CytotoxicityCytotoxicity
Mitzi Nagarkatti
Professor, Dept. Microbiology and Immunology
Massey Cancer Center
533 Medical Sciences Building
Tel. # 827-1555
Cytotoxicity = Cell killing or lysisCytotoxicity = Cell killing or lysis
EffectorsComplement (C’)
Macrophages (M)GranulocyteNatural Killer (NK) cells Cytotoxic T lymphocytes (CTL)
C’
AgAb
Target CellsTarget Cells
TumorsVirally infected cellsCells infected with intracellular bacteria
MacrophageMacrophageMonocyte - bloodAlveolar Ms–lung Histiocytes – connective tissueKupffer cells – liver Mesangial cells – kidney Microglial cells – brain
Function of macrophagesFunction of macrophagesPhagocytosisAntigen Processing and PresentationCytotoxicity
– Direct Cytotoxicity– Antibody dependent cell-mediated cytotoxicity
(ADCC)
Target
Fc receptor
M
Ag
Ab
Normal Ms are not lytic
Activated Ms are lytic
Activation mediated by Th12 signals required: Cytokine - Interferon- (IFN-)
CD40L
MTarget
MTarget
Mechanisms of Macrophage-Mechanisms of Macrophage-mediated cytotoxicitymediated cytotoxicity
Reactive oxygen intermediates (ROIs): O2-,
OH-, H2O2
Reactive nitrogen intermediates (RNIs): NO, NO2
Tumor necrosis factor-Lysosomal enzymes
Activation of Cytotoxic T lymphocytesActivation of Cytotoxic T lymphocytes
CTL Precursor
Ag Activated
CTL
IL-2
TCR
CD8
IL-2R
Activation Proliferation,Differentiation
Target
MHC Cl. I
Ag
CTL ActivationCTL Activation
Ag + Class I MHC on infected cells/graftsCTL
Ag + Dendritic cells (Class I) – cross priming of naïve CD8+ cells
Ag + APC + CD4+ Th CTL
Characteristics of Effector CTLCharacteristics of Effector CTLIncreased 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
Mechanism of CTL-mediated killing of Mechanism of CTL-mediated killing of targets targets
Pathways
FasL
Perforin/Granzyme
Cytokines: IFN-, TNF- and TNF-
Fas FasL
Ag
MHC
TCR
Perforin
GranzymeCTL
Perforin
Supramolecular Activation Cluster (SMAC)Supramolecular Activation Cluster (SMAC)
CD43/CD45
MHCp:TCR
LFA-1:ICAM-1
Exocytic vesicles
cSMAC(central)
pSMAC(Peripheral)
Microtubules
CD28:CD80/CD86
Outside of SMAC
Immunologic Synapse between CTL and target cellImmunologic Synapse between CTL and target cell
Natural Killer CellsNatural Killer Cells
Innate immunity Large granular lymphocytesKill virus infected cells and tumor cellsLysis non-MHC restrictedCD3-, TCR-, Ig-
Present in SCID (severe combined immunodeficiency disease) mice
Asialo GM1+, NK1.1+
Intermediate affinity IL-2R+ FcR(CD16)+, Mediate ADCC
Natural Cytotoxicity ReceptorsNatural Cytotoxicity ReceptorsAltered-self Hypothesis Activating Receptor: Contain immunoreceptor
tyrosine-based activation motif (ITAM) Inhibitory Receptor: Contain Immunoreceptor
tyrosine-based inhibitory motif (ITIM)
ITAMITIMITIM
NK Cell ReceptorNK Cell Receptor– KLR (Killer cell lectin-like receptor): CD94, NKG2– KIR (Killer cell Ig-like receptor): Ly49
Human: NKG2A and B are inhibitoryNKG2C and D are activating
Mouse: Ly49H is activatingOther 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
Beneficial and Deleterious Beneficial and Deleterious Effects of CytotoxicityEffects of Cytotoxicity
Protection against – Tumors – Virus-infected cells– Intracellular bacteria– Parasites– Fungal infections
Cause – Autoimmune disorders – Transplant rejection– Immunopathology
Cell DeathCell DeathApoptosis
(Programmed cell death) Nuclear and cytoplasmic
condensation Membrane blebs DNA fragmentation
(early, 180bp multiples) Apoptotic bodies Phagocytosis Localizes infection
Ex. Development3H-thymidine-release assay
Necrosis
(Pathologic cell death) Cell Swelling and lysis Inflammation DNA fragmentation
(late, varying size) Spreads infection
Ex. Stress51Cr-release assay
Role of ApoptosisRole of ApoptosisEmbryogenesisOrganogenesisCytotoxic Lymphocyte killing of targets
– FasL – Perforin/Granzyme
Fas FasL
Ag
MHC
TCR
Perforin
GranzymeCTL
In the Periphery
Memory
Resting T Cell
Ag +
-Activated T Cell
CyclingT Cell
Ag
AgIL-2
Activation induced cell death (AICD)
T Cell DevelopmentT Cell Development
Thymus – Central Tolerance• Death by Neglect• Positive Selection: T cells with low affinity
for self MHC • Negative Selection: Deletion of autoreactive
T cells
BoneMarrow
Thymus
MatureT cells
Spleen, LN, Peripheral blood
Apoptosis SignalingApoptosis Signaling
FasL (CD95L)
Fas (CD95)Procaspase 9
Caspase 9
Procaspase 3
Caspase 3
Cytochrome c
Receptor mediated Pathway Mitochondrial Pathway
Bcl-2
Procaspase 8 Caspase 8
Activates DNase, Inactivate DNA repair enzymesDNA fragmentation
FADD (Fas associated death domain)
Apaf-1
Molecules involved in apoptosisMolecules involved in apoptosis
Tumor necrosis factor receptor (TNFR) superfamily:
Fas (CD95)
TNFR (I and II)
Ligands: Induce apoptosis
FasL (CD96L)
TNF – and LymphotoxinLT TRAIL (TNF-related apoptosis inducing ligand)
Bcl-2 family
Bcl-2, Bcl-xL –Inhibit apoptosis
Bax, Bad – Pro-apoptotic
Apoptosis and DiseaseApoptosis and DiseaseLack of apoptosis Cancer Autoimmunity:
– MouseLpr – CD95 deficientGld – CD95 Ligand defectiveLprcg – CD95 intracellular death domain defect
– HumanType Ia Autoimmune lymphoproliferative disease
(ALPS) – CD95 defectType Ib ALPS – CD95L defectType II ALPS – CD95 signaling defect
Induction of apoptosisAIDS and other viral infectionsNeurodegenerative disorder
Suggested ReadingSuggested Reading
Immunobiology: The Immune System in Health and Disease by Janeway et al. 6th ed., 2005. Pg 89-95; 341-361;717.
Exam FormatExam Format
Multiple ChoiceEssaysBoth
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