Human natural killer cells in health and disease.
ABSTRACT Natural killer (NK) cells are an essential component of the innate immune system and play a critical role in tumor immune surveillance. NK cells express their own repertoire of receptors (NKRs) that bind to major histocompatibility class I or class I-like molecules. The balance of signals from stimulation or inhibition of NKRs determines the ability of NK cells to lyse specific targets. In haploidentical stem cell transplantation with purified stem cells, NK cell alloreactivity (killer immunoglobulin-like receptor [KIR] mismatch) has been demonstrated to reduce the risk of relapse in acute myeloid leukemia. There is a need for adequately powered prospective randomized studies to determine the usefulness of NK cells as adoptive immunotherapy, optimal NK cell doses and timing of administration. Further studies are required to determine optimal selection of donors and recipients, both on NKR matching/mismatching, undergoing haploidentical and unrelated hematopoetic stem cell transplantation.
- SourceAvailable from: Esin Aktaş-Çetin[Show abstract] [Hide abstract]
ABSTRACT: Natural killer (NK) cells are essential members of innate immunity and they rapidly respond to a variety of insults via cytokine secretion and cytolytic activity. Effector functions of NK cells form an important first line of innate immunity against viral, bacterial and parasitic infections, as well as an important bridge for the activation of adaptive immune responses. The control of NK-cell activation and killing is now understood to be a highly complex system of diverse inhibitory and activatory receptor-ligand interactions, sensing changes in MHC expression. NK cells have a functional role in innate immunity as the primary source of NK-cell-derived immunoregulatory cytokines, which have been identified in target organs of patients suffering from autoimmune diseases, and play a critical role in early defense against infectious agents. This review focuses on recent research of NK cells, summarizing their potential immunoregulatory role in modulating autoimmunity and infectious diseases.Expert Review of Clinical Immunology 07/2009; 5(4):405-20. · 2.89 Impact Factor
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ABSTRACT: Although reduced natural killer (NK) cell levels have been reported consistently in patients with coronary artery disease (CAD), the clinical significance and persistence of this immune perturbation is not clarified. In this study we characterized the NK cell deficit further by determining (i) differentiation surface markers and cytokine profile of NK cell subsets and (ii) ability to reconstitute NK cell levels over time. Flow cytometry was used to analyse NK cell subsets and the intracellular cytokine profile in 31 patients with non-ST elevation myocardial infarction (non-STEMI), 34 patients with stable angina (SA) and 37 healthy controls. In blood collected prior to coronary angiography, the proportions of NK cells were reduced significantly in non-STEMI and SA patients compared with controls, whereas NK cell subset analyses or cytokine profile measurements did not reveal any differences across groups. During a 12-month follow-up, the proportions of NK cells increased, although not in all patients. Failure to reconstitute NK cell levels was associated with several components of metabolic syndrome. Moreover, interleukin (IL)-6 levels remained high in patients with sustained NK cell deficit, whereas a decline in IL-6 (P < 0·001) was seen in patients with a pronounced increase in NK cells. In conclusion, we found no evidence that reduction of NK cells in CAD patients was associated with aberrations in NK cell phenotype at any clinical stage of the disease. Conversely, failure to reconstitute NK cell levels was associated with a persistent low-grade inflammation, suggesting a protective role of NK cells in CAD.Clinical & Experimental Immunology 01/2014; 175(1):104-12. · 3.41 Impact Factor
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ABSTRACT: INTRODUCTION: NK cells represent a distinct lymphocyte population with extensive cytolytic activity and a variety of other functions, including regulation of hemopoiesis, suppressor functions and immunoglobulin production. Recently, reports suggest that NK cells also display potent regulatory functions via secretion of cytokines or cell-contact-dependent mechanisms. Thus NK cells may regulate innate and adaptive immune responses and play a role in immune homeostasis. AREAS COVERED: NK cells play important roles in viral infections, autoimmunity, pregnancy, cancer and bone marrow transplantation. Although the role of NK cells in allergic diseases is poorly described, recent findings suggest their role in allergy. EXPERT OPINION: Recent developments in the study of NK cell subsets have support their role in allergic diseases that contribute to allergen-specific immune suppression, allergen-specific T(H)1 cell generation as well as IgE and other Ig production.Expert opinion on biological therapy 03/2011; 11(7):833-41. · 3.22 Impact Factor
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, March 1994, p. 125-133
Copyright (C 1994, American Society for Microbiology
Vol. 1, No. 2
Role of Human Natural Killer Cells in Health and Disease
THERESA L. WHITESIDE' 2,3* AND RONALD B. HERBERMAN1' 24
Pittsburgh Cancer Institute1 and Departments ofPathology,2 Otolaryngology,3and Medicine, 4 University of Pittsburgh,
Natural killer (NK) cells, the CD3- CD56+ CD16+ subset of peripheral blood lymphocytes, have long been
known to be involved in non-major histocompatibility complex-restricted natural immunity to virally infected
and malignant target cells. The association of abnormalities in NK cell numbers or functions with a broad
spectrum of human diseases has been more clearly defined in recent years as a result of the improved
knowledge of NK cell physiology and advances in monitoring of NK cell functions in health and disease. The
ability to reliably measure changes in NK activity and/or numbers during the course of disease or response to
treatment has focused attention on the role of the NK cell in disease pathogenesis. The improved
understanding ofNK cell deficiency in disease has opened a way for therapies specifically designed to improve
NK cell function. The therapeutic use of biologic response modifiers capable of augmenting NK cell activity in
vivo and of adoptive transfer of highly enriched, activated autologous NK cells in diseases such as cancer and
AIDS is being evaluated. The importance ofNK cells in health and the consequences of NK cell deficiency or
excess are likely to be more extensively monitored in the future.
Natural killer (NK) cells are a subset of lymphocytes with
the distinct morphologic features of large granular lympho-
cytes (LGL) and important biologic functions. They are distin-
guishable from T and B lymphocytes by surface phenotype,
cytokine profile, and the ability to mediate spontaneous cyto-
toxicity, without prior sensitization, against a broad range of
targets, including tumor cells and virally infected targets (22,
63). In human peripheral blood, NK cells account for about 5
to 15% of circulating lymphocytes, but in some organs, e.g., the
liver, they represent up to 45% of tissue-infiltrating lympho-
While it has been widely recognized that NK cells mediate
natural immunity, their role in human health has generally
been underestimated. Current evidence indicates that de-
creased or absent NK cell numbers or activity is often associ-
ated with the development or progression of cancer, acute or
chronic viral infections, autoimmune diseases, immunodefi-
ciency syndromes, and psychiatric illness. The ability to reliably
measure NK activity in human body fluids or tissues and to
enumerate cells which express NK cell-associated surface
markers has contributed considerably toward a better defini-
tion of NK cell involvement in human diseases and their
From recent evidence, it appears that the NK cell partici-
pates either directly or indirectly in multiple developmental,
regulatory, and communication networks of the immune sys-
tem. Today, the NK cell is increasingly frequently viewed as a
remarkably efficient effector cell which is not only equipped for
killing but is also capable of rapid response to exogenous or
endogenous signals by producing a variety of cytokines and
factors involved in interactions between immune and nonim-
mune cells. In this review, we summarize the functional
characteristics and the physiologic role of human NK cells and
evaluate potential therapeutic approaches based on NK cell
*Corresponding author. Mailing address: Pittsburgh Cancer Insti-
tute, W1041 Biomedical Science Tower, 200 Lothrop St., Pittsburgh,
PA 15213-2582. Phone: (412) 624-0096. Fax: (412) 624-0264.
upregulation or their adoptive transfer to patients with cancer
or other diseases.
Morphologic and phenotypic characteristics of NK cells.
NK cells, which belong to a subset of lymphocytes referred to
as LGL, have average diameters of 7 to 8 ,um in the resting
state and 10 to 12 ,um in the activated state. On May-
Grunwald-Giemsa-stained smears of peripheral blood mono-
nuclear cells (PBMNC), LGL are easily recognizable not only
by the reniform nucleus but also by the presence in the
cytoplasm of numerous azurophilic granules. To enumerate
NK cells in blood or body fluids, it is not sufficient to depend
on morphology, however, because activated T lymphocytes
may acquire the same appearance. To distinguish NK cells
from other lymphocytes, it is necessary to use monoclonal
antibodies (MAbs), which recognize distinctive surface mark-
ers expressed on NK cells, for staining and flow cytometry.
Mature, circulating NK cells express the CD3- CD56+ CD16+
CD2dim phenotype and are distinguishable from T cells by the
lack of the T-cell receptor or of rearranged T-cell receptor
genes, which retain the germ line configuration in mature NK
cells (28). Unlike B cells, NK cells do not express surface
Fc-yRIII, the Fc receptor for IgG, NK cells may be positive for
surface-bound Ig (40). Surface markers expressed on NK cells
or activated NK cells include interleukin-2 (IL-2) receptors
(41); other types of Fc receptors (45, 46); 1 and I2 integrins
(74); various activation antigens, e.g., HLA-DR, transferrin
receptor (CD71), CD69, and the activation-inducing molecule
Leu23 (47); and putative clonotypic NK receptor(s) for binding
to target cells (38). The nature of this NK receptor remains
undefined and controversial today.
Many of the surface molecules expressed on NK cells are
present on other hematopoietic cells, and therefore, what
distinguishes NK cells from other PBMNC is a unique combi-
nation of several markers, e.g., CD56, CD16, and CD2, as well
as the absence of certain other markers, such as CD3, CD14,
and surface Ig. Not all NK cells express the consensus pheno-
type defined above, and subsets of, for example, CD56+
CD16- or CD56-CD16+ NK cells have been recognized and
126WHITESIDE AND HERBERMAN
TABLE 1. Expression ofPIand 12 integrins on human resting NK
cells and NK cells activated in vitro by IL-2a
Mean % positive cells ± SEM (mean
fluorescence intensity ± SEM)
Fresh NK cells
(n = 9)
NK cells +
IL-2 (n = 10)
85 ± 3(85 ± 11)
6 ± 1 (NAC)
8 ± 2 (NA)
94 ± 2 (141 ± 15)
85 ± 4 (89 ± 8)
43 ± 5 (61 ± 6)
96 ± 1 (343 ± 34)
94 ± 5 (349 ± 43)
85 ± 5 (369 ± 32)
93 ± 2 (257 ± 56)
89 ± 6(175 ± 30b)
87 ± 3" (165 ± 39)
91 ± 5b (123 ± 20)
98 ± 1 (249 ± 12b)
83 ± 7 (148 ± 19b)
9 ± 4d (84 ± 8)
99 ± 1 (927 ± 137b)
97 ± 3 (287 ± 25)
85 ± 3 (341 ± 39)
98 ± 1 (428 ± 49")
aNK cells were purified by negative selection with magnetic beads from the
peripheral blood of normal donors. They were incubated in the presence of IL-2
(300 IU/ml) for 6 days, stained with fluorescein isothiocyanate-conjugated MAbs
to the surface antigens listed, and evaluated by flow cytometry. The data were
provided by H. Rabinowich, Pittsburgh Cancer Institute.
bSignificantly increased (P < 0.002) compared with fresh (resting) NK cells.
'NA, not available.
d Significantly decreased (P < 0.04) compared with fresh (resting) NK cells.
may represent functionally distinct subsets of NK cells (29, 40).
Surface receptors on NK cells are up- or downmodulated,
depending on the cellular activation state. Among various
classes of surface molecules expressed on NK cells, three have
been particularly extensively investigated in recent years: re-
ceptors for IL-2 (IL-2R), Fc receptors (FcR), and adhesion
The IL-2R is a complex of at least three distinct polypeptide
chains, ot (p55), 1 (p75), and y (p64), expressed on the surface
of lymphoid cells (62). Each of these component chains can
bind IL-2 independently of the others, but the interaction of
the ( plus y or ox plus ( plus y chains leads to the formation of
the high-affinity (10-9to 10-l M) receptors. Most resting NK
cells (>50%) constitutively express the
IL-2R, and only a small subset (<10%) ofNK cells (CD56bright
CD16-) in peripheral blood express functional high-affinity
IL-2R (68). Shortly after activation with exogenous IL-2,
however, most NK cells rapidly upregulate expression of the (
chain and express IL-2Rot de novo (9). In the presence of IL-2,
cx chains (p55) are rapidly shed from the NK cell surface
(soluble IL-2R), so that mostly intermediate-affinity (C) IL-2R
are detectable on NK cells exposed to IL-2 in vitro or in vivo
in patients receiving IL-2 therapy (68). WhileIL-2RPplays an
initial role in the IL-2-induced intracellular signalling path-
induced signal transduction, and control of the rate of disso-
ciation of IL-2 from the receptor complex (62, 65).
Among the surface molecules involved in signal transduction
on NK cells are the FcR CD16, CD32, and the receptor for
IgM (36, 40, 44). The presence on NK cells of the latter two
types of FcR has been confirmed recently (36, 44). It is known
that CD16 in association with the zeta chain is an essential
signal-transducing complex similar to the T-cell receptor-CD3
complex in T cells (4). Upon ligand binding, the CD16-zeta
complex induces transcription of genes that encode proteins
relevant to NK cell functions (3). The FcR are responsible for
antibody-dependent cellular cytotoxicity mediated by NK cells.
A broad range of cellular adhesion molecules (CAMs) are
detectable on circulating NK cells (Table 1). Among these
CAMs, the (2 integrins have been shown to be important in
(p75) chains of
is essential for IL-2 internalization, IL-2-
FIG. 1. NK cells obtained from the peripheral blood of normal
individuals are able to kill NK-sensitive tumor cell lines such as K562
but generally do not kill freshly isolated tumor cells. NK cells
constitutively express IL-2RP (p75) and, in the presence of IL-2,
develop into lymphokine-activated killer (LAK) cells capable of killing
a broad array of fresh or cultured tumor cell targets.
signal transduction and activation of NK cells (51), while the
B1 integrins, specifically VLA-4 and VLA-5 (receptors for
fibronectin) and VLA-6 (a receptor for laminin), participate in
NK cell binding to solid substrates, extracellular matrix (ECM)
components, and cell targets (15). NK cells also express
adhesion proteins like laminin and fibronectin, and antibodies
to these proteins have been reported to inhibit NK cytotoxicity
without affecting NK-target cell binding (57). If confirmed,
these findings would suggest that antifibronectin MAbs inter-
fere with the lytic process at a postbinding stage (57). Other (1
integrins are not expressed on fresh NK cells but are induced
after cellular activation (Table 1). We have recently demon-
strated that the receptor for vitronectin(4(3integrin), which
mediates cell adhesion to the ECM protein vitronectin, is
present and serves as a signal-transducing molecule on NK
Functional characteristics of NK cells. The ability to spon-
taneously lyse a broad range ofvirally infected targets or tumor
cells is the best-known functional attribute of NK cells. The
mechanisms and molecular basis of NK cell target recognition
and interactions between NK cells and their targets are still
poorly understood. Lysis of targets by NK cells involves several
steps occurring in sequence as follows: (i) recognition of target
cells by as yet unknown mechanisms; (ii) binding ofNK cells to
targets (conjugate formation), probably involving various
CAMs on both effectors and targets; (iii) NK cell activation,
leading to rearrangements of cytoplasmic granules and release
of pore-forming enzymes (degranulation); and (iv) injury and
lysis of target cells. The NK cell is a selective killer which does
not harm normal "self' but eliminates NK-susceptible targets
without a need for antigen processing or presentation by major
histocompatibility complex (MHC) molecules (63).
The selective target cell repertoire of NK cells is not
completely understood. For example, as illustrated in Fig. 1,
only activated NK cells (e.g., lymphokine-activated killer cells)
kill fresh tumor cells, while the cytotoxic repertoire of resting
NK cells is restricted to certain tumor cell lines. It has been
thought that NK cell-mediated killing is not MHC restricted,
but this concept has been challenged recently (38). Moretta
and colleagues have provided evidence that NK cells show
alloantigen specificity and that, by using MAbs EB6 and
GL183, it is possible to define distinct alloantigen-reactive
CLIN. DIAGN. LAB. IMMUNOL.
clones of NK cells (37). Thus, receptors specific for alloanti-
gens encoded by the HLA-C or a closely linked gene are
thought to be present on NK cells (37). The role of the MHC
class I complex in the susceptibility of targets to NK-mediated
lysis has been controversial. Transfection of MHC tumor cells
with the MHC class I gene confers resistance to lysis by NK
cells (59), and this resistance has been mapped to the ox1 and °2
domains of HLA-A2 molecules (60). It thus appears that
expression of class I MHC molecules on target cells confers
protection against NK cell lysis, while the absence of MHC
molecules enhances susceptibility to lysis, as if NK cells were
able to sense this absence ("missing-self hypothesis"). The
possibility that a negative signal is delivered to NK cells by
class I MHC molecules or MHC-associated ligands, inducing
protection from NK-mediated lysis, provides an explanation of
why NK cells are not harmful to self targets (i.e., normal tissue
cells) and is consistent with the presence of alloantigen recep-
tors on NK cells.
While immunosurveillance depends on the ability of the NK
cell to recognize and kill its target, other functions of NK cells
may be biologically and physiologically even more important.
Among lymphocytes, NK cells are the first to respond to
activation by IL-2 and possibly other signals. In response to
IL-2, a small subset of NK cells (10 to 30%) rapidly acquire the
ability to adhere to plastic or other solid surfaces (67) and
upregulate surface expression of IL-2R and other activation
antigens as well as expression of mRNA for a variety of
cytokines (64). We have named these cells A-NK cells, A for
the adherence and activation that characterize this early
response to IL-2 (67). While all NK cells undergo activation
and expansion in the presence of IL-2 and/or other cytokines
(IL-4, IL-6, or IL-12), A-NK cells proliferate significantly
better than nonadherent NK cells, perhaps because they
receive a double stimulatory signal, i.e., IL-2 and adherence to
plastic, and achieve very high levels of antitumor activity in
vitro (67). The optimal conditions for NK cell proliferation in
culture have not been defined so far, and IL-2 and IL-12
(natural killer stimulatory factor), both of which support NK
cell growth in vitro, are not sufficient for optimal proliferation
of NK cells (52). Apparently, costimulatory signals delivered
by, e.g., leukocyte-conditioned medium, irradiated B-lympho-
blastoid cell lines, or as yet undiscovered new NK cell growth-
promoting factors, are required for optimal NK cell prolifera-
tion in culture (52).
Activated NK cells, especially the subset of A-NK cells,
become highly mobile and develop membrane structures called
podosomes, which facilitate their movement along solid sur-
faces and binding to tissue cells or ECM (35). Activated NK
cells show significantly increased integrin-mediated adhesion
to fibronectin- or laminin-coated plates (49) and binding to
human umbilical vein-derived endothelial cells cultured on
type I collagen (1). Associated with this increased adhesion
and mobility of activated NK cells are changes in expression of
activation markers and CAM on the cell surface (47, 49),
upregulation of mRNA levels for cytokines (64), increased
adhesiveness to human umbilical vein-derived endothelial cells
and transendothelial migration (74), as well as highly aug-
mented cytotoxicity (67). It has been demonstrated that not
only do IL-2- or phorbol ester-activated NK cells adhere better
to ECM components or cell surfaces but these changes are
associated with changes in the phosphorylation of the
subunits of the,BI integrins VLA-4, -5, and -6 (25). Signal
transduction via integrins on NK cells is not always associated
with increased expression of these CAMs on the cell surface; in
fact, it may only be associated with conformational changes of
CAMs, which are sufficient for induction of an activation signal
TABLE 2. Cytokines, cellular enzymes, and factors known to be
produced by activated NK cells
IL-1, IL-2, IL-3, IL-4,
or macrophage colony-
stem cell factor type 1
Hematopoietic cell growth
Tumor necrosis factors
Transforming growth factor P
Natural killer cytotoxic factor
Other growth factors
Proteases and peptidases
A2, C-yl, C_y2, D
Granzymes A and B
rich in RGDS)
Chondroitin sulfate A
(25). Alterations in CAMs on activated NK cells are under
intense investigation at this time, in order to elucidate the
interactions of NK cells with ligands on vascular endothelium,
ECM, tumor cells, and other tissue cells. These interactions
determine both the entry and effectiveness ofNK cells in tissue
and for those reasons are of great biologic importance.
Activated NK cells produce a spectrum of cytokines (Table
2). This ability of NK cells to produce hematopoietic cell
growth factors, interferons, interleukins, tumor necrosis factors
a and ,B, transforming growth factor, and other growth factors
(43, 64), coupled with their ability to respond rapidly to
exogenous signals by upregulating mRNA production for
various cytokines within minutes (67) and increasing migration
to tissue sites (50, 74), is responsible for the importance of the
NK cell as a mediator or effector of the intercellular commu-
nication network. Upon activation, NK cells also express and
upregulate the receptors for a variety of chemotactic factors,
cytokines, growth factors, and hormones, including neuropep-
tides (33), which allows them to remain responsive to signals
generated not only within the immune system but also else-
where in the body. As indicated in Table 2, NK cells are also
capable of producing many enzymes, some of which are
associated with the cellular membrane and are likely involved
in cell-to-cell interactions; others, which are intracellular, may
be released during NK cell-mediated
produced during this process is natural killer cytotoxic factor,
which is probably released from intracytoplasmic granules and
participates in the lysis ofNK cell-sensitive targets (6). Overall,
the NK cell has the potential to register changes rapidly and
respond spontaneously to signals generated in its immediate
environment or at distant locations in the body without a need
for antigen presensitization.
The interactions of NK cells with Ig, mediated by the FcR,
probably play a crucial role in regulation of NK cell functions.
Both stimulatory and inhibitory signals can be received and
transduced via the Fc-yIIIRs (10, 61), depending on whether
monomeric, cross-linked, or antigen-complexed IgG molecules
are involved, and the intracellular pathways engaged in pro-
lysis. One cytokine
VOL. 1, 1994
WHITESIDE AND HERBERMAN
cessing and directing these signals are now under intense
investigation (56). The possibility that different types of FcR
expressed on NK cells cooperate with each other as well as
with other receptors on NK cells in the handling of exogenous
signals has to be considered one explanation for the cell's
ability to channel various stimuli into a response which best
reflects the requirements imposed by its external microenvi-
ronment. Binding of serum IgM to the Fc,uR on NK cells,
which has been shown to result in downregulation of gamma
interferon (IFN-y) mRNA expression (74), may serve the
essential regulatory function of inhibiting the nondiscrimina-
tory capacity of NK cells to kill a variety of cellular targets,
including normal hemopoietic and other tissue cells. The
ability ofNK cells to produce IFN-,y in response to IL-2 or viral
challenge (64) is important in host defense against infectious
agents. It may be physiologically desirable to downregulate this
function in the absence of such agents, and serum IgM may be
responsible for this downregulation via the FcpR on NK cells.
In the presence of target-bound antibodies, especially those of
the IgG3 isotype, expression of FcR on the NK cell surface
provides it with an additional opportunity for binding to the
target and initiating the lytic process.
The majority of circulating and tissue NK cells in healthy
humans are in a resting state, i.e., they are not in cycle or
proliferating. However, these resting NK cells are prepared to
respond immediately to signals and thus are remarkably well
suited to mediate the first line of defense against various
pathogens (17, 63). While this swift responsiveness to antigen-
independent stimuli is advantageous for the effector cell, it also
requires a regulatory "check and balance" system. The func-
tions of NK cells have to be carefully regulated because of the
potential for inappropriate cytokine release or damage to
normal cells or tissues. Both autocrine and paracrine types of
regulatory mechanisms are probably involved, and the func-
tions of NK cells may be controlled at the level of develop-
ment, differentiation, activation, and availability of NK cells in
the microenvironment as well as via the modulatory influences
of other mononuclear cells (73). For example, the develop-
ment of NK cells from lymphoid cell precursors in the bone
marrow into NK cell precursors depends on soluble factors
produced by the stromal cells in the marrow (17) and various
cytokines produced by T or B lymphocytes (32). NK cell
maturation from NK cell precursors present in the blood,
spleen, and other lymphoid organs is also dependent on a
mixture of cytokines derived from T and B lymphocytes,
including IL-2, which has been shown to interact with colony-
stimulatory factors, a group of hematopoietic growth factors
supporting the proliferation and differentiation of precursor
cells into mature blood cells (43, 63, 73).
The presence of NK cells at the site of tissue injury is a
function of their availability, i.e., the number in the environ-
ment and the ability to proliferate and migrate. The latter is
clearly determined by cytokines and chemotactic factors pro-
duced locally (74). The ability of NK cells to proliferate
appears to depend not only on the local concentration of IL-2
but also on the putative NK cell proliferation-inducing fac-
tor(s), which is thought to be distinct from IL-12 (natural killer
stimulatory factor). The production of proliferation-inducing
factor by activated human PBMNC has been inferred from the
observation that irradiated concanavalin A-activated PBMNC
or lymphoblastoid B cell lines are necessary for NK cell
proliferation in culture (52). There is a general agreement that
neither recombinant IL-2 nor IL-12 is sufficient to optimally
support the proliferation of purified human NK cells in culture
(47, 52) and that a unique growth factor may be required for
NK cell growth. Finally, evidence has accumulated that mono-
cytes are involved in the regulation of NK activity. Thus,
human monocytes maintained in short-term culture have been
shown to strongly modulate the NK activity of fresh autologous
or allogeneic PBMNC (11). On the other hand, monocytes
cultured for 5 to 7 days acquire the morphologic and pheno-
typic features of macrophages and significantly and consis-
tently upregulate NK activity. This modulation of NK activity
was dependent on macrophage viability, cellular integrity, and
ability to synthesize RNA and proteins, indicating that a
macrophage-derived cytokine might be responsible for NK cell
A number of cytokines and various subsets of mononuclear
cells as well as other cells present in a particular tissue
environment modulate NK functions. In vivo, NK cell devel-
opment, growth, activation, and proliferation may be orches-
trated by events which occur at a particular tissue site. The
ability of NK cells to promptly and efficiently respond to such
events qualifies them as excellent mediators of regulatory and
Biologic role of NK cells. NK cells are involved not only in
defense against pathogens and elimination of metastases but in
a variety of other biologically significant interactions (53, 63,
73). The antiviral activities of NK cells have been well docu-
mented in animal models of viral pathogenesis (69). In hu-
mans, changes in systemic NK activity after viral challenge are
consistent with the pattern of initial activation of NK cells
within the first 2 days and suppression of this activity around
day 5 to 7 after infection, followed by a return to the baseline
level (58). There
eliminating tissue cells infected by mycoplasma or bacteria (13,
73). NK cells also participate in regulatory interactions be-
tween immune cells and nonimmune cells. For example, NK
cells are capable of directly upregulating polymorphonuclear
leukocytes to kill Candida albicans (13). NK cells produce
neutrophil-activating factors, which allow polymorphonuclear
leukocytes to more effectively kill C. albicans and possibly
other infectious organisms (13).
The involvement of NK cells in the regulation of hemato-
poiesis has been investigated extensively. Because the NK cell
is able to produce a spectrum of cytokines, including colony-
stimulating factors, it plays a role in regulation of hematopoi-
etic differentiation (12). NK cells may also play an important
role in determining the outcome of bone marrow transplanta-
tion and generation of the graft-versus-host disease (39, 76).
Although controversy has long existed about the beneficial
versus the detrimental effects of human NK cells on bone
marrow progenitors, recent studies indicate that the transfer of
donor IL-2-activated NK cells enhances engraftment after
allogeneic transplantation, possibly because these cells serve as
a source of multiple cytokines necessary for immunologic
reconstitution (39). On the other hand, it has been generally
expected that transferred allogeneic NK cells would not con-
tribute to the graft-versus-host process and, in fact, might
ameliorate it via the release of immunosuppressive cytokines
(39). However, in a recent report, graft-versus-host disease-like
lesions were induced by xenogeneic transplantation of human
IL-2-activated NK cells into mice with severe combined immu-
nodeficiency (76). The role of NK cells in graft-versus-host
disease remains unclear at present and is under careful scru-
tiny in both experimental and clinical transplantations.
NK cells are known to be present in the decidua in the first
trimester of pregnancy, and although their role in reproduction
is not well understood, they may play a trophic and/or regula-
tory role in the growth of the fetal-placental unit.
Substantial evidence for the involvement of NK cells in
interactions of the immune system with the neuroendocrine
is evidence that NK cells participate in
CLIN. DIAGN. LAB. IMMUNOL.
axis has accumulated. NK cells express receptors for neuroen-
docrine hormones as well as neural adhesion molecules on
their surface, and it is likely that they play an important role in
modulating behavioral changes that accompany stressful life
events (30). The current view is that NK cells participate either
directly or indirectly in multiple developmental, regulatory,
and effector functions of the immune system. In addition, they
appear to be responsible for activities at the interphase be-
tween the immune system and other systems, e.g., reproductive
or neurologic. The biologic role of NK cells is not restricted to
immune surveillance against infectious agents or tumor metas-
involvement in a variety of essential biologic processes ranging
from reproduction to senescence (73).
Measurements of NK cells. In humans, the number of NK
cells and NK activity are generally measured in the peripheral
blood. The measurement of NK cell number involves staining
PBMNC with MAbs to mark NK cells and then determining
the percentage of positive cells by flow cytometry (71). The
percentage is then converted into the absolute number of NK
cells by comparison with the simultaneously obtained differen-
tial lymphocyte count. NK activity is measured in a short-term
in vitro assay with 5'Cr-labeled K562 leukemia cells as targets
and PBMNC as effector cells. These assays have, in the past,
required isolation of PBMNC from peripheral blood, but
today, both can be, and probably should be, performed on
unseparated peripheral blood. Whole-blood NK cell assays
probably provide a more precise measurement of the effects of
various factors present in the blood on NK activity than do
conventional assays performed with isolated PBMNC. Both
types of NK cell assays have been described previously (14, 70),
and they can be reliably performed in a clinical laboratory but
only when sufficient quality control measures are taken to
establish reproducibility and to minimize daily variability. This
becomes particularly important when serial measurements of
NK activity are needed, as in monitoring of patients during the
course of their disease or during immunotherapy. The criteria
for acceptability of the NK cell assay have been reviewed (71).
Although the number and activity of NK cells are generally
determined in the peripheral blood, NK cells are widely
distributed in human tissues. The spleen, liver, and lungs
appear to contain considerable numbers of NK cells (73). In
contrast, the lymph nodes contain relatively few NK cells, as
determined by flow cytometry with anti-NK cell MAbs, and the
NK activity of cells freshly obtained from lymph nodes is
generally much lower than that of NK cells in the blood and is
often undetectable (73). Little is known about the distribution
of NK cells in other human tissues. Tumor-infiltrating lympho-
cytes from certain tumors, e.g., glioblastoma, renal cell or
ovarian carcinoma, and some others, appear to contain more
NK cells (10 to 20%) than those from melanoma, for example
(unpublished observations). The bone marrow, which is the
source of NK precursor cells, contains very few mature NK
cells. Cells with NK surface markers have been detected in the
human thymus, although NK activity is usually not detectable
and their function at this site of T-cell maturation remains
unknown (73). All lymphoid tissues and bone marrow appear
to contain NK cell precursors, because considerable NK activ-
ity can be induced from lymphoid tissue cells after incubation
with IL-2. A new MAb, 8A2, which might detect NK cell
precursors and thus be useful in localization of these precur-
sors in various human tissues, has recently been described (66).
NK activity and the number of NK cells in tissues can be
measured, but such measurements require dissociation of
these tissues with enzymes and separation of mononuclear
cells. Both the percentage of NK cells and level of NK activity
it is viewed as a much more broadly based
appear to be higher in human liver than in peripheral blood,
and NK cells isolated from this organ are predominantly in an
activated state (19). In contrast, NK cell activity measured in
fresh mononuclear cells separated from a variety of human
tumors, including those in the liver, is low or undetectable,
suggesting that NK cells might be absent or functionally
suppressed in the tumor microenvironment (75).
Both the level of NK activity and number of NK cells vary
substantially among normal individuals, and normal ranges for
both need to be established for every clinical laboratory by
testing a large population of normal volunteers. In our labo-
ratory, the normal range of NK activity, defined in terms of
80% middle range, is 55 to 350 LU2(J/107 effector cells, based
on assays performed with many hundreds of normal donors
(unpublished data and reference 71). The percentage of circu-
lating NK cells defined by two-color flow cytometry as CD3
CD56+ lymphocytes in the circulation of these healthy donors
is 12% ± 6% (mean ± standard deviation). Enumeration of
NK cells by flow cytometry cannot substitute for the assess-
ment of cytotoxic activity. The correlation between the number
of circulating NK cells and NK activity for normal individuals
is significant but not particularly strong (71), probably because
NK cells may vary considerably in their state of activation. For
this reason, assessments of both the number of NK cells and
their activity are necessary to adequately evaluate natural
immunity or monitor its changes during disease or therapy.
In general, NK activity is a stable trait for a given individual,
and normal individuals fall into groups with low, moderate, or
high NK activity, based on repeated (at least three) NK activity
measurements over time. It thus appears that among normal
individuals, it is possible to define low and high responders,
and although this distinction is not clearly understood, it might
be biologically important because of indications that individu-
als with persistently low NK activity may be prone to more
frequent upper respiratory infections and less able to deal with
stressful events (71). Serial measurements of NK activity have
not been widely performed in the past because of a require-
ment for rigorous control of day-to-day reproducibility to
ensure that the changes observed reflect true biologic activity
and not differences in the assay. Nevertheless, serial measure-
ments of NK activity are necessary to be able to correlate
changes in NK activity with,
response to treatment. Also, if NK cells are to be examined for
their biologic importance in human disease and to determine
whether NK activity can be used as a prognostic or even
diagnostic parameter for patients with various diseases, longi-
tudinal NK measurements must be reliably performed.
NK cells in disease. The role of NK cells in human disease
has been reviewed by us recently (71). Briefly, human diseases
with an associated NK abnormality can be categorized into
those with low or absent NK activity (i.e., NK cell deficiency)
and those in which NK activity appears to be excessive. In
either category, abnormalities in NK activity can be transient
or persistent. Transient decreases or increases in NK activity
relative to the normal baseline level defined for a given
individual accompany a variety of events and diseases, e.g.,
circadian variations, exercise, stressful situations, common
colds, and more severe viral infections (71). A normal NK cell
response to a viral infection, for example, appears to be a rapid
and significant rise which occurs within 24 h of viral challenge,
followed by a decrease on days 5 to 7 and a gradual return to
the baseline level (58). Frequently, but not always, the number
of circulating NK cells parallels changes in NK activity (71).
Thus, transient changes from the baseline in NK cell activity
appear to be physiologically normal responses to life events.
On the other hand, persistently low or high levels of NK
e.g., disease progression or
VOL. 1, 1994
130WHITESIDE AND HERBERMAN
TABLE 3. Examples of abnormalities in NK activity or cell number associated with clinical symptoms or increased disease risk in humans
Associated symptoms or risks
Persistently low activity/numberAcquired or congenital
immunodeficiencies, including AIDS
Deficiency of the CD11/CD18 CAM
X-linked lymphoproliferative syndrome
Breast cancer (at diagnosis)
Head and neck cancer (prior to
Other viral/bacterial infections
Higher incidence of cancer and increased frequency
and severity of infections
Increased risk for lymphoma
Increased susceptibility to viral infections
Dissemination of metastases
Higher than normal incidence of malignancy
Increased susceptibility to Epstein-Barr virus
Higher rates of recurrence
Higher frequency, severity, and duration
Higher frequency, severity, and duration
Possibly more active disease, increased frequency of
Low NK syndrome
Chronic fatigue syndrome
Fatigue, dullness, fever
Extreme fatigue, listlessness, increased frequency of
More severe symptoms
Unable to cope with daily hassles, fatigue
Variable, including severe, disseminated, and life-
threatening viral infections; recurrent warts, CIS,c
and pulmonary abnormalities; erythrocyte aplasia
NK cell lymphoproliferation
Chronic LGL proliferation
Acute LGL proliferation
LGL lymphocytosis, cytopenia, splenomegaly
Consistent with aggressive leukemia/lymphoma
ae.g., cytomegalovirus, Epstein-Barr virus, herpesvirus.
bRare; only three cases described.
CIS, carcinoma in situ.
activity are likely to be associated with disease. NK activity
appears to be a more sensitive marker of disease progression
than the absolute number of NK cells.
A considerable amount of evidence has accumulated to
substantiate the involvement of the NK cell in many human
diseases (53, 71). Table 3 is a list of human diseases which have
been associated with persistent alterations in NK activity
and/or NK cell number. It is essential to realize that while the
association between low or high NK activity and disease has
been convincingly established in many cases, for the most part,
such an association does not imply that the NK cell abnormal-
ity is related to the pathogenesis. It is likely that abnormally
high or low NK activity is a result of disease rather than its
cause in many cases and that it may be an epiphenomenon not
at all related to the disease process itself. On the other hand,
it is necessary to recognize that, in some instances, experimen-
tal evidence has been obtained for a direct and, perhaps, causal
relationship between low NK activity and disease. For exam-
ple, mice depleted of NK cells by treatment with the anti-NK
cell antibody anti-asialoGM,after surgical removal of a tumor
developed significantly and overwhelmingly more lung metas-
tases than control animals which were not treated with the
antibody and had a normal number of NK cells (16). The
susceptibility of mice to cytomegalovirus infection increases
significantly in the absence of NK cells, and resistance to
cytomegalovirus can be restored with adoptively transferred
NK cells (7). In humans, a positive correlation has been
observed between sensitivity to infections and depressed NK
activity (2). Also, a patient whose persistently low or absent
NK activity was accompanied by insidious, severe, and fre-
quent viral infections has been described (5). However, to be
able to causally associate the deficit in NK activity with the
disease pathogenesis, it will be necessary to demonstrate not
only that the deficit precedes disease onset and that the degree
of deficit can be linked to disease severity, but that correction
of the deficit leads to amelioration of symptoms or recovery.
Thus, if therapeutic transfers of NK cells to animals bearing
established tumor metastases could be demonstrated to result
in regression of these metastases and prolonged survival, then
a strong argument could be made for the role ofNK cells in the
control of metastatic disease. Studies ongoing in our laboratory
indicate that therapeutic intrasplenic transfers of IL-2-acti-
vated NK cells and IL-2 to nude mice bearing established
hepatic metastases ofhuman gastric carcinoma indeed result in
nearly complete elimination of liver metastases and in signifi-
cantly prolonged survival of the animals (unpublished data).
As shown in Table 3, chronically low levels of NK activity
occur not only in cancer, particularly when there are large
tumor burdens or disseminated metastases, but also in a variety
of immunodeficiency syndromes, severe, life-threatening viral
infections, autoimmune diseases, and behavioral disorders
(71). Chronically high levels of NK activity are associated with
lymphoproliferative syndromes, including aggressive LGL leu-
kemia (26), and in some hepatic disorders (18). In addition,
CLIN. DIAGN. LAB. IMMUNOL.
low NK activity may not only be a risk factor for malignancy
but have a prognostic significance in predicting relapse, re-
sponse to treatment, and survival time free of metastasis in
patients with cancer (71). In general, patients with low NK
activity appear to be at higher risk of infections, to have more
prolonged diseases, or to suffer more severe symptoms than
those whose NK activity remains normal. On the other hand,
excessive levels of NK activity are relatively rare, and the
biologic significance of chronically high NK activity is not clear
at this time.
NK cells in therapy. From the evidence reviewed above that
chronically low levels ofNK activity or number in patients with
cancer or other diseases may be associated with more severe
symptoms or increased risk of disease progression, augmenta-
tion of NK activity in disease may be of benefit to the patient.
Therapy aimed at augmenting NK activity could be particularly
advantageous for patients with cancer or immunodeficiencies.
Such therapy is available today, and it generally consists of
attempts to increase NK activity in vivo by the administration
of agents with known NK-potentiating activity or adoptive
transfer of activated autologous NK cells either locally or
systemically to patients deficient in NK activity.
A variety of agents, generally referred to as biologic re-
sponse modifiers (BRMs), are known to increase the activa-
tion, proliferation, or cytotoxicity of existing NK cells; other
BRMs promote NK cell extravasation and accumulation in
tissues, e.g., lung and liver, resulting in higher local cytotoxic
activity. A partial list of commonly used BRMs includes a
spectrum of cytokines such as interferons, IL-2, and IL-12;
bacterial products such as OK432 (picibanil); plant lectins such
as lentinan; MAbs that bind to triggering structures on NK
cells (e.g., anti-CD16 MAb); and interferon inducers such as
polyribonucleotides. The effects of BRMs, especially cytokines,
on signal transduction in NK cells, their mobility in tissues or
ECM, and their ability to lyse tumor cells or virally infected
targets are under intense investigation at this time.
Among the best-known activators of NK cells are IL-2 and
IFN-ot (20, 21). These two cytokines have been used exten-
sively not only for patients with malignancies but also for those
with viral infections. For example, IFN-a has been therapeu-
tically effective in a proportion of patients with chronic hepa-
titis B infection (24), and patients with human immunodefi-
combination with zidovudine (34). For patients with advanced
malignancies unresponsive to other treatments, both IL-2 and
IFN-ot therapies have resulted in durable responses (27, 55).
Systemic or locoregional therapy with IL-2, even at low or
moderate doses, leads to well-documented increases in the
number of circulating NK cells and NK activity (8). Although
the extent of this IL-2-induced stimulation of NK activity in
vivo could not be directly linked to clinical response, it may be
at least partly responsible for the antitumor effectiveness of
Immunotherapy with adoptively transferred activated NK
cells and cytokines has been used mainly for patients with
advanced malignancies in the hope of capitalizing on the
well-documented antimetastatic activity of NK cells (23, 72).
The earliest clinical trials were performed with lymphokine-
activated killer cells and high-dose IL-2 in patients with
metastatic melanoma or renal cell carcinoma (54). The results
of these trials have been only mildly encouraging in that the
rate of objective clinical responses achieved was about 20 to
30% (54). However,
sponses, some of long duration (>2 years), were achieved in
patients with metastatic disease unresponsive to conventional
therapies. Lymphokine-activated killer cells are obtained by
it is important to note that these re-
incubating PBMNC in the presence of 6,000 IU of IL-2 per ml
and generally contain a substantial proportion of activated NK
cells in addition to activated T lymphocytes (42). More re-
cently, clinical trials with purified NK cells, selected and
expanded in vitro from patients' PBMNC, have been done on
the hypothesis that transfer of a selected subset of highly
activated antitumor effector cells may be therapeutically more
effective and require fewer cells and possibly lower doses of
cytokines to support their viability and in vivo activity. The
results of two preliminary phase I trials with patients with
metastatic melanoma or renal cell carcinoma demonstrated
that immunotherapy with selected, highly purified, and in
vitro-expanded subsets of NK cells is feasible, well tolerated,
and, in some cases, effective for metastasized disease (23, 72).
Since the mechanism of the antitumor activity of systemically
transferred, in vitro-activated NK cells is not known and may
depend on the ability of these effector cells to reach or localize
to tumor metastases, locoregional delivery of purified activated
NK cells together with IL-2 to the liver via the hepatic artery is
currently being evaluated at the Pittsburgh Cancer Institute for
patients with colon cancer metastatic to the liver.
Increasingly often, bone marrow transplantation or periph-
eral stem cell transfer has been used for the treatment of
malignancies. The transfer of activated NK cells after trans-
plantation is based on the rationale that NK cells have potent
antitumor effects and thus can help eliminate minimal residual
disease. Purified and activated human NK cells are being used
for the therapy of patients with cancer after high-dose chemo-
therapy and peripheral blood stem cell transplantation (31).
Preliminary results from clinical trials evaluating this therapy
suggest that systemic transfer of A-NK cells plus IL-2 within 2
to 3 days after peripheral blood stem cell transplantation may
not only help eliminate minimal residual disease but facilitate
hematopoietic recovery (31).
While both in vivo augmentation of NK activity with BRMs
and adoptive transfer of activated NK cells are promising new
therapies, additional clinical and basic studies are needed to
build on the progress achieved to date and to acquire a better
understanding of the interactions between activated NK cells
and their targets in vivo.
Summary. The NK cell plays a major role in human health
and disease. Although persistent abnormalities in NK cell
activity or number appear to be associated with a wide
spectrum of human diseases, evidence for the causal associa-
tion of abnormally low NK activity with pathogenesis is so far
available only from a limited number of experimental models.
For diseases characterized by NK cell deficiency, therapy with
adoptively transferred purified and in vitro-activated subsets of
NK cells or with BRMs, which can restore or augment NK
activity in vivo, offers an opportunity to demonstrate that the
NK cell contributes to immunopathogenesis. Future research
and clinical efforts should be directed at achieving a better
understanding of the interactions between the NK cell and its
various targets, dissecting the functional repertoire of the NK
cell, improving the monitoring of various NK cells in tissues,
organs as well as the peripheral blood, and devising clinical
trials in which hypotheses relevant to the role of the NK cell in
various human diseases can be reliably evaluated.
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