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Introduction: Phenotype and physiology:

Natural killer cells are large lymphocytes with properties of both lymphocytes and monocytes. NK
cells spontaneously kill a variety of target cells in a MHC unrestricted manner, without priming or
activation. NK cells are in a lymphocyte lineage separate from B cells and T cells with a large and slightly
kidney shaped nucleus. They are recognised as Large Granular Lymphocytes (LGL) in blood whose
cytoplasm contains 10-12 large basophilic granules. Also present in secondary lymphoid organs, they are
recognised by their ability to kill certain tumor cells without having had prior exposure to them, called
natural cytotoxicity. As assessed by NK specific markers like CD56, they comprise 10-15% of the
lymphocytes in human blood and lack idiotypic receptors for antigen typical of B-cells (Ig) and T-cells
(TCR). About 3-5% of human lymphocytes are positive for cell surface TNF-α and are cytolytically active.
A similar fraction of lymphocytes are CD56+ve and contain a number of large granules that stain for
perforin and granzymes. Other unique markers for NK cells are CD11b (C3bR), CD16 (FcγRIII) and CD57
(NCAM). NK cells come from the bone marrow with a generation time of about 5 days where they
differentiate from CD34+ precursors into agranular NK phenotype cells. NK cells circulate in the blood and
spleen where they develop the granules and cytolytic activity but few are found in normal lymph nodes and
peripheral tissues. NK activity rises to adult values shortly after birth and is stable throughout life with the
activity in males slightly higher than females. Systemic NK activity is easily suppressed by a number of
factors including surgery, cytotoxic drugs, estrogens, progesterone, stress, smoking and many other factors.

Role(s) of NK cells:
Natural killer cells mediate the immediate killing of tumor cells, virally infected cells and tissue graft
cells. Eliminating NK cells in vivo increases the rate of growth of tumours and the severity of viral diseases.
Rapidly proliferating cells like bone marrow cells and tumor cells are most easily killed and slowly growing
differentiated cells are poor targets. As such NK-cells cause cellular resistance in irradiated hybrid recipients
to grafts of parental bone marrow (B6D2F1 rejects C57Bl/6 and DBA/2 marrow). Therefore, NK cells may
also have a role to play in the negative regulation of endogenous proliferative responses.

More recently, natural killer cells have been shown to be capable of producing a variety of cytokines
including interferon-γ, which may enhance the activation of Th1 responses leading to enhanced T-cell
mediated immunity, which may help the development of immune resistance to viral pathogens. Natural killer
cells can produce a surprising variety of cytokines, including interferons-α and IFN-γ, TNFα, IL-4, IL-1 and
possibly CSF. It is further possible that depending on the relative concentrations of the cytokines expressed,
that the NK cell could indirectly influence the nature of the immune response by inducing Th0
differentiation into either Th1 or Th2.

Mechanism of NK cytotoxicity:
Natural killer cells can directly kill targets to which they are capable of adhering within 1-4 hours
without prior activation, priming or assistance by cytokines. Adherence can be by antibody or complement
component C3b as NK cells have FcγR and C3bR that allow them to produce antibody-dependent cellular
cytotoxicity. Therefore, NK cells are also known as "K" cells when they participate in certain
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) reactions against opsonized microbes or tumor
cells. NK cells express membrane receptors for IgG (FcγRIII / CD16) and inactivated third component of
complement (iC3b Receptor or CR3 / CD11b). Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
reactions are effective against opsonized microbes, parasites or tumor cells. Further, if the NK cells are
"glued" to targets by lectins like concanavaline A, they are also capable of killing the target cells. The actual
receptors used by NK cells to attach to target cells are lectin-like transmembrane receptors. Most of these
receptors, many of which can mediate antagonistic effects on cytotoxicity, are type II membrane proteins
with a C-type lectin motif, which preferentially bind MHC-I antigens. In the mouse, these receptors are
associated with a complex of genes in the distal arm of mouse chromosome 6 near Prp. Homologues of this
NK gene complex that control NK function exist in the mouse, rat (Chromosome 4) and human
(Chromosome 12). These receptors are conserved and therefore presumably important for overall health.
The gene complex is multigenic (many similar genes with 60-90% homology but different reactivities) and
may have a limited number of alleles for each gene locus, the expression of which is selected in an unknown
manner without allelic exclusion except within a locus. Therefore, NK cells can simultaneously express
several different lectin-type receptors and show specificity for multiple target cell antigens and different
target cells. Similarly, not all NK cells will express all the same NK receptors so the population is also
heterogeneous with respect to specificity for target cells.

Natural killer cell receptors:

Killing is non-restricted with respect to Histocompatibility Complex (MHC) but can detect MHC-I.
Cells can also recognise subtle surface changes such as cell surface proteins, their nature and charge and
degree of sialylation of glycoproteins. Natural killer cell activating receptors (NKAR) are C-type lectins and
Ig-like receptors which recognise as yet undefined target cell epitopes and express an immunoreceptor
tyrosine-activating motif (ITAM) in their cytoplasmic domain (YxxL(x)6-8YxxL). Cross-linking of NKA-
Receptors increases Ca++ mobilisation, receptor phosphorylation, increased NKAR expression in NK and
T-cells and increased cytotoxic activity in NK and T-cells. Natural killer cell inhibitory receptors (NKIR)
are also C-type lectins and Ig-like receptors that recognise conserved monomorphic MHC-I epitopes and
express an immunoreceptor tyrosine inhibitory motif (ITIM) in their cytoplasmic domain. The receptors
containing the ITIM motif (V/IxYxxL) activate the phosphatases SHP-1 and SHP-2 inhibits kinase-
mediated activation, blocks signalling and globally inhibits cytotoxicity. Therefore, ITIMs are dominant
over ITAMs though ITIM-ve receptors that lack YxxL or have a defective YxxL motif may allow
cytotoxicity to occur even if they do not express ITAMs. Antibodies to either the NKIR or the MHC ligand
will block the negative signal and allow (activate) killing. Interestingly, the gene for SHP-1 is also found on
mouse chromosome 6 and human chromosome 12, near the NK gene complex. The presence of peptide in
the MHC groove may be important for recognition by NKARs and NKIRs. NKARs and NKIRs are
members of a multigene family, the expression of which in individual NK-cells would appear to be random
and clonal. Single NK-cells appear to express single NKAR and NKIR receptors, the latter being selected
for reactivity to one of the MHC-I antigens expressed by the host.

NK induced target cytolysis:

Antibody Dependent Killer (K) and Natural Killer (NK) cells (ADCC) kill by extracellular
cytotoxicity by binding to a target cell and secreting cytolysins which unidirectionally kill the target cell.
Once the target is bound by an NKAR and no NKIR is activated, the cytotoxic reaction occurs. The
interaction of cell adhesion molecules between NK and the target cell may tighten the attachment. The first
step is a magnesium dependent movement of the cytoplasmic organelles (Golgi and granules) of the NK cell
to face the target cell. The secretion of the granule contents into the intercellular space is a calcium
dependent step that results in the preferential insertion of perforin pores into the target cell membrane. The
granule contents appear to be micro-vesicles that may target perforin and internal granzymes to the target
cell. If sufficient perforin is present, target cell osmotic lysis is rapid (1-4 hours), otherwise the granzyme
initiated apoptosis requires 10-24 hours for DNA fragmentation and complete cell destruction.

Evidence of Major Role for NK in Tumor Immunosurveillance:

The primary function of NK cells is surveillance that is effective against low numbers of targets,
high tumor specific antigen (TSA) epitope densities, and low MHC expression augments lysis. NK cells can
also activate monocytes via release of IFNγ but there is no memory or long term resistance so recurrence is
common. NK and MΦ responses are non-adaptive, with low proliferation and expansion potential.
Activation is inhibited by PGE2, IL-10 that suppresses their activity during chronic disease.
Immunodeficient hosts show a normal incidence of malignant disease. B or T cell deficient animals resist
cancers (cancer incidence is normal but growth may be faster). T and B combined immunodeficiency is
associated with increased NK. NK deficient animals have increased incidence of malignant disease.
Depletion of NK increases disease. Boosting of NK decreases disease. Transfusion with enriched NK or
LAK decreases disease.

Cancer Immunotherapy by Cytokines Augments NK Activity:

Tumor therapies that augment natural resistance mediated by NK cells shows much promise as a
cost-effective treatment. Currently such treatments are only utilised in advanced malignant disease.
Experimental protocols are not in clinical use today. Primarily involve natural killer cells and macrophages
(non-specific effectors). Progressive malignant disease or persistent viral diseases have, by definition,
evaded the non-specific resistance and specific immune mechanisms of the host.

Lymphokine activated killers (LAK) (See NK section) are induced by the direct administration of
soluble cytokines: IL-2 and/or IFNα. NK cells activated to become LAK cells with IL-2, used in LAK
therapy successful using PBL or tumor infiltrating lymphocytes (TIL) effectors. Aspirin or indomethacin
inhibits PG synthase inhibits PGE and may be effective therapy when combined with LAK therapy.
Advantages: Well tolerated by patients, effective against a wide variety of primary and secondary cancers.
Disadvantages: Cytokines are expensive; Rapidly cleared from the circulation; Toxic side effects are

Review: Function of Natural Killer Cells

1. Natural cytotoxicity - kill some tumor cells and some virally-infected cells in the absence of
specific recognition of viral or tumor-associated antigens

2. First line of defence against spontaneously arising tumor cells and against viral infection. Kill cells
with low or absent expression of MHC on cell membranes.
NK cell similarity to T cells:
- Express another T cell marker, CD2
- Express low affinity IL-2R and proliferate in response to high doses of IL-2.
- Express a low-affinity receptor for IgG; FcγRIII = CD16
- Express the transmembrane signalling chain of the CD3 complex; ζ (zeta)
BUT: NK cells have no smIg or TCR-like idiotypic receptor for the recognition of specific antigen; they
may also be triggered to proliferate or to generate cytolytic function in the presence of cytokines
alone (Type I IFNs, IFN-γ, IL-2, TNF-α). Recognition involves either an Ig-like or lectin-like
receptor for target cell monomorphic autologous MHC-I epitopes or carbohydrate (α3 domain)
respectively. These receptors either activate or inhibit killing depending on their cytoplasmic cell-
activation motifs (ITAM/ITIM).

3. Activated by the T-cell derived cytokine Interleukin-2 (IL-2), produced during a specific immune
response, to become Lymphokine Activated Killer (LAK) Cells.

LAK cells are effective in the treatment of malignant melanoma or renal cell carcinoma in
approximately 20-40% of cases tested:
i. Patient peripheral blood LGL + IL-2 ----> LAK cells
ii. LAK cells + additional IL-2 ---> transfused to patient.
iii. In vivo localisation and proliferation of LAK Cells.
iv. Enhanced Tumor Cytotoxicity, mediated by LAK cells.
v. Serious side effect due to IL-2 toxicity (vascular leaking due to TNF and nitric oxide).