Download Cytotoxic T Lymphocytes (CTLs) and NK Cells Effector T cells

After activation, naïve T cells differentiate
into effector and memory T cells
Cytotoxic T Lymphocytes
(CTLs) and NK Cells
After activation, T cells remain in lymph nodes for 5-6 days
CTLs do not require costimulation to kill infected targets
Effector T cells
Cell
Function
Released
Effector
Molecules
Membrane
Effector
Molecules
Th1
Th2
Cytokines to
activate CTLs
and macrophages
Activate B cells
TNF
IL-2,
IFN TNF ,
GM-CSF
IL-4, IL-10, CD40L
CTL
Kill Target cells
Perforin,
Granzymes
Fas Ligand
Chromium Release Assay
Experiments demonstrating CTL killing of target cells are usually done with a
chromium (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 targets
infected with virus that are
radioactively labeled with
51Cr that will be released
when cell is lysed.
Chromium attaches to
proteins in the cytoplasm
of 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.
How do CTL kill target cells?
CTL Killing
Perforin
Perforin/
Granzyme B
Pathway
• Contained in CTL granules
Directional
release of
granules
(in red).
CTL Killing
• Pore forming protein.
• Pokes holes in target cell membrane.
• Homologous to C9.
CTL Killing
Granzymes
• A family of proteases which are involved in
induction of apoptosis in target cells after entry
through perforin pores.
• Granzyme = granule enzyme
Perforin forms polymers that
poke holes in membranes to allow
enzymes inside cell.
• Granzyme B is most active granzyme.
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CTL Killing
Perforin/Granzyme pathway
CTL Killing
Selective (antigen specific) killing happens in minutes.
CTLs can be serial killers (repeatedly killing).
Antigen specific killing of infected target cells allows
for reduced bystander killing of uninfected cells.
Why don't CTLS kill themselves?
Perforin deficient mice can still kill
virally-infected target cells. How?
Virus
Virus
Wildtype
Perforin KO
Cathepsin B-- a protease
which cleaves perforin!!!
Harvest
splenocytes
51Cr
labeled targets
Harvest
splenocytes
Anti-cathepsinB
control
Fas/Fas Ligand Pathway
Specific killing of
virally infected targets
Specific killing of
virally infected targets
Fas Induced Apoptosis
Cascade of Caspases
Proteases that cut at C-terminal side of an
aspartate.
Pro-enzyme form becomes active through
cleavage into subunits.
Proteolytic cascade must be activated for eventual
CTLs express Fas ligand interact with Fas
expressed on the target cell surface.
DNA fragmentation.
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Fas Induced Apoptosis
Bcl-2 Proteins Control Apoptosis
Pro-apoptosis and anti-apoptosis proteins
Bcl-2 proteins can inhibit or activate apoptosis.
Proteolytic cascade must be activated and not
inhibited by anti-apototic bcl-2 proteins.
(Target cell has to want to commit suicide).
Summary
Naïve CD8 T cells are activated in secondary lymphoid
organs and differentiate into CTL effector cells.
CTL
Pathways of
Cytotoxicity
CTLs can kill targets independently of costimulation.
Once a CTL encounters a target cell it releases cytotoxic
granules containing perforin and granzymes.
Perforin forms pores in the membrane of the target cell
allowing granzymes to enter the cell.
Granzymes induce apoptosis in the target cell by cleaving
caspases.
CTL also express FasL and can kill targets via Fas expressed
on target cells.
But CTLS are not enough…..
Viruses are tricky!
NK Cells Detect "Missing Self"
Protection Against Viruses
Immune evasion mechanism of viruses to decrease Class I MHC.
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
•CMV
•CMV
US2
US3
US6
ER to cytosol
Retain in ER
Blocks TAP
NK cells preferentially kill cells that have lost expression of MHC class I.
C M V i n fe ct e d
a n ti-c la ss I
cIg
N o n -in fe c te d
a n ti-c la ss I
CMV infection down-regulated MHC class I on human fibroblasts
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NK Cells Detect "Missing Self"
NK cells
Immune surveillance for Tumors
Tumor cells often have
decreased expression
of Class I MHC to escape
T cell recognition.
NK cells kill tumor cells.
Distinct lineage of lymphocytes.
Do not rearrange α,β,γ or δ TCR.
CD3-,CD56+ in humans.
CD3-,NKR-P1+ (NK1.1) in rodents
Effector functions include cell-mediated
cytotoxicity & cytokine secretion.
NK and T cell Development
NK Cells - Distribution
TCR
Re arrange me nt
Ste m Ce ll
T / NK Prog e nitor
Thymus
CD34 ++
Pre -T
Pre -T
CD3 +
CD3 +
CD3+
CD4 + o r CD8 +
CD34 +
CD3-
Mature T
CD4+, CD8+
Normal NK cells in
scid mice and mice
without RAG1 or
RAG2
Bone Marrow
Stroma
IL-2, IL-7,
stem cell factor (SCF)
~5% lymphocytes in spleen
Rare in uninfected lymph nodes
Pre-NK
Thymus not required
for development.
~5-20% peripheral blood lymphocytes
Mature NK
>90% of lymphocytes in placenta
CD3 +
CD16+ , CD56+
CD3-, CD4-, CD8-
NK Cells - Effector Functions
Cell mediated-cytotoxicity
– Perforin granzyme pathway
– Secreted or membrane TNF-α
Antibody-dependent cellular cytotoxicity
(ADCC)
Antibody-dependent cellular
cytotoxicity (ADCC)
Cells that perform
ADCC must have
FC receptors to
bind Ig molecules
and trigger killing
of target cell.
Cytokine secretion
– Early γ-interferon production
– Secretion of TNF-α, LT-α, GM-CSF, IL-5, MCSF, IL-3, IL-10, IL-13.
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Cytokine secretion of NK cells
Role for IFN-γ
Natural Killer (NK) Cells
Part of Early Immune Response
Viruses
Varicella zoster virus & CMV are lifethreatening in humans lacking NK cells.
Bacteria
NK cells protect against intracellular bacteria
which tend to infect macrophages. (e.g.
Listeria Toxoplasma, Leishmania)
NK are lymphocytes without traditional antigen receptors
How do they get activated?
NK cell Activating Receptors
Have ITAMs
• 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
–
Intracellular signal of
lymphocyte receptors
through ITAMs.
Immunoreceptor
Tyrosine based
Activating motifs
(ITAMs).
+
NK cell Activating Receptors
Have ITAMs
Ligand
Class I MHC Specific NK Inhibitory Receptors
Killer Inhibitory Receptors(KIR)
10-12 genes
Extensive allelic polymorphism
KIR genes found in primates, but
not rodents
Ly49 ~10 genes
Extensive allelic polymorphism
Deleted from the human genome.
mouse
human
STIMULATORY
RECEPTOR
(FcR, NKR-P1,
NKG2D)
KIR
CD94
NKG2A/C/E
Ly49
-
+ -
DAP12
Immunoreceptor
tyrosine-based
Activation motif
(ITAM)
YxxL x 2
Y
X
X
L
Y
X
X
L
P
Protein
Tyrosine
Kinase
e.g. SYK
KIR2DL KIR3DL
Ig SF
C-type lectinrelated family
= Immunoreceptor tyrosine-based inhibitory motif (ITIM)
Phosphorylation
of substrates
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ITIMs on Lymphocyte
Receptors
ITIM Signaling through Inhibitory
Receptors
MHC
Inhibitory
receptor
Inhibitory receptors have
ITIMs to prevent activation.
(Act in opposition to ITAMs)
Immunoreceptor tyrosine-based
Inhibitory motif= ITIM
V
X
Y
X
X
L
PROTE IN
TYROSIIN E
PHOSPHATASE
E.g.
P
Shp-1
V/IxYxxL/V
De-phosphorylation of
signaling molecules
NK cell activation is regulated by integrated
positive and negative signals
Very little known about NK activating receptors and their ligands
+
NK Cell
Activate
No
No
Yes
Yes
Ligands for NK cell activating receptors
Target
Inhibit
No
Yes
No
Yes
Outcome
No killing
No killing
Killing
No killing/killing
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 been
identified yet…
NK Cells kill cells expressing activating ligands but need to have
inhibitory receptors to protect MHC expressing cells.
Inducible Ligand Model
NK cell
Normal Cell
MHC
–
+
No killing
Infected Cell
NK cell
MHC
Ligand
–
Killing
+
NK Cell- Opposing Signal
Model
Ligand induced by stress or infection
In these situations, activating receptor can overcome inhibitory signal
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Summary
NK cells activation is controlled by the balance between
activating and inhibitory receptors.
Inhibitory receptors bind MHC class I molecules and
prevent inappropriate lysis of self cells.
NK cells are activated by “missing self”, which can occur
when viruses or tumor cells downregulate MHC class I to
avoid recognition by CTL.
Some ligands for activating receptors are constitutively
expressed. Others are induced upon viral infection or
stress.
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