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Cytotoxic T Lymphocytes(CTLs) and NK Cells

After activation, naïve T cells differentiateinto effector and memory T cells

After activation, T cells remain in lymph nodes for 5-6 days Effector T cells

Cell Function Released Effector Molecules

Membrane Effector Molecules

Th1 Cytokines to activate CTLs and macrophages

IL-2, IFN TNF , GM-CSF

TNF

Th2 Activate B cells IL-4, IL-10, CD40L

CTL Kill Target cells Perforin, Granzymes

Fas Ligand

CTLs do not require costimulation to kill infected targets Chromium Release AssayExperiments demonstrating CTL killing of target cells are usually done with achromium (51Cr) release assay, which measures target cell lysis in 4-8 hours.

Infect mouse with virus to generate CTLs specific

for virus infected targets.

Wait until mouse has generated immune response.

Use Spleen as source of CTLs.

Incubate with syngeneic targetsinfected with virus that are radioactively labeled with51Cr that will be released

when cell is lysed.

Chromium attaches toproteins in the cytoplasmof target cells

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Chromium Release CTL Assay

•Target cells mixed with

effector cells at various ratios.

•Measure release of 51Cr into media.

•Express as percentage lysis

relative to non-specific effector cell.

Perforin/Granzyme BPathway

Directional release ofgranules (in red).

How do CTL kill target cells? CTL Killing Perforin

• Contained in CTL granules• Pore forming protein.• Pokes holes in target cell membrane.• Homologous to C9.

Perforin forms polymers thatpoke holes in membranes to allowenzymes inside cell.

CTL Killing CTL Killing Granzymes

• A family of proteases which are involved ininduction of apoptosis in target cells after entrythrough perforin pores.

• Granzyme = granule enzyme

• Granzyme B is most active granzyme.

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CTL Killing Perforin/Granzyme pathway

Antigen specific killing of infected target cells allows for reduced bystander killing of uninfected cells.

CTL Killing Selective (antigen specific) killing happens in minutes.

CTLs can be serial killers (repeatedly killing).

Why don't CTLS kill themselves?

Cathepsin B-- a proteasewhich cleaves perforin!!!

control

Anti-cathepsinB

Wildtype Perforin KO

Virus Virus

51Cr labeled targetsHarvest splenocytes

Harvest splenocytes

Perforin deficient mice can still killvirally-infected target cells. How?

Specific killing ofvirally infected targets

Specific killing ofvirally infected targets

Fas/Fas Ligand Pathway

CTLs express Fas ligand interact with Fasexpressed on the target cell surface.

Fas Induced Apoptosis Cascade of Caspases

Proteases that cut at C-terminal side of anaspartate.

Pro-enzyme form becomes active throughcleavage into subunits.

Proteolytic cascade must be activated for eventualDNA fragmentation.

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Fas Induced Apoptosis Bcl-2 Proteins Control ApoptosisPro-apoptosis and anti-apoptosis proteins

Bcl-2 proteins can inhibit or activate apoptosis.

Proteolytic cascade must be activated and notinhibited by anti-apototic bcl-2 proteins.

(Target cell has to want to commit suicide).

CTLPathways ofCytotoxicity

Summary

Naïve CD8 T cells are activated in secondary lymphoidorgans and differentiate into CTL effector cells.

CTLs can kill targets independently of costimulation.

Once a CTL encounters a target cell it releases cytotoxicgranules containing perforin and granzymes.

Perforin forms pores in the membrane of the target cellallowing granzymes to enter the cell.

Granzymes induce apoptosis in the target cell by cleavingcaspases.

CTL also express FasL and can kill targets via Fas expressedon target cells.

But CTLS are not enough…..Viruses are tricky!

Class I MHC inhibition by viruses•Virus Protein Effect on class I•Adenovirus E3-k19 Retain in ER•HSV-1,2 ICP47 Blocks TAP•EBV EBNA1 Block peptides•CMV US2 ER to cytosol•CMV US3 Retain in ER•CMV US6 Blocks TAP

NK Cells Detect "Missing Self"Protection Against Viruses

N on-in fec ted

anti-c lass I

C MV in fected

anti-c la ss IcIg

CMV infection down-regulated MHC class I on human fibroblasts

Immune evasion mechanism of viruses to decrease Class I MHC.

NK cells preferentially kill cells that have lost expression of MHC class I.

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NK Cells Detect "Missing Self"Immune surveillance for Tumors

Tumor cells often have decreased expression of Class I MHC to escape T cell recognition.

NK cells kill tumor cells.

NK cells

Distinct lineage of lymphocytes.Do not rearrange α,β,γ or δ TCR.CD3-,CD56+ in humans.CD3-,NKR-P1+ (NK1.1) in rodentsEffector functions include cell-mediated

cytotoxicity & cytokine secretion.

CD3+

Stem Cell T / NK Prog enitor Pre-T Pre-T Mature T

Thymus

CD34++ CD34+

CD16+, CD56+

Mature NKPre-NK

CD4+ or CD8+

CD3+ CD3+

CD3+

TCRRearrange ment

CD3- CD4+, CD8+

NK and T cell Development

Bone MarrowStromaIL-2, IL-7, stem cell factor (SCF) CD3-, CD4-, CD8-

Thymus not requiredfor development.

Normal NK cells inscid mice and micewithout RAG1 orRAG2

NK Cells - Distribution

~5-20% peripheral blood lymphocytes~5% lymphocytes in spleenRare in uninfected lymph nodes>90% of lymphocytes in placenta

NK Cells - Effector Functions

Cell mediated-cytotoxicity– Perforin granzyme pathway– Secreted or membrane TNF-α

Antibody-dependent cellular cytotoxicity(ADCC)

Cytokine secretion– Early γ-interferon production– Secretion of TNF-α, LT-α, GM-CSF, IL-5, M-

CSF, IL-3, IL-10, IL-13.

Antibody-dependent cellularcytotoxicity (ADCC)

Cells that performADCC must haveFC receptors to bind Ig moleculesand trigger killing of target cell.

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Cytokine secretion of NK cellsRole for IFN-γ

VirusesVaricella zoster virus & CMV are life-threatening in humans lacking NK cells.

BacteriaNK cells protect against intracellular bacteria

which tend to infect macrophages. (e.g.Listeria Toxoplasma, Leishmania)

Natural Killer (NK) CellsPart of Early Immune Response

NK are lymphocytes without traditional antigen receptors

How do they get activated?

• NK cells express both activating and inhibitory receptors• Inhibitory receptors recognize MHC class I (self) on target cells• Activating receptors recognize ligands upregulated on infected

cells or tumor cells

+–

NK cell Activating ReceptorsHave ITAMs

Intracellular signal oflymphocyte receptorsthrough ITAMs.

Immunoreceptor Tyrosine basedActivating motifs (ITAMs).

NK cell Activating ReceptorsHave ITAMs

STIMULATORYRECEPTOR

(FcR, NKR-P1,NKG2D)

YXXL

Ligand

P

Phosphorylation of substrates

Immunoreceptortyrosine-based

Activation motif(ITAM)

YxxL x 2

e.g. SYK

ProteinTyrosineKinase

YXXL

DAP12+ - -

Class I MHC Specific NK Inhibitory Receptors

humanmouse

Ig SF

C-type lectin-related family

= Immunoreceptor tyrosine-based inhibitory motif (ITIM)

KIR CD94 NKG2A/C/E Ly49

KIR2DL KIR3DL

Ly49 ~10 genesExtensive allelic polymorphismDeleted from the human genome.

Killer Inhibitory Receptors(KIR)10-12 genesExtensive allelic polymorphismKIR genes found in primates, butnot rodents

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ITIMs on Lymphocyte Receptors

Inhibitory receptors have ITIMs to prevent activation.

(Act in opposition to ITAMs)

ITIM Signaling through InhibitoryReceptors

VXYXXL

PPROTE IIN

TYROSIINE PHOSPHATASE

Shp-1E.g.Immunoreceptor tyrosine-based

Inhibitory motif= ITIM

V/IxYxxL/VDe-phosphorylation ofsignaling molecules

Inhibitoryreceptor

MHC

NK cell activation is regulated by integratedpositive and negative signals

TargetNK Cell

+

-

Activate Inhibit Outcome No No No killing No Yes No killing Yes No Killing Yes Yes No killing/killing

NK Cells kill cells expressing activating ligands but need to have inhibitory receptors to protect MHC expressing cells.

Ligands for NK cell activating receptors

Very little known about NK activating receptors and their ligands

NKG2D (activating receptor)Recognizes “MHC-like” ligands (β2m-independent) MIC-A, MIC-B (humans)

Rae-1 family (mice)

These ligands are induced during viral infection and cellular stress

Ligands for many of the activating receptors have not beenidentified yet…

NK Cell- Opposing SignalModel

+

–MHC

MHC –

+

No killing

Normal Cell

Infected Cell

Ligand

Killing

Ligand induced by stress or infectionIn these situations, activating receptor can overcome inhibitory signal

NK cell

NK cell

Inducible Ligand Model

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SummaryNK cells activation is controlled by the balance betweenactivating and inhibitory receptors.

Inhibitory receptors bind MHC class I molecules andprevent inappropriate lysis of self cells.

NK cells are activated by “missing self”, which can occurwhen viruses or tumor cells downregulate MHC class I toavoid recognition by CTL.

Some ligands for activating receptors are constitutivelyexpressed. Others are induced upon viral infection orstress.