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