (Apo-3/DR-3)
• DR-6 DR-6L
• Via death domain containing proteins
• Death domain containing adaptor proteins not
  required

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In Vivo Significance of NK/CTL Induction of
Apoptosis in Target Cells

• While many envision the primary in vivo
  significance of death receptor induced apoptosis
  to be immunoregulatory, i.e. elimination of
  thymocytes, there is ample evidence of other in
  vivo functions by effector cells
• FasL utilized by NK cells for tumor elimination
  in vivo. Interestingly, many tumors lack Fas,
  but NK cells up-regulate Fas via IFNg secretion
  and then kill them in a Fas-dependent fashion
• Cytokine therapy with IL18 suppresses tumor
  metastases via NK cell associated FasL-mediated
  cytolysis
• TNFa-/- mice are defective in rejection of
  NK-sensitive tumors in the peritoneum
• LTa-/- mice lack capacity for NK cell-mediated
  tumor rejection or protection for pulmonary
  metastases
• Fas lpr or FasL gld mutations in mice results
  in development of plasmacytoid tumors
• Fas mutations in humans result in development of
  multiple neoplasias including multiple myeloma

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