REVIEWS NK cell development, homeostasis and function: parallels with CD8+ T cells
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REVIEWS
NK cell development, homeostasis and
function: parallels with CD8+ T cells
Joseph C. Sun* and Lewis L. Lanier‡
Abstract | Natural killer (NK) cells survey host tissues for signs of infection,
transformation or stress and, true to their name, kill target cells that have become
useless or are detrimental to the host. For decades, NK cells have been classified as
a component of the innate immune system. However, accumulating evidence in mice
and humans suggests that, like the B and T cells of the adaptive immune system,
NK cells are educated during development, possess antigen-specific receptors,
undergo clonal expansion during infection and generate long-lived memory cells.
In this Review, we highlight the many stages that an NK cell progresses through
during its remarkable lifetime, discussing similarities and differences with its close
relative, the cytotoxic CD8+ T cell.
A productive immune response against pathogen inva- Development of NK cells
Perforin and granzymes
The cytolytic molecules that sion consists of a concerted effort from many effector NK cells have traditionally been classified as innate
are secreted by NK cells and cell types of haematopoietic origin. Both innate and immune cells because of their ability to rapidly
cytotoxic CD8+ T cells. adaptive immune cells contribute to the recognition and respond against target cells in the absence of prior
Perforin subunits assemble removal of foreign pathogen materials as well as infected sensitization, as well as because of the previous belief
into a pore-forming structure
in the membrane of the target
host cells. The best-known cell types responsible for the that they are cells with a short lifespan. By contrast,
cell, and this allows granzymes direct killing of infected cells are natural killer (NK) B and T cells are designated cells of the adaptive
(a family of proteolytic cells and cytotoxic CD8+ T cells (also known as CTLs). immune system because they generate long-lived
enzymes) to activate caspases These professional killer cells are defined based on their progeny following the activation of a naive precursor
and induce apoptosis in the
cytolytic machinery, and the killing of their targets is and can ‘remember’ previous encounters with antigen.
target cell.
mediated predominantly via perforin and granzymes. NK Although considered as innate immune cells, NK cells
cells and CD8+ T cells both originate from a common constitute the third major lineage of lymphocytes, the
lymphoid progenitor (CLP) and require cytokine signals other lineages of which are B and T cells4,5. Unlike B
through cytokine receptors that contain the common and T cells, individual NK cells lack a unique antigen
γ-chain (γc; also known as IL‑2Rγ) for their survival recognition receptor and do not use recombination-
and homeostasis. During infection, both NK cells and activating gene (RAG) enzymes for rearrangement of
CD8+ T cells become activated through antigen-specific their receptor genes, although transient expression of
receptors and by pro-inflammatory cytokines (such as RAG proteins and even incomplete V(D)J recombi-
interleukin‑12 (IL‑12) and type I interferons (IFNs)), nation have been observed in a low frequency of NK
*Immunology Program,
Memorial Sloan-Kettering
and produce large amounts of IFNγ1. Although they cells during their development 6–10. NK cells are pre-
Cancer Center, New York, have been classified as innate immune cells, there is sent in normal numbers in mice deficient in RAG1 or
New York 10065, USA. accumulating evidence from both mice and humans RAG2 (REFS 11,12).
‡
Department of Microbiology that NK cells share some attributes with the B cells and Early studies suggested that NK cells, like B cells
and Immunology and the
T cells of the adaptive immune system. For example, and myeloid-lineage cells, develop primarily in the
Cancer Research Institute,
University of California San NK cells are ‘educated’ and selected during their devel- bone marrow. Ablation or disruption of an intact bone
Francisco, San Francisco, opment, their receptors exhibit antigen specificity, they marrow microenvironment abrogated the develop-
California 94143, USA. undergo clonal expansion during infection and they gen- ment and function of NK cells 13,14. Unlike T cells,
Correspondence to L.L.L. erate long-lived memory cells2,3. Here, we discuss the NK cells do not require the thymus for their develop-
e-mail: lewis.lanier@ucsf.edu
doi:10.1038/nri3044
many stages that an NK cell progresses through during ment and, thus, exist in normal numbers in athymic
Published online its remarkable lifetime and compare it with its close nude mice 15–17 . However, a small population of
26 August 2011 relative, the cytotoxic CD8+ T cell. CD127‑expressing NK cells has been recently found to
NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | O CTOBER 2011 | 645
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
arise in the thymus through a GATA-binding protein 3 Analogous processes in NK cell and CD8+ T cell devel‑
(GATA3)-dependent pathway, independently from opment. Developing CD8+ T cells undergo processes
Clonal expansion T cell precursors18. In humans, a population of CD34+ such as positive selection and negative selection through
The process whereby individual haematopoietic precursor cells was reported to develop interactions between their T cell receptor (TCR) and
B and T cells that express
unique antigen-specific
into CD56hi NK cells in lymph nodes19. Furthermore, MHC class I molecules expressed on thymic epithelial
receptors encounter their a recent examination of NK cell ontogeny suggested cells and dendritic cells (DCs). Similarly, developing NK
cognate ligands and rapidly that NK cells can also develop in the liver 20, and this cells are educated or selected through engagement of
proliferate to give rise to a perhaps explains why phenotypically immature NK their receptors with various MHC class I ligands (FIG. 1).
large number of progeny that
cells exist in the liver of adult mice21. It is not entirely Immature human and mouse NK cells express inhibi-
express the same antigen-
specific receptor (that is, clear whether these thymic‑, lymph node- and liver- tory killer cell immunoglobulin-like receptors (KIRs)
clones). derived populations represent distinct NK cell lineages and LY49 receptors, respectively, during their devel-
or merely consist of predominantly less-mature periph- opment, and this is essential for establishing efficient
NK1.1 eral cells that originated from the bone marrow. The ‘missing-self’ recognition29,30. Engagement of these recep-
(Also known as KLRB1C and
NKRP1C). This activating
bone marrow is certainly the site where NK cell devel- tors with cognate MHC class I molecules results in the
receptor associates with the opment has been best characterized, and many of the generation of functional NK cells in the periphery 31–35,
adaptor molecule FcεRIγ and is cues that NK cells receive from bone marrow stromal a process akin to the positive selection of developing
expressed on all NK and and haematopoietic cells during their full functional T cells (FIG. 1). In addition, NK cells can vary in their
NKT cells in C57BL/6 mice.
maturation are discussed in this Review. responsiveness, or ‘tune’ their capability to react, and
Although crosslinking of the
receptor using an In vitro studies conducted with mouse and human this is dependent on the number of inhibitory receptors
NK1.1‑specific mAb (clone cells have demonstrated that NK cells can be derived for autologous MHC class I molecules that they express.
PK136) induces NK cell- from early haematopoietic cells cultured with stro- Indeed, both mouse and human NK cell responsiveness
mediated cytotoxicity and mal elements, such as IL‑7, IL‑15, stem cell factor and increases quantitatively with each additional self-MHC-
effector cytokine secretion,
the in vivo function of NK1.1
FLT3 ligand (reviewed in REF. 4). Like with thymocytes, specific inhibitory receptor 36–38 (FIG. 1). Inhibitory NK
and nature of its ligand(s) studies using the OP9 stromal cell culture system con- cell receptors contain immunoreceptor tyrosine-based
remain unknown. firmed that functional NK cells could develop in vitro inhibitory motifs (ITIMs) in their cytoplasmic tail, and
on stromal cells expressing specific Notch ligands, these motifs can recruit SH2 domain-containing protein
NKG2D
such as members of the Delta and jagged families22–24. tyrosine phosphatase 1 (SHP1; also known as PTPN6)
(Natural killer group 2,
member D). An activating Interestingly, following culture of early-stage thymo- and SH2 domain-containing inositol 5‑phosphatase
receptor constitutively cytes with OP9 cells and jagged 1, large percentages (SHIP)39. Failure to engage inhibitory receptors during
expressed on NK cells and (as much as 50%) of the recovered cells expressed the development — because of either a lack in inhibitory
activated CD8+ T cells that NK cell receptor NK1.1 (REFS 22–24). Furthermore, the receptor expression or an inability to interact with MHC
recognizes a family of induced
host ligands on the surface of
cytokine IL‑15 was shown to be crucial for the develop- class I molecules — results in peripheral NK cells that
stressed cells. DNA instability ment of NK cells25, as mice deficient in either IL‑15 or are hyporesponsive31–35.
and damage arising from the IL‑15 receptor α-chain (IL‑15Rα) had a dramatic Rather than ‘death by neglect’ — a process thymo-
radiation, chemicals, infection reduction in NK cell numbers26,27. cytes undergo if they cannot appropriately engage their
or transformation can lead to
In vivo reconstitution studies have identified the CLP TCRs with MHC class I molecules — analogous NK cell
cellular stress and the
upregulation of NKG2D ligands in mouse and human bone marrow as the pluripotent populations that cannot ligate their inhibitory receptors
(namely, the retinoic acid cell type that specifically gives rise to mature NK, B and do not die and are still exported to the periphery, albeit
early-inducible protein 1 (RAE1) T cells, but not myeloid-lineage cells28. However, unlike as anergic cells31–35. Notably, NK cells that lack an inhibi-
family, histocompatibility the well-established stages of thymocyte development tory receptor for autologous MHC class I molecules can
antigen 60 (H60) and murine
UL16‑binding protein-like
— CD4–CD8– double-negative (DN) stages 1–4, the respond normally in inflammatory settings33,40,41 and
transcript 1 (MULT1) in CD4+CD8+ double-positive (DP) stage and the CD4+ or more robustly against viral infection and leukaemias
mice, and the MHC class I CD8+ single-positive (SP) stage — NK cell development than their counterparts that express self-specific inhib-
polypeptide-related sequence and differentiation has been assigned several arbitrary itory receptors for MHC class I42–44. Recently, mature
(MIC) and UL16‑binding protein
stages based on sequential acquisition of NK cell- NK cells have been shown to undergo a ‘re-education’
(ULBP) families in humans).
specific markers and functional competence. The ear- process whereby functional competence is reset follow-
LY49 receptors liest NK cell progenitor is defined as a non-stromal ing adoptive transfer into a different MHC class I envi-
A family of NK cell receptors in bone marrow cell that expresses CD122 (the shared ronment 45,46. In these studies, anergic NK cells placed
mice that contains inhibitory β-chain of IL‑2R and IL‑15R)4,5 but is without any in an MHC class I‑sufficient setting acquired the abil-
members that bind to MHC
class I molecules and inhibit
lineage-specific markers. In C57BL/6 mice, the earliest ity to produce effector cytokines, and functional NK
NK cell responses and NK cell-defining markers are NK1.1 and NKG2D, fol- cells lost their effector capabilities in an environment
activating members that lowed by the inhibitory and activating LY49 receptors21. devoid of MHC class I molecules45,46. Together, these
predominantly associate with The functional capabilities of NK cells, such as cytotoxicity results suggest that continuous engagement of inhibi-
the adaptor molecule DAP12
and secretion of cytokines (namely, IFNγ and tumour tory receptors with MHC class I molecules is required
to initiate effector functions
following encounter with target necrosis factor (TNF)), are acquired last in the current to dictate NK cell responsiveness. Interestingly, in both
cells that express cognate developmental schemes21. Although this model provides a mice and humans, activated and memory CD8+ T cells
ligands. The ligands for the framework for studying NK cell development, the precise also express inhibitory receptors for MHC class I47, per-
majority of activating LY49 rate-limiting cues that maturing NK cells receive at each haps because of a need to tightly regulate these respon-
receptors are currently
unknown; however, some are
stage to ultimately achieve functional competence, along sive T cell populations. A recent study, in which ligation
likely to be virally encoded with the genetic and epigenetic programming that occurs, of inhibitory KIRs by MHC class I molecules could be
components. require further investigation. instantly induced through a photochemical approach,
646 | O CTOBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
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survive and mature into Figure 1 | Education of developing NK cells and thymocytes. a | In the bone marrow, developing natural killer (NK)
single-positive (CD4+ or CD8+) cells interact with MHC class I molecules on stromal and haematopoietic cells via inhibitory receptors, and the number
T cells based on an appropriate
of interactions determines the degree of responsiveness. b | Developing NK cells that interact0CVWTG4GXKGYU^+OOWPQNQI[
with self or viral ligands
degree (low to intermediate) of
stimulation through the T cell
via activating receptors will become anergic or hyporesponsive. c | In the thymus, developing T cells interact with
receptor. self-peptide–MHC complexes on epithelial and haematopoietic cells via T cell receptors (TCRs), and the affinity or
avidity of these interactions determines survival and export to the periphery.
‘Missing-self’
A hypothesis proposed by Klas
Karre suggesting that NK cells
preferentially recognize and kill
demonstrated that clustering and triggering of activat- developing thymocytes, activating receptor ligation with
host cells that have lost ing NK cell receptors immediately ceases following cognate viral or self ligands leads to anergy and a partial
expression of self MHC class I inhibitory receptor ligation, and the NK cells collapse repertoire deletion in developing NK cells49–52 (FIG. 1).
molecules — in other words, their actin cytoskeleton to strongly retract from the Altogether, these tolerance mechanisms exist to ensure
cells that are ‘missing self’.
source of stimulation48. that mature NK cells do not attack healthy self tissues.
Uninhibited by immunoreceptor
tyrosine-based inhibitory motif Developing NK cells can also be influenced by sig-
(ITIM)-containing receptors for nals received through activating receptors, which pair Lineage specification in NK cells. Over the past dec-
MHC class I molecules, NK cells with immunoreceptor tyrosine-based activation motif ades, lineage-specifying transcription factors have been
can target virally infected or (ITAM)-containing adaptor molecules that recruit identified in B cells and T cells (including αβ T cells, γδ
transformed cells that
downregulate MHC class I
spleen tyrosine kinase (SYK) and ζ‑chain-associated pro- T cells and NKT cells), and this has provided impor-
molecules to evade detection tein kinase of 70 kDa (ZAP70) to mediate signalling 39. tant information about the genesis of these immune cell
by CD8+ T cells. In an analogous manner to the negative selection of subsets. B cell development is driven by transcription
NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | O CTOBER 2011 | 647
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
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Figure 2 | Factors that influence the development, homeostasis and survival of NK cells and T cells. A comparison of
the lineage-specifying and external signals required for the maturation of natural killer (NK) cells and T cells from the common
0CVWTG4GXKGYU^+OOWPQNQI[
lymphoid progenitor (CLP) is shown. The CLP uses factors such as Notch and runt-related transcription factor (RUNX) family
members to give rise to immature T cells, whereas inhibitor of DNA binding 2 (ID2) and E4BP4 specify NK cell lineage
precursors. Notch, interleukin‑7 (IL‑7) and T cell receptor (TCR) signals dictate the selection of developing T cells in the
thymus, whereas IL‑15 and inhibitory receptor signals promote the proliferation and education of developing NK cells in the
bone marrow. Factors that influence the survival and function of T and NK cells during antigen-driven versus homeostatic
proliferation are shown, including cytokine signals, cell surface receptor signals and various other unknown signals.
factors such as paired box protein 5 (PAX5), E2A (also T follicular helper cells, forkhead box P3 (FOXP3) for
known as TCF3) and early B cell factor (EBF)53, whereas regulatory T (TReg) cells, retinoic acid receptor-related
specific runt-related transcription factor (RUNX) and orphan receptor-γt (RORγt) for TH17 cells, and T‑bet
Notch family members promote T cell development 54,55 and eomesodermin for effector CD8+ T cells57,58.
(FIG. 2). Within the T cell lineage, CD4+ or CD8+ lineage The transcription factors PU.1, Ikaros, ETS1 and
commitment in DP thymocytes occurs under the guid- inhibitor of DNA binding 2 (ID2) have independently
ance of factors such as GATA3 and THPOK (also known been shown to be important for NK cell lineage deri-
as cKROX) for CD4+ T cell development and RUNX1 vation from the CLP (FIG. 2), as mice deficient in these
and RUNX3 for CD8+ T cell development 54,56. In addi- factors have substantially reduced numbers of peripheral
tion, transcription factors that dictate the differentiation NK cells59,60. It should be noted that these factors also
of mature naive T cells into effector cells have also been influence the development of other lymphocyte lineages
characterized, including T‑bet for T helper 1 (TH1) cells, in addition to NK cells59,60. Similarly, NK cell develop-
GATA3 for TH2 cells, B cell lymphoma 6 (BCL‑6) for ment is impaired in mice deficient in TOX (thymocyte
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© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
selection-associated high-mobility group box protein)61, other organs80. NK cells are found only at low frequency
a shared DNA-binding factor that is also required for in the lymph nodes, even though the majority (>90%)
TReg, NKT and CD4+ T cell differentiation. A recent of resting NK cells in mouse spleen and blood express
report using ID2–GFP reporter mice showed that the L‑selectin (also known as CD62L), which mediates
earliest known NK cell precursor highly expresses ID2 naive T cell recruitment into lymph nodes from the cir-
and IL‑7R, before expression of common NK cell mark- culation. In mice deficient for L‑selectin or L‑selectin
ers62. Other factors — such as T‑bet, eomesodermin, ligands, NK cells are not properly recruited to lymph
interferon-regulatory factor 1 (IRF1) and GATA3 — nodes and, thus, cannot control tumour metastasis
have been shown to be important for the acquisition of to secondary lymphoid organs in a mouse melanoma
full NK cell function by using gene-deficient mice, bone model81. The general migration of NK cells between
marrow chimaeras and conditional deletion mutants63–66. the circulation, lymphoid organs and non-lymphoid
An indirect role for IRF1 in NK cell development was organs has not been completely characterized, although
discovered when IL‑15 expression was observed to be many chemokines have been implicated in NK cell
defective in mice lacking this transcription factor 67. localization. These include CC-chemokine ligand 3
Interestingly, genetic deletion of the transcription fac- (CCL3; also known as MIP1α), CCL4, CCL5, CCL19,
tor BCL‑11B in conditional knockout mice resulted in CXC‑chemokine ligand 12 (CXCL12), CXCL16 and
the reversion of maturing DN2 and DN3 thymocytes CX3C-chemokine ligand 1 (CX3CL1). In addition, there
to killer cells that were described as NK cell-like68–71. is regulated expression of adhesion molecules (such as
Thus, perhaps in the absence of T cell-determining fac- α2 integrin, α4 integrin, macrophage receptor 1 (MAC1;
tors the NK cell lineage becomes the default pathway of also known as αMβ2 integrin) and DNAX accessory
lymphocyte programming. molecule 1 (DNAM1)) and chemokine receptors (such
as CC‑chemokine receptor 1 (CCR1), CCR5, CCR7,
NK cell development requires E4BP4. Recently, the basic CXC-chemokine receptor 3 (CXCR3), CXCR4, CXCR6
leucine zipper transcription factor E4BP4 (also known and CX3C-chemokine receptor 1 (CX 3CR1)) during
as NFIL3) was proposed as a NK cell lineage-specifying NK cell development and homeostasis in mice and
factor (FIG. 2). E4BP4‑deficient mice showed a severe loss humans82. Recently, the lipid sphingosine-1‑phosphate
in mature NK cell numbers, whereas B and T cells were (S1P) has been implicated in NK cell retention and
found at normal numbers72–74. E4BP4 is implicated in a egress from sites such as the bone marrow and lymph
wide range of biological processes, including repression nodes, with expression of the dedicated S1P receptor
of viral promoter sequences and regulation of the mam- on NK cells (namely, S1P receptor 5) observed to be
malian circadian clock75, and it was shown to function regulated by T‑bet 83,84.
downstream of IL‑15R in immature NK cells, as ex vivo In the steady state, NK cells are generally localized
addition of IL‑15 did not rescue NK cell development to the red pulp of the spleen and the sinusoidal regions
in E4BP4‑deficient progenitor cells72. Experimental evi- of the liver 85,86; however, during viral infection they
dence suggests that E4BP4 controls the expression of infiltrate the splenic white pulp and liver parenchyma
GATA3 and ID2 (REF. 72), and ID2 is known to inhibit near infected foci87–89. Thus, NK cell homeostasis leads
E protein transcription factors (such as E2A) and to to the presence of these cells in both lymphoid and
thereby inhibit B cell development while promoting the non-lymphoid tissues, where they are poised to rapidly
maturation of NK cells76. It should be noted that, like respond against pathogen invasion. The precise mecha-
the promiscuous role of T‑bet in both CD4+ TH1 and nisms by which NK cells traffic between and within
CD8+ T cell differentiation, E4BP4 has been shown to organs during homeostasis and following infection
influence the development of certain DC subsets, the remain to be revealed.
regulation of TH2 cell responses and class switching
in B cells77–79. Because the survival of memory CD8+ Continuous maturation of peripheral NK cells. Recent
T cells is also dependent on IL‑15 signals, further work evidence suggests that both NK cells and T cells con-
is needed to determine whether E4BP4 has a role in the tinue to mature in the periphery following egress from
generation of memory T cell subsets. In addition, the the bone marrow and thymus, respectively. Using
conditional deletion of E4BP4 in mature and activated reporter mice to identify recent thymic emigrants,
NK cells will determine whether this factor continues the maturation of T cells was shown to continue post-
to have a role in NK cell survival and function beyond thymically, with progressive acquisition of phenotypic
their development. Nonetheless, there exists a clear role changes and immune functions90. Similarly, continuous
for transcription factors, such as E4BP4 and ID2, in the NK cell maturation in the periphery has been charac-
development of NK cells. terized by the expression of surface markers such as
CD11b and killer cell lectin-like receptor subfamily G,
Peripheral NK cell localization and homeostasis member 1 (KLRG1), and loss of CD27 and TNF-related
Trafficking of mature NK cells. Once they leave the bone apoptosis-inducing ligand (TRAIL)91–94.
marrow, NK cells that have gained functional competence IL‑18 has recently been described to ‘prime’ NK cells
have the capability to respond robustly against infec- either during their development or in the periphery (FIG. 2),
tion and provide immunosurveillance against tumours. as resting splenic NK cells that were unable to receive
During basal homeostasis, mature peripheral NK cells IL‑18 signals were found to be defective in cytotoxicity
reside in the blood, spleen, liver and lung, and various and cytokine secretion following ex vivo stimulation95–97.
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IL‑18 has also been implicated in the basal homeostasis IL‑15 using mutant IL‑15–Fc fusion proteins or a solu-
of γδ T cells, suggesting that IL‑18 commonly influ- ble form of IL‑15Rα has shown efficacy in the treat-
ences other innate-like lymphocytes before activation98. ment of arthritis and in improving allograft transplant
During herpes simplex virus type 1 (HSV‑1) infection, survival in mice116–118.
mice deficient in IL‑18 are more susceptible than control
mice, as they show a higher viral burden and decreased NK cells ‘fill the space’ during lymphopenia.
survival, and their NK cells have profound defects in IFNγ Immunocompetence means having a sufficient num-
production and cytotoxicity 99. ber of functional immune cells ready to respond against
Interestingly, because mature NK cell responsiveness pathogen invasion. Thus, cells of the immune system
can be continuously modulated as a result of constant undergo homeostatic, or space-driven, proliferation dur-
interactions between inhibitory receptors and MHC ing times of lymphopenia to restore overall cell numbers
class I molecules45,46, the NK cell population exhibits (FIG. 2). Like B and T cells, NK cells can rapidly expand
a novel versatility that is less apparent in T cells. Thus, in number when placed in a lymphopenic environment.
an individual NK cell can adapt to new environments Adoptive transfer of mature splenic NK cells into mice
to either gain or lose functional competence, thereby deficient in both RAG and γc (which lack B, T and NK
maintaining tolerance to self while responding against cells) or into mice treated with sublethal doses of radia-
pathogens and tumours. Additional signals that mature tion results in rapid NK cell division, as measured by
peripheral NK cells require in the steady state for sur- 5,6‑carboxyfluorescein diacetate succinimidyl ester
vival, basal turnover and maintenance of function need (CFSE) labelling or 5‑bromodeoxyuridine (BrdU) incor-
further investigation. poration101,103,104,119. In these studies, NK cell homeostatic
proliferation mimicked that of CD8+ T cells and was
IL‑15 maintains NK cell survival. Cytokines of the γc more rapid than that of B cells and CD4+ T cells119. Like
family are crucial for the development of lymphoid lin- CD8+ T cells, NK cells become phenotypically activated
eage cells. Indeed, mice deficient in γc have defective during homeostatic proliferation, and this involves the
maturation in the B, T and NK cell compartments. IL‑2 upregulation of markers such as CD11a, CD44, CD122
and IL‑15, along with other pro-inflammatory cytokines, and LY6C120–122. In addition, homeostatically proliferating
promote clonal expansion of T cells, with elevated or NK cells and CD8+ T cells can rapidly degranulate and
prolonged levels of IL‑2 leading to apoptosis of effector secrete IFNγ following antigen receptor triggering. This
T cells25. IL‑7 and IL‑15 then protect memory precursor rapid IFNγ secretion is due to increased Ifng transcript
cells from cell death during the contraction phase of the expression, as demonstrated in NK cells and CD8+ T cells
T cell response100. IL‑15 also has an important role during from yeti mice (in which expression of yellow fluores-
NK cell development and continues to be crucial in the cent protein (YFP) is driven by the Ifng promoter)119.
homeostasis and survival of peripheral NK cells25 (FIG. 2). This heightened ability of NK cells and CD8+ T cells to
In adoptive transfer experiments, the half-life of mature respond to antigens may represent an evolved mechanism
NK cells was found to be approximately 1 week; however, that ensures the host can effectively defend itself during
in the absence of IL‑15, nearly all transferred NK cells lymphopenia, when it is most susceptible to pathogens.
rapidly disappeared within 48 hours 101–104. Memory
CD8+ T cells, which have been likened to NK cells, also Homeostatic proliferation generates long-lived NK cells.
depend on IL‑15 for their homeostasis and survival25. Mature peripheral NK cells were previously considered
DCs and macrophages mediate trans-presentation of to be terminally differentiated effector cells incapable of
IL‑15 (REFS 105–107), as Cre-induced specific deletion self-renewal. However, initial studies that evaluated adop-
of Il15ra in these professional antigen-presenting cells tive transfer of NK cells into lymphopenic hosts did not
resulted in the loss of both NK cells and memory CD8+ investigate the long-term consequences of homeostatic
T cells, although T cells were more severely affected proliferation. In a recent study, homeostatic proliferation
than NK cells108. Transgenic expression of IL‑15R spe- of NK cells was demonstrated to result in an unexpected
cifically in DCs or treatment of mice with soluble IL‑15 longevity of the transferred mature NK cells119, which
and IL‑15Rα complexes also promotes the expansion were tracked using a congenic marker. The homeostasis-
of NK cell populations, resulting in elevated numbers driven NK cells were found to reside in both lymphoid
and function109,110; however, transgenic overexpression and non-lymphoid organs for more than 6 months, and
of IL‑15 leads to fatal leukaemia111. IL‑15 signalling in they were able to self-renew and slowly turn over in the
NK cells acts by increasing the expression and activ- steady state, in a similar manner to memory T cells119.
ity of BCL‑2 and its family members104,112,113 and, at the Furthermore, these long-lived NK cells retained their
same time, suppressing the transcription factor fork- functionality many months after initial transfer and
head box O3A (FOXO3A) and the pro-apoptotic factor responded robustly to viral infection119. However, unlike
BCL‑2‑interacting mediator of cell death (BIM)113. long-lived CD8+ or CD4+ T cells that have undergone
Given the known influence of IL‑15 on immune homeostatic proliferation and retain a ‘memory-like’
cells, this cytokine has become an immunotherapeutic phenotype and function (FIG. 2), the expanded NK cell
target. For example, administration or overexpression population reverted to a quiescent phenotype with func-
of IL‑15 has been shown to protect mice against a vari- tions equivalent to those of resting NK cells, as both
ety of infections by boosting the effector functions expanded and resting NK cell populations responded with
of cytotoxic NK and T cells114,115. Conversely, blockade of comparable kinetics against viral challenge119.
650 | O CTOBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
Homeostatically driven CD8 + T cells have been Interestingly, a similar clonal-like expansion has
shown to require CD4+ T cell help for protective capa- been observed in human NK cell responses against
bility 123, and they deferred to ‘true’ antigen-experienced viral infection. The NKG2C-bearing NK cell subset
memory CD8+ T cells when mice were challenged with is highly enriched within the total NK cell popula-
pathogens 124. Similar studies remain to be done to tion in humans who are seropositive for human cyto-
investigate the requirements for robust homeostatic megalovirus (HCMV)130,131. In these studies, NKG2C+
proliferation of NK cells, and to compare the func- NK cells typically constituted less than 1% of the
tions of space-driven versus antigen-driven NK cells. total NK cell population in the peripheral blood of
Nonetheless, the ability of mature NK cells to self-renew HCMV-seronegative donors, whereas the percentage
and possibly persist in the host for months or years of NKG2C+ NK cells in seropositive donors ranged
will be of clinical importance for the reconstitution of from less than 5% to as much as 50–60%. A challenge
immune compartments during viral infections such as arises in the analysis and interpretation of these data
HIV, or during NK cell adoptive immunotherapy for the sets because the time of HCMV infection or reactiva-
treatment of certain cancers (BOX 1). tion is unknown, which explains the large differences
in NKG2C+ NK cell percentages between individuals.
Dynamic response of NK cells during viral infection One recent study documented a B and T cell-deficient
During viral infection, homeostasis is perturbed and newborn who presented with complications stem-
mature NK cells become activated, proliferate robustly and ming from HCMV infection. At a time point at which
contribute to both innate and adaptive immunity 125. NK viral loads were increasing, greater than 80% of this
cells, together with CD8+ T cells, directly target infected child’s NK cell population expressed NKG2C, suggest-
cells during viral invasion, and deficiency of NK cells in ing a prolific expansion of this subset 132. Longitudinal
mice and humans results in susceptibility to many viral studies have demonstrated a clonal-like expansion of
infections and severe clinical outcomes1,126. Like CD8+ human NK cell subsets during HCMV infection in
T cell responses against various pathogens, a clonal-like immunosuppressed recipients of solid-organ trans-
expansion has been described in the LY49H‑bearing sub- plants who have reactivated or become acutely infected
set of NK cells during mouse cytomegalovirus (MCMV) with HCMV133. In these patients, NKG2C+ NK cells
infection127 (FIG. 3). In certain mouse strains (such as undergo a substantial proliferation within the first
C57BL/6), LY49H+ NK cells can undergo a 3- to 10‑fold week after virus detection, and the elevated frequency
expansion in absolute numbers (measured in the spleen of these NKG2C+ NK cells persists after the virus has
and liver) over the course of 6–7 days after viral infec- been controlled133.
tion127,128. Although this expansion is not as prolific as the Interestingly, a hantavirus outbreak in northern
several-thousand-fold expansion measured in antigen- Sweden in 2007 allowed clinicians to prospectively
specific T cell responses129, the adoptive transfer of small investigate the immune response to infection in a
numbers of wild-type NK cells into LY49H‑deficient hosts setting where initial viral infection was well docu-
demonstrated that, during infection, LY49H+ NK cell mented 134 . This study demonstrated that human
populations are fully capable of 100‑fold and 1000‑fold NKG2C+ NK cells rapidly proliferate following hanta-
expansions in the spleen and liver, respectively 128. virus infection134, and the kinetics and magnitude of
this response strikingly mimic the clonal-like expan-
sion of mouse LY49H+ NK cells during MCMV infec-
Box 1 | NK cells in the clinic: promising therapeutic potential tion128. Of note, this proliferation of NKG2C+ NK cells
in hantavirus-infected individuals occurred in HCMV-
Reconstitution of the host immune system with haematopoietic stem cell transplants
— in humans with natural immunodeficiency syndromes (such as patients with severe
seropositive but not HCMV-seronegative patients, sug-
combined immunodeficiency (SCID)) or after radiation or chemotherapy in patients gesting that these NKG2C+ NK cells may have been
with cancer — can result in homeostatic proliferation of natural killer (NK) cells and primed by prior recognition of HCMV. Together,
other haematopoietic cell types. In the past decade, the adoptive transfer of these mouse and human studies demonstrate that NK
in vitro-cultured and -activated human NK cells has been applied towards the cell clones have proliferative capacities that were not
treatment of certain cancers174. Although adoptive NK cell therapy shows promise in previously appreciated.
the clinic, many questions arise in light of the novel findings on the longevity of NK
cells following homeostatic proliferation. For example, are there detrimental side Signals for NK cell and T cell activation. Some of the
effects (such as graft-versus-host disease) or other unforeseen dangers to healthy host cues that promote NK cell activation and proliferation
tissues associated with having a long-lived population of self-renewing NK cells poised
during viral infection have been characterized, whereas
to kill cellular targets? Conversely, are there unexpected benefits associated with
adoptive NK cell immunotherapy beyond the destruction of cancers such as
others remain to be investigated. Triggering of TCRs
leukaemias? Although it is thought to be a potent but transient therapy in humans, the on T cells by viral peptides bound to MHC molecules
adoptive transfer of NK cells may allow these cells to persist for months to years in the constitutes ‘signal 1’ in T cell activation135 and, similarly,
patient, mediating continuous surveillance against the recurrence of cancer. in the case of MCMV infection, recognition of cognate
Furthermore, in patients who require haematopoietic stem cell transplants, co-transfer antigen through the LY49H activating NK cell receptor
of long-lived NK cells might protect against opportunistic pathogens (such as human promotes NK cell activation and clonal proliferation
cytomegalovirus reactivation) over the several months it takes for B and T cells to fully (FIG. 3a). During MCMV infection of C57BL/6 mice,
reconstitute the immune system. The use of mouse models to understand the nature of only virus-specific LY49H+ NK cells kill infected cells
NK cells undergoing homeostatic proliferation will provide insights that will be crucial bearing the viral m157 glycoprotein and expand signifi-
to the implementation of NK cell-based treatments in the clinic.
cantly in overall numbers to provide protection against
NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | O CTOBER 2011 | 651
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
C #PVKIGPURGEKȮETGURQPUGU 5KIPCN
the virus127,128,136. The NK cell subsets that do not express
2TQKPȯCOOCVQT[ 1WVEQOG LY49H can respond in a nonspecific ‘bystander’ manner,
E[VQMKPGU 'CTN[ PQE[VQMKPGUGETGVKQP but do not undergo a clonal-like expansion or provide
.CVG ENQPCNGZRCPUKQP
0CKXG CPFE[VQVQZKEKV[ any protection against MCMV127,128,136 (FIG. 3b).
%&
6EGNN %[VQMKPG
T cells require co-stimulation through CD28 as
TGEGRVQT ‘signal 2’ for the activation of effector function135, but
6%4 %& it is not clear whether NK cells require this signal for
5KIPCN
6%4s/*%
full effector function in vivo during viral infection. Early
/*% 5KIPCN
KPVGTCEVKQP ENCUU+ $ %QUVKOWNCVKQP in vitro studies suggested that, although resting NK cells
expressed CD28 at much lower levels than naive T cells,
CD28 triggering on NK cells was required for optimal
&GPFTKVKE +. cytokine secretion and proliferation, but not for cyto-
EGNN 6[RG++(0U toxicity 137. These findings can now be revisited in vivo
8KTWU by measuring virus-specific responses in NK cells from
mice lacking CD28.
5KIPCN #EVKXCVKPI In certain mouse models of infection, T cells have
#EVKXCVKPI 5KIPCN
NKICPF %QUVKOWNCVKQP been shown to require ‘signal 3’ — in the form of the
0-EGNN
TGEGRVQTs #EVKXCVKPI TGSWKTGF! pro-inflammatory cytokines IL‑12 and type I IFNs —
NKICPF TGEGRVQT
for optimal activation and proliferation129. The early
KPVGTCEVKQP
nonspecific NK cell response against MCMV, charac-
4GUVKPI
0-EGNN 1WVEQOG terized by the secretion of IFNγ and TNF, but not IL‑2,
5KIPCN 'CTN[ E[VQMKPGUGETGVKQP is also dependent primarily on the IL‑12 and type I IFNs
2TQKPȯCOOCVQT[ CPFE[VQVQZKEKV[ produced by conventional DCs (cDCs)138–140 (FIG. 3). In
E[VQMKPGU .CVG ENQPCNNKMGGZRCPUKQP
addition to cDCs, plasmacytoid DCs (pDCs) contrib-
ute to NK cell-mediated protection by secreting large
D #PVKIGPPQPURGEKȮETGURQPUGU amounts of type I IFNs following the triggering of
Toll-like receptors (TLRs) and intracellular sensors of
%[VQMKPG viral nucleic acids; indeed, specific pDC ablation leads
TGEGRVQT 1WVEQOG to decreased NK cell activity and increased MCMV
/GOQT[ 'CTN[ E[VQMKPGUGETGVKQP
%& 6EGNN /GOQT[ CPFE[VQVQZKEKV[ titres141. Interestingly, the crucial IL‑15 signals required
%& 6EGNN .CVG PQENQPCNGZRCPUKQP for NK cell development, basal homeostasis and survival
are not essential for NK cell activation, proliferation or
function during acute MCMV infection, as the antiviral
NK cell response is primarily driven by IL‑12 even in
mice lacking γc142. Conversely, IL‑12 and type I IFNs are
&GPFTKVKE not thought to have a role during NK cell development,
EGNN as both cytokine- and cytokine receptor-deficient mice
+.
have normal NK cell numbers and phenotypes143–145.
In addition to directing the nonspecific NK cell
6[RG+
8KTWU +(0U response during MCMV infection, there is evidence that
pro-inflammatory cytokines are crucial for the prolifera-
R&% tion and effector function of antigen-specific LY49H+
NK cells139 (J.C.S. and L.L.L., unpublished observa-
tions). This is similar to ‘signal 3’ for T cell activation.
Interestingly, like resting NK cells, memory CD8+ T cells
1WVEQOG
have been described to possess the ‘innate-like’ ability
4GUVKPI 'CTN[ E[VQMKPGUGETGVKQP to produce IFNγ and proliferate following exposure
0-EGNN CPFE[VQVQZKEKV[ to pro-inflammatory cytokines in the absence of TCR
.CVG PQENQPCNGZRCPUKQP
signals and co-stimulation146,147. Altogether, these data
suggest that parallel mechanisms of signal integration
Figure 3 | Antigen-specific and -nonspecific responses of NK cells and CD8+ T cells through activating receptors, co-stimulatory receptors
during infection. a | During viral infection, natural killer (NK) cells and CD8+ T cells mount and cytokine receptors facilitate a productive effector
0CVWTG4GXKGYU^+OOWPQNQI[
specific responses following antigen receptor triggering. T cell receptor (TCR)–MHC response in both NK cells and T cells.
interactions, co-stimulation and pro-inflammatory cytokines (such as interleukin‑12
(IL‑12) and type I interferons (IFNs)) are the three signals thought to promote the NK cell memory. In addition to clonal expansion, much
activation and clonal expansion of naive CD8+ T cells. Similarly, resting NK cells receive
evidence in recent years suggests that NK cells possess
signals via activating receptors and pro-inflammatory cytokine receptors; it is unclear
whether co-stimulatory molecules have an important role in NK cell activation and other features of adaptive immunity, such as longev-
expansion. The short- and long-term functional outcomes are shown. b | IL‑12 and type I ity and immune memory, manifested as the ability to
IFNs can directly act on memory CD8+ T cells and resting NK cells in the absence of mount recall responses (BOX 2). Following the prolific
antigen receptor triggering, and this leads to nonspecific ‘bystander’ responses. The expansion phase of LY49H+ NK cell populations dur-
short- and long-term functional outcomes are shown. pDC, plasmacytoid dendritic cell. ing MCMV infection, the effector NK cells undergo
652 | O CTOBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
apoptosis 1–2 weeks later 127,128,148. This resembles the The mechanisms behind the trafficking of effector NK
contraction phase observed when effector T cells cells to peripheral organs and the precise signals required
undergo activation-induced cell death149, and is most to maintain long-lived NK cells at those sites are currently
similar to the contraction phase of the CD4+ T cell unknown. Memory LY49H+ NK cells were shown to be
response, during which a gradual but continuous decline self-renewing and could undergo secondary and tertiary
in cell numbers is observed150. expansion, with each round of expansion followed by
IL‑7 and IL‑15 dictate the homeostasis of effector contraction and a subsequent increase in the levels of
T cells during the contraction phase, but the cytokines LY49H (measured by mean fluorescence intensity)161.
that influence the resolution of the effector NK cell Interestingly, following acute HCMV and hantavirus
response have not been defined, although it is likely that infection, NKG2C+ NK cells became NKG2Chi during
they also include IL‑15 (FIG. 2). The survival of effector clonal proliferation and expressed the CD57 marker on
T cells during the contraction phase and the transition to long-lived cells after the contraction phase, providing a
memory cells is dictated by anti- and pro-apoptotic fac- possible memory marker for human NK cells133,134. The
tors of the BCL‑2 family (such as BCL‑2 and BIM, respec- higher expression of virus-specific receptors — LY49H
tively)151–155, and evidence suggests that these factors also and CD94–NKG2C in mouse and human NK cells,
regulate the contraction phase of the NK cell response respectively — following infection suggests a form of
to MCMV infection (N. A. Bezman, J.C.S. and L.L.L., ‘avidity maturation’ in NK cell receptors that have previ-
unpublished data). Although early IL‑21 signalling has ously engaged their cognate ligands, and this is similar
been described to be important for the functional matu- to what is observed in CD8+ T cells during infection162.
ration and activation of NK cells156–158, a regulatory role
for IL‑21 may exist in NK cells during proliferation, such Reactivity of naive, effector and memory NK cells and
that IL‑21 restricts the NK cell response while promot- CD8+ T cells. The spontaneity of killing and the speed
ing T cell responses159. This dichotomous effect of IL‑21 of effector function mediated by NK cells led Eva Klein
on NK cells is not unprecedented, as another γc family to first coin them “natural killers” in 1975 (REF. 163),
cytokine, IL‑2, can act to promote T cell proliferation, but and frequent comparisons are drawn to activated CD8+
can also induce apoptosis, depending on the phase and T cells2,125 (but not naive CD8+ T cells). However, recent
context of the T cell response25,160. studies using the MCMV model have shown that NK
Following the contraction of effector NK cell popula- cells can also exist as both resting cells and activated
tions, long-lived memory NK cells can be detected for (effector) cells, distinguished by their ability to degranu-
several months in adoptive transfer studies128. Memory late and secrete effector cytokines (such as IFNγ)127,128,136.
NK cells were found to reside in both lymphoid and CD8+ T cells can exist in at least three states of reactiv-
non-lymphoid organs and could mediate rapid and ity depending on their ability to kill and secrete effector
potent effector functions when stimulated ex vivo 128. cytokines during ex vivo TCR triggering. Naive CD8+
Box 2 | The evidence for NK cell memory
Immunological memory is a classical hallmark that distinguishes adaptive immune B and T cells from all other cells of the
haematopoietic lineage. However, evidence for immune memory exists in invertebrates — including crustaceans, flies,
beetles and mosquitoes and more primitive species (such as tunicates, sea urchins and sea sponges) — which lack
lymphocytes that possess rearranged antigen receptors175. Early transplantation studies that demonstrated the natural
killer (NK) cell-mediated rejection of parental bone marrow cells transferred into F1 progeny recipients (a process termed
hybrid resistance) first suggested that immune memory could also reside in NK cells. Interestingly, when the F1 offspring
had been previously primed with parental bone marrow cells, they rejected a second graft from the same parent more
efficiently176, implicating NK cell-mediated recall responses in graft rejection. Previous studies had also suggested that
NK cell memory may be responsible for enhanced cytokine secretion following exposure to pro-inflammatory cytokines
or for more potent tumour-specific responses following stimulation177–179.
More recent studies have demonstrated that NK cells mediate contact hypersensitivity responses to chemical haptens,
as these responses occur in recombination-activating gene (RAG)-deficient mice (which lack B and T cells), but not in
mice deficient for both RAG and γc (which also lack NK cells)180. Secondary responses were only elicited by the hapten to
which mice had been originally exposed and not by a different hapten180. The contact hypersensitivity observed in the
RAG-deficient mice was mediated by an NK cell population that expressed CXC-chemokine receptor 6 (CXCR6) and was
resident in the liver181. During mouse cytomegalovirus infection, adoptively transferred NK cells bearing the receptor
LY49H proliferate 100–1000‑fold in lymphoid and non-lymphoid tissues. This results in a long-lived pool of memory
NK cells that are capable of more potent effector function, self-renewal, recall response and greater protection against
viral challenge128,161. In a subsequent study, exposure to the pro-inflammatory cytokines interleukin‑12 (IL‑12) and
IL‑18 in vitro also led to the generation of memory NK cells, and these cells were capable of more robust interferon-γ
production following activating receptor stimulation compared with resting NK cells182. In humans, acute infection with
human cytomegalovirus (HCMV) results in the preferential expansion of NK cell populations that express the activating
receptor NKG2C (natural killer group 2, member C)133. Similarly, acute hantavirus infection led to the prolific expansion
and long-term persistence of NKG2C‑expressing NK cell populations134. In this study, greater numbers of NKG2C+
NK cells were observed at the peak of expansion in HCMV-seropositive patients than in HCMV-seronegative patients,
suggesting that a more robust response was measured in this HCMV-responsive NK cell subset134.
NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | O CTOBER 2011 | 653
© 2011 Macmillan Publishers Limited. All rights reservedREVIEWS
5VCVGQHTGCEVKXKV[
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.;%NQY %GPVTCNOGOQT[ 'ȭGEVQTOGOQT[
%& 6EGNN %& 6EGNN
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%&.JK %&.NQY
.;%JK .;%JK
4GUVKPI #EVKXCVGF
0-EGNN GȭGEVQT 0-EGNN
-.4)NQYOKF
%&.JK
.;%NQYOKF
/GOQT[ -.4)JK
0-EGNN %&.NQYOKF
.;%OKFJK
-.4)JK
%&.NQY
.;%JK
%[VQVQZKEKV[
CPFE[VQMKPG s
UGETGVKQP
Figure 4 | Comparing the reactivity of NK cells and CD8+ T cells during differentiation. The state of reactivity of
0CVWTG4GXKGYU^+OOWPQNQI[
resting natural killer (NK) cells, naive CD8+ T cells and activated (effector) and memory NK and CD8+ T cells is marked by
the expression of activation and differentiation markers. These markers include killer cell lectin-like receptor subfamily G,
member 1 (KLRG1), CD62L and LY6C. The relative capacities of these subsets to mediate cytotoxicity and secrete effector
cytokines are indicated.
T cells have low reactivity, activated (effector) CD8 + KLRG1 and low CD62L expression) and in function
T cells have high reactivity and memory CD8+ T cells (high IFNγ production following stimulation)128 (FIG. 4).
have intermediate reactivity (FIG. 4). We believe that a In this comparison between responding NK cells and
similar model can be applied to NK cell responses CD8+ T cells, the lymphocyte with the greatest state
against viral infection; however, a naive NK cell is in a of reactivity is the activated (effector) NK cell, which
more responsive state than a naive CD8+ T cell. Naive secretes cytokines spontaneously 127,128,136 and does not
resting NK cells have the capability to respond to acti- require ligation of activating receptors for its effector
vating receptor triggering, and their Ifng transcript function ex vivo. Although NK cells and CD8+ T cells dif-
levels are similar to those of memory CD8+ T cells, as fer fundamentally in the generation of antigen recogni-
detected using IFNγ–YFP reporter mice164. The equiva- tion receptors, their similar nature and response suggests
lent Ifng transcript levels in resting NK cells and mem- an evolutionary ancestry between these two cytotoxic
ory CD8+ T cells translate into functional similarities cell lineages that have been distinctly categorized as
between these two cell types, as both can secrete IFNγ innate and adaptive167.
following stimulation with pro-inflammatory cytokines
alone127,146,147. Memory NK cells have a greater respon- Concluding remarks
siveness than resting NK cells, and ligation of activating The precise mechanisms of NK cell development and
receptors on memory NK cells leads to greater cytokine homeostasis are actively being investigated. The discov-
production and degranulation (measured by lysosome- ery of NK cell memory generates many new questions,
associated membrane protein 1 (LAMP1) expression) on some of which also remain to be answered in the study
a per cell basis128. Thus, perhaps resting NK cells possess of immune memory in T cells. Moreover, questions that
a comparable state of reactivity to memory CD8+ T cells, T cell biologists have raised (and answered, in many
and likewise memory NK cell reactivity is similar to that circumstances) in the previous decades can now be
of activated or effector CD8+ T cells (FIG. 4). applied towards understanding how NK cells can also
The overall memory CD8+ T cell compartment has become long-lived cells capable of recall responses. For
been described to contain at least two subsets: central instance, what molecular and cellular interactions are
and effector memory T cell populations165,166. By con- important for optimal NK cell homeostasis and memory
trast, memory LY49H+ NK cells appear to be a homo- generation? Crucial cues from DCs and CD4+ T cells
geneous population that closely resembles the effector are required for proper generation and maintenance of
memory CD8+ T cell subset, both in phenotype (high memory CD8+ T cells129,168. Does the viral glycoprotein
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