Natural shown to be critical for NK cell development

killer Cell Biology

Natural killer (NK) cells were originally described in terms
of function in 1971 when Cudkowizc and Bennett observed that mice that had
received lethal irradiation were capable of rejecting allogeneic or parental
strain bone marrow cell (BMC) allografts.(1, 2) NK cells are of lymphoid origin
and are found in the peripheral blood, (constitute
approximately 10% of the lymphocytes in human peripheral blood), spleen, and BM, as
well as other tissues. They are radio-resistant, large, granular
lymphocytes that represent
an important arm of innate immunity and are thought to play a critical role in
the immune surveillance against tumors and virally infected cells.(3)They are
regulated by a number of receptors with opposite function that finely tune
potent effector functions such as cytolytic activity and production of
cytokines playing a major role in inflammation and regulation of both innate
and adaptive immune responses(4-9)

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NK cells can
kill in a rapid manner and this NK cell-mediated cytotoxicity occurs primarily
through the perforin/granzyme-dependent pathway, although NK cells can also use
Fas ligand (FasL) and tumor necrosis factor related apoptosis inducing ligand
(TRAIL) to kill target cells (10). NK cells also have the ability to secrete a wide
range of cytokines upon activation (11) and these cytokines can either promote
or inhibit hematopoiesis, for example, GM-CSF and TGF-?, respectively(10-12).

also play a key role in the differentiation of NK cells. Interleukin (IL)-2,
IL-15, and IL-21, are capable of inducing proliferation and activation of NK
cells. However, only IL-15 and fms-like tyrosine kinase 3 (flt3) ligand have
been shown to be critical for NK cell development and maintenance (13-18). Stem
cell factor (SCF) and fetal liver kinase ligand (flk2L) have also been shown to
be important in NK cell differentiation (19)

function of NK cells is also mediated by cytokines.IL-12 and IFN-?/? exert
potent stimulatory effects on NK cells, and IL-12 and IL-18 in combination is
particularly effective in augmenting NK cell function (20, 21). IL-2 has also
been shown to significantly activate NK cells, and adoptive immunotherapy of
IL-2 activated NK cells after autologous BMT has been used in patients with cancer
with acceptable toxicities.(22) The predominant cytolytic targets of NK cells are uncommon cells
that have downregulated expression of class I MHC(MHC-I), which is expressed on
nearly every healthy cell of the body.(23) MHC-I
loss is a fairly common mechanism by which tumors and virus-infected cells can
evade recognition by the T-cell receptor of cytolytic T cells, and NK cells.(24,25)


Recently, NK cells were officially classified as the
prototypical members of the group 1 innate lymphoid cells (ILCs), which are
defined by their capacity to secrete IFN-? but not type 2 cytokines (IL-4 and
IL-13), IL-17, or IL-22. Group 1 ILCs are thus distinct from group 2 ILCs,
which produce IL-13, and group 3 ILCs, which can produce IL-17, IL-22, or both.(26,27)
Human NK cells are classically defined as CD56+CD3- cells, distinguishing them from CD56+CD3+ cells, which consist of a mixed population of
NK-like T cells and antigen-experienced T cells that have upregulated several
NK cell markers.(28)

Two major subsets
of NK cells are found in human subjects that can be distinguished by their
levels of CD56 expression, namely CD56dim and CD56bright.(28) CD56dim
NK cells are fully mature, make up approximately 90% of the NK cells in
peripheral blood, and predominantly mediate cytotoxicity responses. In
contrast, CD56bright cells are more immature, make up approximately
5% to 15% of total NK cells, and have been considered primarily as cytokine
producers while playing a limited role in cytolytic responses. Although CD56bright
NK cells are more efficient at producing cytokines overall, the CD56dim
NK cells can also contribute significantly to early cytokine production because
they comprise a significantly greater fraction of the total NK cell pool and
can more rapidly secrete cytokines.(29-31)

NK Cell


identification of MHC class I-specific inhibitory and activating receptors in
addition to non-MHC class I specific activating and inhibitory receptors has
grown substantially in the two past decades. This has led to a tremendously complex
set of receptors responsible for innate recognition of foreign, abnormal, or
virally infected cells by NK cells, and these receptors have become relevant
with respect to allogeneic BMT and malignant cancer therapies.(1) The first
class of MHC class I-specific receptors was identified in mouse and is termed
Ly49. In any given mouse, an NK cell population can express a variable
combination of the 20 or more Ly49 receptors characterized. Some of these
receptors have different affinities for the different MHC class I

The next
class of receptors are the killer cell Ig-like receptors (KIRs) which
specifically recognize groups of HLA-C (p58 or KIR2DL1-KIR3DL3)(4-7), HLA-B
(p70 or KIR3DL1) and HLA-A alleles (p140) (8-13) Both the Ly49 and KIR
multi-receptor families contain members that have either activating or
inhibitory action.(14)

Another class
of MHC class I-specific receptors expressed both in humans and in mice is
comprised of the C-type lectin molecule CD94, which is covalently associated
with a member of the NKG2 family. Like Ly49 and KIR receptors, these receptors
also have been shown to exert inhibitory (NKG2A or NKG2B) or activating (NKG2C)
signals upon binding Qa-1b (mouse) or non-classical HLA-E (human) molecules on
targets (15-18).


NK cells also
have Ig-like transcript (ILT) receptors that interact with HLA-G (to protect
the fetus and placenta from rejection)(19). Another group of NK cell receptors
comes from a more diverse family of receptors of NK-cell-specific Ig-like
molecules that are known as natural cytotoxicity receptors, or NCRs. NCRs
include NKp30, NKp46, and NKp44 as well as NKG2D. NKG2D is a member of the NKG2
family expressed by NK cells and cytotoxic lymphocytes (CTLs) (20-23). most NK
cells can express the Fc?RIII (CD16) molecule, which recognizes the Fc
component of bound Ig molecules and initiates cytolysis by the antibody
dependent cellular cytotoxicity (ADCC) pathway (24), thus giving the NK cell
another method of target recognition.(1)

natural killer cells have 5 main categories of cell surface receptors. Activating  receptors (e.g. CD16, NKp46, NKG2D, NKG2C,
KIR-S, Ly9), Inhibitory receptors (e.g. KIR-L, NKG2A), chemotactic receptors
(e.g. CCR2,CCR5,CXCR1,CXCR4,CXCR6), Cytokine receptors(e.g.  IL-1R, IL-15R,IL-18R), Adhesion
receptors(e.g. ?1 integrins).



Allogeneic bone marrow
transplantation (BMT) has proven to be an effective treatment for hematologic
malignancies and some solid tumors.(1) However, the high incidence of
graft-versus-host disease (GVHD) as a complication of this treatment has
limited the overall effectiveness of BMT.(2) GVHD is mediated by the activation
and proliferation of alloreactive T cells leading to tissue damage in the host,
primarily in the gastrointestinal tract, liver, and skin (3) causing
significant morbidity and mortality.

One of the major challenges of
allo-Stem Cell Transplantation(SCT) is to reduce the incidence and severity of
GVHD while boosting the graft-versus-leukemia (GVL) effect. In the setting of
allo-SCT, the reconstitution of natural killer (NK) cells is of notable
interest due to their known capability to induce GVL without GVHD.(4) Studies of the role of NK cells in bone marrow engraftment
demonstrated that host NK cells persisting after conditioning can contribute to
graft rejection (5) while donor NK cells can promote hematopoietic
engraftment (6).


The first study suggesting a
relationship between NK cells and GvHD development was reported by Lopez and
coworkers from the Sloan Kettering Cancer Center showing a significant
association between GvHD development and pre-transplant levels of NK cell
activity, as measured by cytotoxic assays performed using herpes simplex virus
type 1-infected fibroblast as target cells, in peripheral blood of a small and
heterogeneous cohort of 13 patients undergoing different protocols of HCT.(7) Livnat et al. (8) and Dokhelar et al. (9) addressed the same
issue assessing NK cell activity against the K562 leukemic cell line both
before and after hematopoietic cell transplantation (HCT) and obtained contradictory results finding either no
relationship (8) or a positive association (9) between early post transplant NK
cell activity and GvHD development. In 2015
Jacobs B. and et al could demonstrate that NK cells gain cytotoxic and
cytokine producing functions early during hematopoietic immune reconstitution
following autologous SCT.(10) In addition to clinical studies, it has been
shown in animal models that IL-2-activated NK cells may efficiently prevent or
even reduce GVHD without any adverse impact on their important GVL effect.(11-13)

After chemotherapy or hematopoietic
stem cell transplantation, NK cells are the first lymphoid cells to recover (14,15).
Surprisingly, such postgrafting regeneration of NK cells does not cause
clinical graft-versus-host disease (GVHD); this has led to the conclusion that
normal nonhematopoietic tissues lack ligands able to activate NK cell lysis.(16)

The concept of an NK-mediated
regulatory function is also supported by the observation that a higher number
of bone marrow NK cells has been associated with a decreased incidence of
chronic GVHD after HLA identical sibling bone marrow transplants in human.(17)Researches
in 2002 and 2004 showed that this regulatory function can be indirect, through
the interplay and molecular crosstalk with dendritic cells (DCs) (18,19). On
the one hand, DCs can prime, further the activation of, augment the expansion
of, and enhance the activities of NK cells through the production of cytokines
such as IL-2, IL-12, IL-15, IFN-a/b, and TNFa.(16) The regulatory function of
NK cells on adaptive immuneresponses appears also to be mediated through direct
lysis of activated T cells (20,21). This pathway has been postulated to play an
important role in the generation of memory T cell repertoire. Several recent
observations suggest that certain subpopulations of NK cells promote allograft
tolerance via a cytolysis-dependent regulatory pathway (22–24). However, little
is known about the effects of NK cells on donor T cells after BMT. In 2010,
Magali Noval Rivas et al showed that NK cells can regulate chronic GVHD by
limiting recipient minor histocompatibility Ag (mHA)-driven proliferation of
donor CD4+ T cells.(16)

According to studies by scientists, the relationship between NK Cell and GVHD can be generally described in two ways: firstly, Nk Cell cytotoxic functions and GvHD prevention: NK cells can suppress GvHD development through their cytotoxic function either directly, by depleting activated alloreactive T cells, or indirectly, by depleting APC and preventing T cell stimulation,T cell killing by NK cells appears to be dependent on both perforin production (25,26,27) and FAS-mediated induction of apoptosis (26, 28, 29),and secondary Nk Cell cytokine production and GvHD induction: Although it is unclear if NK cells production of immunesuppressive

cytokines can prevent GvHD, it is
established that pro-inflammatory cytokine production by NK cells can
contribute to GvHD development. Xun et al. showed In a xenogeneic model that in
vitro interleukin-2 (IL-2)-activated human NK cells producing interferon-?(IFN-?)
and tumor necrosis factor-?(TNF-?) were able to induce acute GVHD upon transfer
into SCID mice (30, 31) What is now important about NK cells and its impact on
GVHD is that researchers are looking for new ways to reduce GVHD with the help
of these cells, which we will continue to mention these


Natural Killer Cell Memory

Immunological memory is a cardinal
feature of adaptive immunity. Although natural killer (NK) cells have long been
considered short-lived innate lymphocytes that respond rapidly to transformed
and virus-infected cells without prior sensitization.(1) It has recently become
appreciated that NK cells can also acquire functional qualities commonly
associated with immunological memory similar to that of T and B cells in
response to pathogens and in non-infectious settings.(2)

Antigen-specific memory NK cell responses were first observed in a
murine model of hapten-induced CHS (3). This model was established through
sensitization via painting a specific hapten, such as 2,4-dinitrofluorobenzene
(DNFB) or oxazolone (OXA), on mouse skin and subsequent challenge with the same
hapten on the ears of the mice, after which the recall responses to the haptens
were measured based on ear swelling.(4,5)

The ability of the immune system to
respond rapidly and provide enhanced protection of the host against a
previously encountered pathogen is defined classically as immunological memory.
Longlived memory cells are generated after initial infection and display
heightened responses upon secondary challenge with the same pathogen. The
process of memory formation in T cells has been well studied and is generally
divided into three distinct phases. Upon exposure to cognate antigen, naive
CD8+ T cells clonally expand and differentiate into effector cells during the
”expansion” phase. This first phase is followed by a rapid ”contraction”
phase, when the vast majority of effector CD8+ T cells undergo apoptosis to
form a small, but stable, pool of surviving cells that then enter the third
”memory” phase. Memory CD8+ T cells persist throughout the host organs and
maintain their longevity through self-renewal until a subsequent encounter with
their cognate antigen, when they exhibit enhanced effector function and host
protection. In an experimental system in which Ly49H+ NK cells were adoptively
transferred into mice lacking this receptor, these Ly49H+ cells underwent
robust antigen-driven expansion after MCMV infection. Similar to activated CD4+
T cells, expanded effector NK cells undergo a slower and sustained contraction
phase to establish a long-lived and self-renewing ”memory” pool of
antigen-specific NK cells that can be recovered many months after infection in
a variety of peripheral tissues. (6)

In the following section, we
summarized the receptors, cytokines and signalling pathways that have been impli­cated
in the development of memory NK cells:


1.      Hapten-specific memory NK cells:
Following sensitization of mice with haptens, hapten-specific memory natural
killer (NK) cells are detected in the liver. The generation of these memory NK
cells is dependent on the cytokines interleukin?12 (IL?12), interferon?? (IFN?)
and IFN?. Antibody-mediated blockade of the NK cell receptor natural killer
group 2, member D (NKG2D) or CXC-chemokine receptor 6 (CXCR6), or molecules
that are involved in NK cell trafficking (such as CD18 and P- and E?selectin)
prevents the development of contact hypersensitivity (CHS) responses in the ear
after hapten challenge.(3,7,8)

2.      MCMV-specific memory NK cells:
During mouse cytomegalovirus (MCMV)-infection, naive NK cells expressing the
LY49H receptor expand with contribution of signalling mediated by the DNAX
accessory molecule 1 (DNAM1) receptor and the inflammatory cytokines IL?12, IL?18
and IL?33. Inflammatory cytokines drive the expression of zinc finger and BTB
domain-containing 32 (ZBTB32) and the microRNA miR?155, which are involved in
the expansion of ‘effector’ NK cells. Following the elimination of the virus,
the BIM and autophagy pathways regulate the contraction of the expanded
populations of NK cells, giving rise to a population of MCMV-specific memory NK
cells. Although MCMV-specific memory NK cells distribute systemically in mice,
memory NK cells specific for vaccinia virus reside in the liver, and influenza
virus-specific memory NK cells are found in the liver and lungs (not shown). In
non-human primates, simian immunodeficiency virus-specific memory NK cells
reside in the spleen and liver.(9,10,11,12,13,14)

3.      Cytokine-induced memory NK cells: In vitro, the
brief exposure of NK cells to the cytokines IL?12, IL?15 and IL?18 results in
the upregulation of IFN?, perforin and granzymes, and the production of high
levels of CD25, the high-affinity ?-chain of the IL?2 receptor, is also
induced. After adoptive transfer, these cytokine-induced memory NK cells
persist long term, and their ability to produce abundant cytokines and express
perforin and granzymes is maintained. The presence of IL?2 (or IL?15) further
increases NK cell numbers and their ability to express IFN?, perforin and granzymes
after adoptive transfer.(15,16,17,18)