Natural Killer NK Cells

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Natural Killer (NK) Cells

• Megan Klein
• MT 432 - Spring 2002
• Honors Option

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NK cells are

• lymphocytes that are derived from the bone
  marrow. They circulate in the blood and contain
  cytolytic granules.
• important in the defense from certain lymphoid
  tumor cell lines and from virally infected cells.
  They are key components of the innate immune
  response in that they act without prior
  activation.

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• NK cells' activity is increased when they are
  exposed to
• IFN
• IFN
• IL-12
• Each NK cell expresses several inhibitory and
  stimulatory receptors. These receptors allow NK
  cells to kill infected cells, while sparring
  uninfected cells.

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ACTIVATION
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• Activation of NK cells results in the release of
  cytotoxic granule contents and the production of
  the cytokines IFN and TNF
• NK cells are activated through Killer Activating
  Receptors (KARs). The most important KARs are
• NKp46
• NKp30
• NKp44
• Each of these receptors contain a () charge in
  their transmembrane domain.

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• This () charge corresponds to a (-) charge in
  the transmembrane domain of adaptor molecules,
  allowing them to interact.
• Adaptor molecules are necessary to internalize
  external signals.
• Each receptor pairs with a specific adaptor
• NKp44 - KARAP/DAP12
• NKp30 - CD3
• NKp46 Fc RI /CD3

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Structures of various KARs, including their
associations with adaptor molecules.
Figure 1 Moretta A., et al. Surface Receptors
Delivering Opposite Signals Regulate the Function
of Human NK Cells. Seminars in Immunology. Vol
12, 2000 pg. 129-138.
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• The adaptor molecules contain immunoreceptor
  tyrosine-based activation motifs or ITAMs.
• ITAMs consist of short homo- or heterodimer
  tails which extend into the cytoplasm.

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KAR
Activating receptor

• When an NK cell comes in contact with a target
  cell, activating receptors on the cell surface
  bind to the KARs. Activating receptors could be
  antibodies, stress inducible MHC class I-like
  molecules (MICA or B), or an as yet unknown
  ligand.

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• Upon binding, a signal is sent through the KAR
  to the ITAM region. The ITAM is phosphorylated.
  It is then capable of recruiting src family
  kinases, thus inducing a signaling cascade 1.
• Protein tyrosine kinase activity leads to the
  recruitment of Syk and ZAP70. These molecules
  subsequently stimulate granule release and
  cytokine production.

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• The granules are released onto the surface of the
  target cell.
• Effector proteins penetrate the cells' membrane
  and once inside the cell, induce programmed cell
  death 2.

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But what if we like this cell and dont want to
kill it??
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INHIBITION
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• NK cells express inhibitory receptors that are
  specific for MHC class I alleles 3.
• In humans these molecules are p58 and p70,
  known as Killer Inhibitory Receptors (KIRs).

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• Unlike KARs, KIRs do not need to associate
  with adaptor molecules.
• KIRs consist of long cytoplasmic domains which
  contain immunoreceptor tyrosine-based inhibition
  motifs (ITIMs).
• ITIMs are longer than ITAMs. These additional
  regions contain the elements used for inhibitory
  signaling.

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• When NK cells contact target cells, MHCI
  molecules on the cell interact with the KIRs.
  Upon receptor engagement, ITIMs are
  phosphorylated. They then recruit the cytoplasmic
  tyrosine phosphatases SHP-1 and SHP-2 4.

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Syk
Syk
ZAP70
ZAP70
inactive
SHP-1
active

• Since SHP-1 is a tyrosine phosphatase it is
  able to dephosphorylate tyrosine kinases,
  inactivating them. Syk and ZAP70 are tyrosine
  kinases.
• SHP-1 dephosphorylates Syk and ZAP70,
  inactivating them.
• This disrupts the ITAM cascade and prevents
  granule release from the NK cell.

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• The effector function of an NK cell is triggered
  by a balance between opposite signals 5. If
  both an inhibitory and activating signal is
  detected the NK cell will not be stimulated to
  kill.

INHIB
ACT
INHIB
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• However, in the absence of appropriate
  interaction between KIRs and MHCI, NK cells can
  exert their cytolytic function 6.
• Upon positive stimulus an NK cell will kill.
  An inhibitory signal MUST be present to "turn
  off" the cell.

ACT alone
granule release
cell death
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Left) ITAM alone leads to activation. Right) ITIM
blocks ITAM action, leading to inhibition.
FIGURE 1 - Long, Eric O. "Regulation of Immune
Responces Through Inhibitory Receptors." Annual
Review of Immunology, 1999. 17 pg. 875-904.
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• By blocking activation signals in this way,
  inhibitory receptors protect NK cells from
• exhausting their granule supply
• producing unnecessary cytokines
• causing unnecessary cell death 7.

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How do you get decreased inhibitory
signaling?Dont all cells have MHC class I?
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Virus-infected and tumor cells are able to alter
MHC-I expression.

• Infected cells can inhibit the synthesis of all
  proteins. This decreases the amount of MHC-I
  produced.
• Viruses can also selectively prevent the export
  of MHC-I molecules.
• In each case, the number of MHC class I
  molecules on the cell surface is decreased.

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• Decreased MHC-I expression results in a
  decrease in the number of KIR/MHC-I interactions.
• What interactions are still present are
  insufficient to inhibit NK cell triggering 8.
• Thus, lack of expression completely, or the
  expression of MHC-I in inadequate amounts,
  reduces inhibition signaling.

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B
A
Activating receptor bound to KAR. When MHC is
present in adequate amounts, NK cell receives
BOTH activating and inhibiting stimuli. Thus,
target cell death is averted.
Activating receptor bound to KAR. Without enough
MHC, KIR cant bind. NK cell only receives
activating stimulus, which results in target cell
death.
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• Infected cells can also change the proteins
  bound in the MHC. One way is by glycosylation. If
  this occurs, the MHC-Iprotein complex the cell
  expresses will be altered. KIRs can't bind to
  these "foreign" complexes, so no inhibitory
  signal will be sent to the cell.

Whats that? I cant bind!
I know you Ill bind.
altered MHCprotein
normal MHCprotein
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Summary

• NK cells recognize decreased and/or altered
  MHC-I expression in target cells. This prevents
  KIRs from interacting.
• Without proper KIR/MHC interaction, no
  inhibitory signals can be sent. Thus, activating
  signals will dominate and NK cells will attack.

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NK CELL GENETICS
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Gene Locations

• MHC genes are located on chromosome at loci
• Inhibitory receptors are members of two
  families.
• The C-type lectin genes are on chromosome
• The Ig superfamily genes are located on
  chromosome
• The major KIR genes are located at

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6p21.3
12
19
19q13.4
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• Activating versions of the receptors belonging
  to the same molecular superfamilies are
  interleaved with their inhibitory counterparts
  9.
• Specifically,
• NKp30 and NKp44 are found at loci
• NKp46 is located at
• The adaptor molecule DAP12 is on chromosome

6p21.3
19
19q3.4
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• Hmmm.
• MHC genes on chromosome 6.
• KIR genes on chromosomes 12 and 19.
• KAR genes on chromosomes 6 and 19.
• Notice any similarities?

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Isnt nature wonderful?!

• These loci similarities suggests that NK
  receptor evolution must be to some extent tied in
  with MHC class I ligands 10.

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Impact of Polymorphism

• MHC molecules and NK receptors are both very
  polymorphic they both show variation at their
  gene loci and thus in its protein product 11.
• This polymorphism creates increased diversity
  and variation in structure.

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• The existence of two sets of polymorphic
  surface molecules that interact with each other
  provides interesting possibilities for
  cooperation 12.
• MHC molecules can display a greater variety of
  proteins, while NK receptors are able to
  recognize more.
• These sets of polymorphic cell surface
  molecules provide alternate ways of recognizing
  disease and influencing immune responces 13.

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WHY NK CELLS ARE IMPORTANT

• NK cells perform their functions by combining
  activation and inhibition signals. This allows
  for the controlled killing of only those cells
  expressing abnormalities.
• NK cells work early in the immune response,
  controlling the spread of viruses and tumors,
  until the big hitters of the adaptive response
  can go to work.

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End Notes

• 1Bakker, Alexander B.H., et al. "NK Cell
  Activation Distinct Stimulatory Pathways
  Counterbalancing Inhibitory Signals." Human
  Immunology. 2000. 61 pg. 21.
• 2Janeway, Charles A., et al. Immunobiology. New
  York Garland Publishing, 2001. pg. 82.
• 3Janeway, pg. 83.
• 4Blery, Mathieu. "Early Signaling Via
  Inhibitory and Activating NK Receptors." Human
  Immunology, 2000. 61 pg. 53.
• 5Moretta, Alessandro, et al. "Activating
  Receptors and Coreceptors Involved in Human NK
  Cell Mediated Cytolysis." Annual Review of
  Immunology, 2001. 19 pg. 198.
• 6Blery, pg. 51.

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• 7Long, Eric O. "Regulation of Immune Responces
  Through Inhibitory Receptors." Annual Review of
  Immunology, 1999. 17 pg. 879.
• 8Moretta, pg. 199.
• 9 Trowsdale, John. Genetic and Functional
  Relationships Between MHC and NK Receptor Genes.
  Immunity 2001. 15 368.
• 10 Trowsdale, pg. 371.
• 11 Janeway, pg. 172.
• 12 Trowsdale, pg. 372.
• 13 Trowsdale, pg. 363.