Cytotoxicity

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Cytotoxicity

• Mitzi Nagarkatti
• Professor, Dept. Microbiology and Immunology
• Massey Cancer Center
• 533 Medical Sciences Building
• Tel. [phone]

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Cytotoxicity Cell killing or lysis

• Effectors
• Complement (C)
• Macrophages (Mf)
• Granulocyte
• Natural Killer (NK) cells
• Cytotoxic T lymphocytes (CTL)

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Target Cells

• Tumors
• Virally infected cells
• Cells infected with intracellular bacteria

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Macrophage

• Monocyte - blood
• Alveolar Mfslung
• Histiocytes connective tissue
• Kupffer cells liver
• Mesangial cells kidney
• Microglial cells brain

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Function of macrophages

• Phagocytosis
• Antigen Processing and Presentation
• Cytotoxicity
• Direct Cytotoxicity
• Antibody dependent cell-mediated cytotoxicity
  (ADCC)

Ag
Target
Mf
Ab
Fc receptor
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• Normal Mfs are not lytic
• Activated Mfs are lytic
• Activation mediated by Th1
• 2 signals required Cytokine - Interferon-g
  (IFN-g)
• CD40L

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Mechanisms of Macrophage-mediated cytotoxicity

• Reactive oxygen intermediates (ROIs) O2-, OH-,
  H2O2
• Reactive nitrogen intermediates (RNIs) NO, NO2
• Tumor necrosis factor-a
• Lysosomal enzymes

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Activation of Cytotoxic T lymphocytes
IL-2
IL-2R
CTL Precursor
Ag Activated CTL
CD8
MHC Cl. I
Target
TCR
Ag
Proliferation, Differentiation
Activation
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CTL Activation

• Ag Class I MHC on infected cells/grafts CTL
• Ag Dendritic cells (Class I) cross priming of
  naïve CD8 cells
• Ag APC CD4 Th CTL

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Characteristics of Effector CTL

• Increased adhesion molecule expression (LFA-1,
  CD2, CD44, CD45RO)
• Decreased expression of L-selectin (prevents
  homing to lymph nodes)
• Expression of VLA-4 (Very Late Ag-4) which
  interacts with VCAM-1 (vascular cell adhesion
  molecule) on endothelial cells leading to
  inflammation
• Production of effector lytic molecules

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Mechanism of CTL-mediated killing of targets

• Pathways
• FasL
• Perforin/Granzyme
• Cytokines IFN-g, TNF-a and TNF-b

Perforin
TCR
Ag
CTL
MHC
Granzyme
Fas
FasL
Perforin
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Supramolecular Activation Cluster (SMAC)
Immunologic Synapse between CTL and target cell
cSMAC (central)
MHCpTCR
CD28CD80/CD86
Exocytic vesicles
pSMAC (Peripheral)
LFA-1ICAM-1
Microtubules
Outside of SMAC
CD43/CD45
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Natural Killer Cells

• Innate immunity
• Large granular lymphocytes
• Kill virus infected cells and tumor cells
• Lysis non-MHC restricted
• CD3-, TCR-, Ig-
• Present in SCID (severe combined immunodeficiency
  disease) mice
• Asialo GM1, NK1.1
• Intermediate affinity IL-2R
• FcR(CD16), Mediate ADCC

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Natural Cytotoxicity Receptors

• Altered-self Hypothesis
• Activating Receptor Contain immunoreceptor
  tyrosine-based activation motif (ITAM)
• Inhibitory Receptor Contain Immunoreceptor
  tyrosine-based inhibitory motif (ITIM)

ITAM
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NK Cell Receptor

• KLR (Killer cell lectin-like receptor) CD94,
  NKG2
• KIR (Killer cell Ig-like receptor) Ly49
• Human NKG2A and B are inhibitory
• NKG2C and D are activating
• Mouse Ly49H is activating
• Other Ly49 is inhibitory
• Recognize
• Nonclassical MHC HLA-E human Qa-1 mouse
• (Classical MHC HLA-A,B,C,D human
• H-2K,I,D mouse

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Beneficial and Deleterious Effects of Cytotoxicity

• Protection against
• Tumors
• Virus-infected cells
• Intracellular bacteria
• Parasites
• Fungal infections
• Cause
• Autoimmune disorders
• Transplant rejection
• Immunopathology

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Cell Death

• Necrosis
• (Pathologic cell death)
• Cell Swelling and lysis
• Inflammation
• DNA fragmentation (late, varying size)
• Spreads infection
• Ex. Stress
• 51Cr-release assay

• Apoptosis
• (Programmed cell death)
• Nuclear and cytoplasmic condensation
• Membrane blebs
• DNA fragmentation (early, 180bp multiples)
• Apoptotic bodies
• Phagocytosis
• Localizes infection
• Ex. Development
• 3H-thymidine-release assay

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Role of Apoptosis

• Embryogenesis
• Organogenesis
• Cytotoxic Lymphocyte killing of targets
• FasL
• Perforin/Granzyme

TCR
Ag
CTL
MHC
Granzyme
Fas
FasL
Perforin
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Activation induced cell death (AICD)

• In the Periphery

Ag

Ag
IL-2
Memory
Resting T Cell
-
Ag
Activated T Cell
Cycling T Cell
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T Cell Development
Spleen, LN, Peripheral blood

• Thymus Central Tolerance
• Death by Neglect
• Positive Selection T cells with low affinity
  for self MHC
• Negative Selection Deletion of autoreactive T
  cells

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Apoptosis Signaling
Receptor mediated Pathway
Mitochondrial Pathway
Bcl-2
Apaf-1
FasL (CD95L)
Cytochrome c
Fas (CD95)
Procaspase 9
FADD (Fas associated death domain)
Caspase 3
Activates DNase, Inactivate DNA repair
enzymes DNA fragmentation
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Molecules involved in apoptosis

• Tumor necrosis factor receptor (TNFR)
  superfamily
• Fas (CD95)
• TNFR (I and II)
• Ligands Induce apoptosis
• FasL (CD96L)
• TNF a and b (Lymphotoxin LT ab)
• TRAIL (TNF-related apoptosis inducing ligand)
• Bcl-2 family
• Bcl-2, Bcl-xL Inhibit apoptosis
• Bax, Bad Pro-apoptotic

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Apoptosis and Disease

• Lack of apoptosis
• Cancer
• Autoimmunity
• Mouse
• Lpr CD95 deficient
• Gld CD95 Ligand defective
• Lprcg CD95 intracellular death domain defect
• Human
• Type Ia Autoimmune lymphoproliferative disease
  (ALPS) CD95 defect
• Type Ib ALPS CD95L defect
• Type II ALPS CD95 signaling defect

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• Induction of apoptosis
• AIDS and other viral infections
• Neurodegenerative disorder

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Suggested Reading

• Immunobiology The Immune System in Health and
  Disease by Janeway et al. 6th ed., 2005. Pg
  89-95 341-361717.

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Exam Format

• Multiple Choice
• Essays
• Both