Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin-beta receptor expression

A crucial step in murine natural killer (NK) cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49 receptor expression are still largely undetermined. It has been shown that interaction between lymphotoxin alpha1beta2 (LTalpha1beta2) and LTbeta receptor (LTbetaR), expressed on lymphoid progenitor cells and nonlymphoid bone marrow stromal cells, respectively, is important for both quantitative and functional NK cell development. Therefore, we have investigated the role of LT-LTbetaR-mediated signaling in Ly49 and CD94/NKG2 receptor acquisition. We show that the NK receptor repertoire of LTbetaR-/- mice can only be partially analyzed because of the residual 129/Ola mouse genetic background, due to a physical linkage of the LTbetaR locus and the loci encoding the Ly49 and CD94/NKG2 receptors. Therefore, we transferred wild-type B6 lymphoid-committed progenitor cells into LTbetaR-/- mice, which differentiated into NK cells with a normal NK cell receptor repertoire. Also, administration of LTbetaR-immunoglobulin (Ig), which acts as a soluble receptor for LTalpha1beta2, resulted in reduced NK cell percentages but did not influence the Ly49 and CD94/NKG2 receptor acquisition on remaining NK cells. These results indicate that LTbetaR-mediated signals are not required for Ly49 and CD94/NKG2 receptor acquisition.     

Implications of CD94 deficiency and monoallelic NKG2A expression for natural killer cell development and repertoire formation

Natural killer (NK) cells are believed to achieve self-tolerance through the expression of self-MHC-specific inhibitory receptors, such as members of the Ly49 and CD94/NKG2 families. Individual Ly49 genes are stochastically expressed by NK subsets and are expressed in a monoallelic fashion, but little is known about the mechanisms underlying CD94/NKG2A expression. We show here that, like Ly49 genes, mouse Nkg2a is stochastically and monoallelically expressed. Thus, a single general mechanism controls expression of all known MHC-specific receptors by mouse NK cells. In addition, we find that DBA/2J mice are naturally CD94-deficient and do not express cell-surface CD94/NKG2A receptors, even on neonatal NK cells. Thus, self-tolerance of neonatal NK cells cannot be attributed to CD94/NKG2A expression. Taken together, the results lead to a reconsideration of current models of NK cell development and self-tolerance.     

Human natural killer cells with polyclonal lectin and immunoglobulinlike receptors develop from single hematopoietic stem cells with preferential expression of NKG2A and KIR2DL2/L3/S2

The stage of progenitor maturation and factors that determine the fate and clonal acquisition of human natural killer (NK) cell receptors during development are unknown. To study human NK cell receptor ontogeny, umbilical cord blood CD34(+)/Lin(-)/CD38(-) cells were cultured with a murine fetal liver line (AFT024) and defined cytokines. In the absence of lymphocyte-stimulating cytokines or when contact with AFT024 was prohibited, NK cell progeny were killer immunoglobulinlike receptor (KIR) and CD94 lectin receptor negative. In contrast, efficient NK cell differentiation and receptor acquisition was dependent on direct contact of progenitors with AFT024 and the addition of interleukin-15 (IL-15) or IL-2 but not IL-7. To address the question of whether receptor acquisition was determined at the stem cell level, single CD34(+)/Lin(-)/CD38(-) progenitors were studied. More than 400 single cell progeny were analyzed from cultures containing IL-15 or IL-2 and NK cells were always polyclonal, suggesting that receptor fate is determined beyond an uncommitted progenitor and that receptor-negative NK cells acquire class I-recognizing receptors after lineage commitment. KIR2DL2/L3/S2 was expressed more than KIR2DL1/S1 or KIR3DL1, and NKG2A was the dominant CD94 receptor, independent of whether the stem cell source contained the respective major histocompatibility complex class I ligand, suggesting a nonrandom sequence of receptor acquisition. The conclusion is that NK receptor fate is determined after NK cell commitment, does not require stromal presentation of human class I alleles, and is clonally stable after expression but dynamic because new receptors are acquired over time. (Blood. 2001;98:705-713)     

Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts

CD94/NKG2C(+) natural killer (NK) cells are increased in healthy individuals infected with human cytomegalovirus (HCMV), suggesting that HCMV infection may shape the NK cell receptor repertoire. To address this question, we analyzed the distribution of NK cell subsets in peripheral blood lymphocytes (PBLs) cocultured with HCMV-infected fibroblasts. A substantial increase of NK cells was detected by day 10 in samples from a group of HCMV(+) donors, and CD94/NKG2C(+) cells outnumbered the CD94/NKG2A(+) subset. Fibroblast infection was required to induce the preferential expansion of CD94/NKG2C(+) NK cells that was comparable with allogeneic or autologous fibroblasts, and different virus strains. A CD94-specific monoclonal antibody (mAb) abrogated the effect, supporting an involvement of the lectinlike receptor. Purified CD56(+) populations stimulated with HCMV-infected cells did not proliferate, but the expansion of the CD94/NKG2C(+) subset was detected in the presence of interleukin-15 (IL-15). Experiments with HCMV deletion mutants indicated that the response of CD94/NKG2C(+) NK cells was independent of the UL16, UL18, and UL40 HCMV genes, but was impaired when cells were infected with a mutant lacking the US2-11 gene region. Taken together the data support that the interaction of CD94/NKG2C with HCMV-infected fibroblasts, concomitant to the inhibition of human leukocyte antigen (HLA) class I expression, promotes an outgrowth of CD94/NKG2C(+) NK cells.     

Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells

The stages of human natural killer (NK) cell differentiation are not well established. Culturing CD34(+) progenitors with interleukin 7 (IL-7), IL-15, stem cell factor (SCF), FLT-3L, and murine fetal liver cell line (EL08.1D2), we identified 2 nonoverlapping subsets of differentiating CD56(+) cells based on CD117 and CD94 (CD117(high)CD94(-) and CD117(low/-)CD94(+) cells). Both populations expressed CD161 and NKp44, but differed with respect to NKp30, NKp46, NKG2A, NKG2C, NKG2D, CD8, CD16, and KIR. Only the CD117(low/-) CD94(+) population displayed cytotoxicity and interferon-gamma production. Both populations arose from a single CD34(+)CD38(-) Lin(-) cell and their percentages changed over time in a reciprocal fashion, with CD117(high)CD94(-) cells predominating early and decreasing due to an increase of the CD117(low/-)CD94(+) population. These 2 subsets represent distinct stages of NKcell differentiation, since purified CD117(high) CD94(-) cells give rise to CD117(low/-)CD94(+) cells. The stromal cell line (EL08.1D2) facilitated the transition from CD117(high)CD94(-) to CD117(low/-)CD94(+) via an intermediate phenotype (CD117(low)CD94(low/-)). EL08.1D2 also maintained the mature phenotype, preventing the reversion of CD117(low/-)CD94(+) cells to the intermediate (CD117(low)CD94(low/-)) phenotype. An analogous population of CD56(+)CD117(high)CD94(-) cells was found in cord blood. The identified stages of NK-cell differentiation provide evidence for coordinated acquisition of HLA-specific inhibitory receptors (ie, CD94/NKG2A) and function in developing human NK cells.     

Mononuclear myeloid-derived "suppressor" cells express RAE-1 and activate natural killer cells

Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patients and tumor-bearing mice and potently suppress T-cell activation. In this study, we investigated whether MDSCs regu-late natural killer (NK)-cell function. We discovered that mononuclear Gr-1(+)CD11b(+)F4/80(+) MDSCs isolated from RMA-S tumor-bearing mice do not suppress, but activate NK cells to produce high amounts of IFN-gamma. Gr-1(+)CD11b(+)F4/80(+) MDSCs isolated from tumor-bearing mice, but not myeloid cells from naive mice, expressed the ligand for the activating receptor NKG2D, RAE-1. NK-cell activation by MDSCs depended partially on the interaction of NKG2D on NK cells with RAE-1 on MDSCs. NK cells eliminated Gr-1(+)CD11b(+)F4/80(+) MDSCs in vitro and upon adoptive transfer in vivo. Finally, depletion of Gr-1(+) cells that comprise MDSCs confirmed their protective role against the NK-sensitive RMA-S lymphoma in vivo. Our study reveals that MDSCs do not suppress all aspects of antitumor immune responses and defines a novel, unexpected activating role of MDSCs on NK cells. Thus, our results have great impact on the design of immune therapies against cancer aiming at the manipulation of MDSCs.     

Expansion of a unique CD57⁺NKG2Chi natural killer cell subset during acute human cytomegalovirus infection

During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94-NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56(dim)CD16(+) NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94-NKG2C receptor and CD57 in CMV(+) donors. These CD57(+)NKG2C(hi) NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57(+)NKG2C(hi) NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57(+)NKG2C(hi) NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C(+) NK cells proliferated, became NKG2C(hi), and finally acquired CD57. Thus, we propose that CD57 might provide a marker of "memory" NK cells that have been expanded in response to infection.     

Imprint of human cytomegalovirus infection on the NK cell receptor repertoire

Expression of the activating CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E was analyzed in peripheral blood lymphocytes (PBLs) from healthy adult blood donors; the expression of other natural killer (NK) cell receptors (ie, CD94/NKG2A, KIR, CD85j, CD161, NKp46, NKp30, and NKG2D) was also studied. Human cytomegalovirus (HCMV) infection as well as the HLA-E and killer immunoglobulin-like receptor (KIR) genotypes were considered as potentially relevant variables associated with CD94/NKG2C expression. The proportion of NKG2C(+) lymphocytes varied within a wide range (<0.1% to 22.1%), and a significant correlation (r = 0.83; P < .001) between NKG2C(+) NK and T cells was noticed. The HLA-E genotype and the number of activating KIR genes of the donors were not significantly related to the percentage of NKG2C(+) lymphocytes. By contrast, a positive serology for HCMV, but not for other herpesviruses (ie, Epstein-Barr and herpes simplex), turned out to be strongly associated (P < .001) with increased proportions of NKG2C(+) NK and T cells. Remarkably, the CD94/NKG2C(+) population expressed lower levels of natural cytotoxicity receptors (NCRs) (ie, NKp30, NKp46) and included higher proportions of KIR(+) and CD85j(+) cells than CD94/NKG2A(+) cells. Altogether, these data support that HCMV infection selectively shapes the natural killer cell receptor (NKR) repertoire of NK and T cells from healthy carrier individuals.     

Dysregulation of signaling pathways in CD45-deficient NK cells leads to differentially regulated cytotoxicity and cytokine production

CD45, a protein tyrosine phosphatase that regulates Src family kinases, is important for regulating T cell and B cell receptor signaling; however, little is known about how CD45 regulates immunoreceptor tyrosine-based activation motif (ITAM)-dependent natural killer (NK) cell receptor signaling and the resulting effector functions. NK cells from CD45-deficient mice are relatively competent for ITAM receptor-induced cell-mediated cytotoxicity, yet completely deficient for cytokine secretion after stimulation with ligands to or antibodies against NK1.1, CD16, Ly49H, Ly49D, and NKG2D. This deficiency in cytokine/chemokine production occurs at the level of mRNA expression. After receptor engagement, extracellular signal-regulated kinase and c-Jun N-terminal kinase activation was markedly perturbed, whereas p38 activation was not substantially affected. The pattern and amounts of basal tyrosine phosphorylation were altered in freshly isolated NK cells and were surprisingly and markedly increased in IL-2-expanded NK cells from CD45-/- mice. These findings indicate that CD45-dependent regulation of ITAM-dependent signaling pathways is essential for NK cell-mediated cytokine production but not cytolytic activity.     

Development of intraepithelial T lymphocytes in the intestine of irradiated SCID mice by adult liver hematopoietic stem cells from normal mice

BACKGROUND/AIMS: We recently reported the adult mouse liver to contain c-kit+ stem cells that can give rise to multilineage leukocytes. This study was designed to determine whether or not adult mouse liver stem cells can generate intraepithelial T cells in the intestine as well as to examine the possibility that adult liver c-kit+ stem cells originate from the fetal liver. METHODS: Adult liver mononuclear cells, bone marrow (BM) cells, liver c-kit+ cells or bone BM c-kit+ cells of BALB/c mice were i.v. transferred into 4 Gy irradiated CB17/-SCID mice. In other experiments, fetal liver cells from Ly5.1 C57BL/6 mice and T cell depleted adult BM cells from Ly5.2 C57BL/6 mice were simultaneously transferred into irradiated C57BL/6 SCID mice (Ly5.2). At 1 to 8 weeks after cell transfer, the SCID mice were examined. RESULTS: Not only BM cells and BM c-kit+ cells but also liver mononuclear cells and liver c-kit+ cells reconstituted gamma delta T cells, CD4+ CD8+ double-positive T cells and CD8 alpha+beta- T cells of intestinal intraepithelial lymphocytes of SCID mice. Injection of a mixture of fetal liver cells from Ly5.1 C57BL/6 mice and adult BM cells from Ly5.2 C57BL/6 mice into Ly5.2 C57BL/6 SCID mice induced both Ly5.1 and Ly5.2 T cells, while also generating c-kit+ cells of both Ly5.1 and Ly5.2 origins in the liver. CONCLUSIONS: Adult mouse liver stem cells were able to generate intestinal intraepithelial T cells of the SCID mice, and it is thus suggested that some adult liver stem cells may indeed be derived from the fetal liver.     

Inhibitory receptors specific for MHC class I educate murine NK cells but not CD8αα intestinal intraepithelial T lymphocytes

The engagement of inhibitory receptors specific for major histocompatibility complex class I (MHC-I) molecules educates natural killer (NK) cells, meaning the improvement of the response of activation receptors to subsequent stimulation. It is not known whether inhibitory MHC-I receptors educate only NK cells or whether they improve the responsiveness of all cell types, which express them. To address this issue, we analyzed the expression of inhibitory MHC-I receptors on intestinal intraepithelial lymphocytes (iIELs) and show that T-cell receptor (TCR)-αβ CD8αα iIELs express multiple inhibitory receptors specific for MHC-I molecules, including CD94/NKG2A, Ly49A, and Ly49G2. However, the presence of MHC-I ligand for these receptors did not improve the response of iIELs to activation via the TCR. The absence of iIEL education by MHC-I receptors was not related to a lack of inhibitory function of these receptors in iIELs and a failure of these receptors to couple to the TCR. Thus, unlike NK cells, iIELs do not undergo an MHC-I-guided education process. These data suggest that education is an NK cell-specific function of inhibitory MHC-I receptors.     

Incomplete restoration of colony-stimulating factor 1 (CSF-1) function in CSF-1-deficient Csf1op/Csf1op mice by transgenic expression of cell surface CSF-1

The primary macrophage growth factor, colony-stimulating factor 1 (CSF-1), is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell surface glycoprotein (csCSF-1). To investigate the in vivo roles of csCSF-1, we created mice that exclusively express csCSF-1, in a normal tissue-specific and developmental manner, by transgenic expression of csCSF-1 in the CSF-1-deficient osteopetrotic (Csf1(op)/Csf1(op)) background. The gross defects of Csf1(op)/Csf1(op) mice, including growth retardation, failure of tooth eruption, and abnormal male and female reproductive functions were corrected. Macrophage densities in perinatal liver, bladder, sublinguinal salivary gland, kidney cortex, dermis, and synovial membrane were completely restored, whereas only partial or no restoration was achieved in adult liver, adrenal gland, kidney medulla, spleen, peritoneal cavity, and intestine. Residual osteopetrosis, significantly delayed trabecular bone resorption in the subepiphyseal region of the long bone, and incomplete correction of the hematologic abnormalities in the peripheral blood, bone marrow, and spleens of CSF-1-deficient mice were also found in mice exclusively expressing csCSF-1. These data suggest that although csCSF-1 alone is able to normalize several aspects of development in Csf1(op)/Csf1(op) mice, it cannot fully restore in vivo CSF-1 function, which requires the presence of the secreted glycoprotein and/or proteoglycan forms.     

Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2(b) bone marrow cell allografts

Subsets of murine natural killer (NK) cells exist that express the Ly-49 family of molecules that recognize different major histocompatibility complex (MHC) determinants. Bone marrow transplantation studies were performed to examine the in vivo functions of 2 of these subsets. Subsets of Ly-49A and Ly-49G2 NK share specificity for the same MHC class 1 ligand, D(d), binding of which results in an inhibitory signal to the NK cell but allows them to lyse H2(b) targets in vitro. We therefore examined the ability of these subsets to reject H2(b) bone marrow cell allografts in lethally irradiated mice. Surprisingly, depletion of Ly-49A(+) NK cells in BALB/c or B10.D2 mice (both H2(d)) had no effect on the rejection of H2(b) BMC. However, Ly-49A depletion did partially abrogate the ability of B10.BR (H2(k)) mice to reject H2(b) allografts. Although depletion of either Ly-49A(+) or Ly-49G2(+) NK cells alone had no effect on the ability of B10.D2 mice to reject H2(b) BMC, depletion of both subsets dramatically and synergistically abrogated rejection. Studies with various B10 congenic mice and their F(1) hybrids indicate that this synergy between Ly49A and Ly4G2 depletion occurs in every instance. Thus, Ly-49A(+) NK cells appear to play a role in the rejection H2(b) bone marrow allografts, but, in most strains of mice studied, Ly-49G2(+) NK cells must also be eliminated. The putative roles of these NK cell subsets in clinical transplantation remains to be elucidated. (Blood. 2000;95:3840-3844)     

Jagged2 promotes the development of natural killer cells and the establishment of functional natural killer cell lines

Emerging evidence indicates that Notch receptors and their ligands play important roles in the development of T cells and B cells. However, little is known about their possible roles in the development of other lymphoid cells. Here we demonstrate that Jagged2, a Notch ligand, stimulates the development of natural killer (NK) cells from Lin(-) Sca-1(+) c-kit(+) hematopoietic stem cells. Our culture system supports NK cell development for 2 to 3 months, often leading to the establishment of continuous NK cell lines. The prototype of such cell lines is designated as KIL. KIL depends on interleukin-7 for survival and proliferation and is NK1.1(+) CD3(-) TCRalphabeta(-) TCRdeltagamma(-) CD4(-) CD8(-) CD19(-) CD25(+) CD43(+) CD45(+) CD49b(-) CD51(+) CD94(+) NKG2D(+) Mac-1(-/low) B220(-) c-kit(+) perforin I(+) granzyme B(+) Notch-1(+), and cytotoxic. Like normal natural killer cells, the T-cell receptor-beta loci of KIL remain in the germ-line configuration. In response to interleukin-2, KIL proliferates extensively (increasing cell number by approximately 10(10)-fold) and terminally differentiates into adherent, hypergranular NK cells. Our findings indicate that Jagged2 stimulates the development of natural killer cells and the KIL cell line preserves most properties of the normal NK precursors. As such, KIL provides a valuable model system for NK cell research.     

A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature

How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56(bright) cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56(dim) cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% +/- 2.8% of the CD56(dim) NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56(dim) NKG2A(-) KIR(-) NK cells lack "at least one" inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-gamma production. Upon culture with IL-15 and a stromal cell line, CD56(dim) and CD56(bright) KIR(-) NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56(bright) cells precede CD56(dim) cells.     

Self-reactive memory-phenotype CD8 T cells exhibit both MHC-restricted and non-MHC-restricted cytotoxicity: a role for the T-cell receptor and natural killer cell receptors

We have recently shown that interleukin-2 (IL-2)-activated CD8(+)CD44(hi) cells from normal mice express both adaptive and innate immune system receptors and specifically kill syngeneic tumor cells, particularly those that express NKG2D ligands. Here we show that CD8+ T cells from antigen-expressing H-Y T-cell receptor (TCR) transgenic mice also exhibit characteristics of both T cells and natural killer (NK) cells. Interaction with cognate self-antigen was required for the optimal expansion of these cells in peripheral lymphoid tissues. Although these cells possess a higher activation threshold relative to naive T cells, they can be activated by cytokine alone in vitro. They also undergo bystander proliferation in response to a bacterial infection in vivo. Interestingly, upon activation, the cells express the NKG2D receptor as well as the DNAX activation protein 12 (DAP12) adaptor protein. We provide evidence that NKG2D can act additively with the TCR in the killing of target cells, and it can also function as a directly activating receptor in non-major histocompatibility complex (MHC)-restricted killing of target cells. These properties of CD8+ T cells from H-Y TCR transgenic mice are remarkably similar to CD8(+)CD44(hi) cells that are found in normal mice. The H-Y TCR transgenic mice provide a well-defined system for characterizing the developmental biology and function of these cells.