Mouse fetal and embryonic liver cells differentiate human umbilical cord blood progenitors into CD56-negative natural killer cell precursors in the absence of interleukin-15

OBJECTIVE: Human natural killer (NK) cell maturation involves the orderly acquisition of NK cell receptors. Our aim was to understand how stromal interactions and cytokines are important in this developmental process. MATERIALS AND METHODS: Human umbilical cord blood (UCB) CD34(+)/Lin(-)/CD38(-) cells were cultured on two murine stromal cell lines (AFT024 and EL08-1D2) in a switch culture to study NK cell development. RESULTS: When human progenitors were cultured on AFT024 with interleukin (IL)-3 and Flt3 ligand (Flt3L) in the absence of interleukin (IL)-15, NK cell differentiation occurred, albeit at low frequency. These conditions favored the accumulation of CD56(-) NK cell precursors (CD34(+)CD7(-), CD34(+)CD7(+), and CD34(-)CD7(+) cells), which are populations rare in adult blood but abundant in fresh UCB. In secondary culture, addition of IL-3 or IL-3 + Flt3L to IL-15 increased the absolute number of CD56(+) NK cells from precursors and the acquisition of CD94 and killer immunoglobulin-like receptors (KIR). To further explore the microenvironment in early NK cell maturation, a cell line derived from murine embryonic liver (EL08-1D2) was studied. NK cell development and KIR acquisition was superior with EL08-1D2, which supported the differentiation of NK cell precursors, NK cell commitment, and proliferation. CONCLUSION: Although the earliest events in NK cell maturation do not require exogenous human IL-15, it is required at a later stage of NK cell commitment. At a minimum, murine stroma, IL-3, and Flt3L are required to recapitulate early NK cell development and differentiation into distinct NK cell precursors. EL08-1D2 induces KIR acquisition suggesting that extrinsic signals in NK cell development are conserved between mouse and man.     

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.     

Expansion and enhanced survival of natural killer cells expressing the killer immunoglobulin-like receptor KIR3DL2 in spondylarthritis

OBJECTIVE: The spondylarthritides (SpA) are strongly associated with possession of HLA-B27. We hypothesized that the expression of abnormal forms of HLA-B27 in SpA may have a pathogenic role through interaction with cells bearing natural killer (NK) receptors, in particular, killer immunoglobulin-like receptor (KIR) KIR3DL2, a receptor for HLA-B27 homodimer (B27(2)). We therefore undertook the present study to determine the number and function of NK and T cells bearing KIR3DL2 in SpA. METHODS: Expression of KIR3DL2 on NK and T cells was quantified in peripheral blood (PB) from 35 patients with SpA and 5 patients with juvenile enthesitis-related arthritis (juvenile ERA); samples were compared with samples from healthy and rheumatoid arthritis (RA) controls. Paired synovial fluid (SF) was studied where available. Expression of other KIRs as well as activation, memory, and homing markers on KIR3DL2+ NK and T cells was quantified. NK cell survival was assessed using the apoptotic markers annexin V and 7-aminoactinomycin D, and cytotoxicity by (51)Cr release assay. RESULTS: In SpA, an increased number of PB and SF NK and CD4+ T cells expressed the KIR3DL2 receptor compared with controls. In ERA, KIR3DL2 expression was increased in PB and SF CD4 T cells (and SF NK cells) compared with RA controls. KIR3DL2+ NK cells had an activated phenotype, and were protected from apoptosis by culture with a cell line expressing B27(2). SpA PB mononuclear NK cells from SpA patients showed greater cytotoxicity than those from controls. CONCLUSION: KIR3DL2 expression on NK cells and CD4 lymphocytes is increased in SpA and ERA. These cells are activated and may have a pathogenic role.     

Effect of low-dose IL-2 immunotherapy on frequency and phenotype of regulatory T cells and NK cells in HIV/HCV-coinfected patients

We evaluated the effect of low-dose IL-2 therapy (daily 1.2 MIU/m(2), subcutaneously) on the number and phenotype of regulatory T cells (T(regs)) and natural killer (NK) cells in HIV/HCV-coinfected patients taking antiretroviral therapy. The frequency and phenotype of circulating T(regs) (defined as CD3(+) CD4(+) CD25(high) or CD3(+) CD4(+) FOXP3(+)) and NK cells (CD3(-) CD16(+)/CD56(+)) were evaluated at baseline and after 12 weeks of treatment. The expression of CD25, CTLA-4, and granzymes A and B by CD4(+) FOXP3(+) cells, as well as the expression of KIR receptors (NKB1, CD158a, and NKAT2) on NK cells, was evaluated. Low doses of IL-2 resulted in the augmented frequency and absolute number of T(regs) in coinfected individuals. FOXP3 levels per cell as well as augmented CD25 and CTLA-4 expression by T(regs) suggested that IL-2 may lead to both expansion and activation of T(regs), although changes in the proportion of CD4(+) FOXP3(+) cells were not associated with changes in HCV viral load and CD4(+) cells between baseline and week 12. NK cell frequency also increased after IL-2 therapy. Interestingly, the pattern of expression of KIR receptors was changed by IL-2 treatment, since the frequency of NK cells expressing NKB1 augmented whereas the frequency of NK expressing CD158a and NKAT2 decreased.     

Under representation of the inhibitory KIR3DL1 molecule and the KIR3DL1+/BW4+ complex in HIV exposed seronegative individuals

The activation of natural killer (NK) cells is modulated by surface molecules. We analyzed NK cells in human immunodeficiency virus (HIV)-exposed seronegative (HESN) individuals by means of molecular typing of HLA B, Cw, and killer cell immunoglobulin-like receptor (KIR) molecules. In HESN individuals, compared with HIV patients, the frequency of the inhibitory KIR3DL1 allele and of the KIR3DL1(+)/Bw4(+) inhibitory complex was reduced, whereas that of the activatory KIR3DS1(+) ligand and the activatory Bw4(+)/3DL1(-)/3DS1(+) complex was increased, resulting in a statistically significant diversion from Hardy-Weinberg equilibrium (KIR3DS1 homozygote) in HESN individuals. The reciprocal equilibrium between inhibitory and activatory NK receptors and their ligands favors NK activation in HESN individuals.     

Single-cell analysis of the human NK cell response to missing self and its inhibition by HLA class I

Natural killer (NK) cells activate quickly in response to pathogens, tumors, and allogeneic hematopoietic cell transplants. Modulating the NK cell response are clonally distributed NK cell receptors that survey cells for change in the expression of major histocompatibility complex (MHC) class I and structurally related ligands. Here the enzyme-linked immunospot (ELISPOT) assay, intracellular cytokine staining (ICS), and short-term culture were used to quantify the response of bulk NK cell populations from human donors to HLA class I-deficient 221 cells and to 221 cells transfected with single HLA class I allotypes. NK cells in cultures containing interleukin-2 (IL-2) or IL-12 exhibited specificities of HLA class I-mediated inhibition that correlated well with those previously defined using NK cell clones in long-term culture and with the frequencies of cells expressing particular inhibitory HLA class I receptors. Culture with IL-12, but not IL-2, gave an increased frequency of cells expressing CD94: NKG2A but no change in killer immunoglobulin-like receptor (KIR) expression. For some heterozygote combinations of KIR3DL1 alleles, ICS can be used to compare the functional properties of the 2 allotypes. Thus, both the low-expressing KIR3DL1*005 and the high-expressing KIR3DL1*002 gave similar inhibitory response on challenge with an HLA-B*5801 ligand. The single-cell assays developed here should facilitate future population study and clinical analysis of human NK cell regulation by MHC class I.     

Expansion and enhanced survival of natural killer cells expressing the killer immunoglobulin‐like receptor KIR3DL2 in spondylarthritis

Objective

The spondylarthritides (SpA) are strongly associated with possession of HLA–B27. We hypothesized that the expression of abnormal forms of HLA–B27 in SpA may have a pathogenic role through interaction with cells bearing natural killer (NK) receptors, in particular, killer immunoglobulin‐like receptor (KIR) KIR3DL2, a receptor for HLA–B27 homodimer (B27₂). We therefore undertook the present study to determine the number and function of NK and T cells bearing KIR3DL2 in SpA.

Methods

Expression of KIR3DL2 on NK and T cells was quantified in peripheral blood (PB) from 35 patients with SpA and 5 patients with juvenile enthesitis‐related arthritis (juvenile ERA); samples were compared with samples from healthy and rheumatoid arthritis (RA) controls. Paired synovial fluid (SF) was studied where available. Expression of other KIRs as well as activation, memory, and homing markers on KIR3DL2+ NK and T cells was quantified. NK cell survival was assessed using the apoptotic markers annexin V and 7‐aminoactinomycin D, and cytotoxicity by ⁵¹Cr release assay.

Results

In SpA, an increased number of PB and SF NK and CD4+ T cells expressed the KIR3DL2 receptor compared with controls. In ERA, KIR3DL2 expression was increased in PB and SF CD4 T cells (and SF NK cells) compared with RA controls. KIR3DL2+ NK cells had an activated phenotype, and were protected from apoptosis by culture with a cell line expressing B27₂. SpA PB mononuclear NK cells from SpA patients showed greater cytotoxicity than those from controls.

Conclusion

KIR3DL2 expression on NK cells and CD4 lymphocytes is increased in SpA and ERA. These cells are activated and may have a pathogenic role.

     

TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16- NK cells with similarities to decidual NK cells

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells, when isolated and cultured in vitro, were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition, Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor.     

BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIRs)

Natural killer (NK) cells decrease in function during chronic myelogenous leukemia (CML) progression from chronic phase to blast crisis, and they can become BCR/ABL(+) late in the disease course. To study this altered function, NK92 cells were transduced with the BCR/ABL oncogene. In contrast to the parental cells, which died when deprived of interleukin 2 (IL-2), p210(+) NK92 cells proliferated and survived indefinitely in the absence of IL-2. BCR/ABL also decreased the natural cytotoxicity of NK92 cells against K562 targets, without affecting IL-2, interferon gamma (IFN-gamma), or tumor necrosis factor alpha (TNF-alpha) production. Although the ABL-specific tyrosine kinase inhibitor imatinib mesylate (STI-571) had no effect on parental NK92 cells, it markedly decreased the growth and survival of IL-2-independent p210(+) NK92 cells. In contrast to the parental cell line, serial analysis of p210(+) NK92 cells detected small populations that clonally expressed one or more killer immunoglobulin-like receptors (KIRs). Unlike the decreased natural cytotoxicity, the function of the activating CD158j receptor remained intact. Southern blotting and hybridization with an enhanced green fluorescence protein (eGFP) probe showed that KIR(-) and KIR(+) NK92 cells were all derived from the same clone, suggesting that KIR acquisition remains dynamic at the maturational stage represented by the NK92 cell line. When tested in primary CD56(+bright) NK cells, p210 induced partial IL-2-independent growth and increased KIR expression similar to findings in NK92 cells. This is the first study to show that BCR/ABL, well known for its effects on the myeloid lineage, can alter the function of lymphoid cells, which may be associated with the defect in innate immunity associated with CML progression.     

Two opposite signaling outputs are driven by the KIR2DL1 receptor in human CD4+ T cells

Inhibitory killer Ig-like receptors (KIR), expressed by human natural killer cells and effector memory CD8(+) T-cell subsets, bind HLA-C molecules and suppress cell activation through recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). To further analyze the still largely unclear role of inhibitory KIR receptors on CD4(+) T cells, KIR2DL1 transfectants were obtained from a CD4(+) T-cell line and primary cells. Transfection of CD4(+) T cells with KIR2DL1 dramatically increased the T-cell receptor (TCR)-induced production of interleukin-2 independently of ligand binding but inhibited TCR-induced activation after ligation. KIR-mediated costimulation of TCR activation involves intact KIR2DL1-ITIM phosphorylation, SHP-2 recruitment, and PKC- phosphorylation. Synapses leading to activation were characterized by an increase in the recruitment of p-Tyr, SHP-2, and p-PKC-, but not of SHP-1. Interaction of KIR2DL1 with its ligand led to a strong synaptic accumulation of KIR2DL1 and the recruitment of SHP-1/2, inhibiting TCR-induced interleukin-2 production. KIR2DL1 may induce 2 opposite signaling outputs in CD4(+) T cells, depending on whether the KIR receptor is bound to its ligand. These data highlight unexpected aspects of the regulation of T cells by KIR2DL1 receptors, the therapeutic manipulation of which is currently being evaluated.     

Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells

Cellular inactivation through killer immunoglobulin-like receptors (KIRs) may allow neoplastic cells to evade host natural killer (NK) cell-mediated immunity. Recently, alloreactive NK cells were shown to mediate antileukemic effects against acute myelogenous leukemia (AML) after mismatched transplantation, when KIR ligand incompatibility existed in the direction of graft-versus-host disease (GVHD). Therefore, we investigated whether solid tumor cells would have similar enhanced susceptibility to allogeneic KIR-incompatible NK cells compared with their KIR-matched autologous or allogeneic counterparts. NK populations enriched and cloned from the blood of cancer patients or healthy donors homozygous for HLA-C alleles in group 1 (C-G1) or group 2 (C-G2) were tested in vitro for cytotoxicity against Epstein-Barr virus-transformed lymphoblastic cell lines (EBV-LCLs), renal cell carcinoma (RCC), and melanoma (MEL) cells with or without a matching KIR-inhibitory HLA-C ligand. Allogeneic NK cells were more cytotoxic to tumor targets mismatched for KIR ligands than their KIR ligand-matched counterparts. Bulk NK populations (CD3(-)/CD2(+)/CD56(+)) expanded 10(4)-fold from patients homozygous for C-G1 or C-G2 had enhanced cytotoxicity against KIR ligand-mismatched tumor cells but only minimal cytotoxicity against KIR ligand-matched targets. Further, NK cell lines from C-G1 or C-G2 homozygous cancer patients or healthy donors expanded but failed to kill autologous or KIR-matched MEL and RCC cells yet had significant cytotoxicity (more than 50% lysis at 20:1 effector-target [E/T] ratio) against allogeneic KIR-mismatched tumor lines. These data suggest immunotherapeutic strategies that use KIR-incompatible allogeneic NK cells might have superior antineoplastic effects against solid tumors compared with approaches using autologous NK cells.     

From the Cover: Peptide antagonism as a mechanism for NK cell activation

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.     

Differential effects of interleukin-3, interleukin-7, interleukin 15, and granulocyte-macrophage colony-stimulating factor in the generation of natural killer and B cells from primitive human fetal liver progenitors

The regulatory roles of a number of early-acting growth factors on the generation of natural killer (NK) cells and B cells from primitive progenitors were studied. Experiments focused on the contributions of granulocyte-macrophage colony-stimulates factor (GM-CSF) and interleukin-3 (IL-3) to the regulation of the early events of lymphopoiesis.Two progenitor populations isolated from human fetal liver were studied, CD38(-)CD34(++)lineage(-) (Lin(-)) cells (candidate hematopoietic stem cells [HSCs]) and the more mature CD38(+)CD34(++)Lin(-) cells. The effects of different cytokines on the generation of CD56(+)CD3(-) NK cells and CD19(+) B cells were studied in serum-deprived cultures in the absence of stroma.NK cells generated in vitro were able to kill NK-sensitive target cells, expressed NK-associated marker CD161 (NKR-P1A), but exhibited little or no expression of CD2, CD8, CD16, CD94/NKG2A, or killer cell inhibitory receptors (KIRs). Among the cytokine combinations tested, kit ligand (KL) and IL-15 provided the best conditions for generating CD56(+) NK cells from CD38(+)CD34(++)Lin(-) cells. However, either flk-2/flt3 ligand (FL), GM-CSF, IL-3, or IL-7 could partially substitute KL. All of these cytokines also supported the growth of NK-cell progenitors from candidate HSC, with the combination of IL-15, KL, GM-CSF, and FL generating the greatest number of CD56(+) cells. B cells were generated from both progenitor populations in response to the combined effects of KL, FL, and IL-7. Both B and NK cells were generated with the further addition of IL-15 to these cultures. The in vitro generated B cells were CD10(+), CD19(+), HLA-DR(+), HLA-DQ(+), and some were CD20(+), but no cytoplasmic or surface immunoglobulin M expression was observed. In contrast with NK lymphopoiesis, GM-CSF, IL-3, and IL-15 had no effect on the generation of B cells from CD38(-)CD34(++)Lin(-) cells, and GM-CSF inhibited B-cell generation from CD38(+)CD34(++)Lin(-) progenitors. These findings indicate a differential regulation of NK and B lymphopoiesis beginning in the early stages of hematopoiesis as exemplified by the distinctive roles of IL-7, IL-15, GM-CSF, and IL-3.     

Co-evolution of KIR2DL3 with HLA-C in a human population retaining minimal essential diversity of KIR and HLA class I ligands

Natural killer (NK) cells contribute to immunity and reproduction. Guiding these functions, and NK cell education, are killer cell Ig-like receptors (KIR), NK cell receptors that recognize HLA class I. In most human populations, these highly polymorphic receptors and ligands combine with extraordinary diversity. To assess how much of this diversity is necessary, we studied KIR and HLA class I at high resolution in the Yucpa, a small South Amerindian population that survived an approximate 15,000-year history of population bottleneck and epidemic infection, including recent viral hepatitis. The Yucpa retain the three major HLA epitopes recognized by KIR. Through balancing selection on a few divergent haplotypes the Yucpa maintain much of the KIR variation found worldwide. HLA-C*07, the strongest educator of C1-specific NK cells, has reached unusually high frequency in the Yucpa. Concomitantly, weaker variants of the C1 receptor, KIR2DL3, were selected and have largely replaced the form of KIR2DL3 brought by the original migrants from Asia. HLA-C1 and KIR2DL3 homozygosity has previously been correlated with resistance to viral hepatitis. Selection of weaker forms of KIR2DL3 in the Yucpa can be seen as compensation for the high frequency of the potent HLA-C*07 ligand. This study provides an estimate of the minimal KIR-HLA system essential for long-term survival of a human population. That it contains all functional elements of KIR diversity worldwide, attests to the competitive advantage it provides, not only for surviving epidemic infections, but also for rebuilding populations once infection has passed.     

Immunological changes in different patient populations with chronic hepatitis C virus infection

AIM: To investigate killer inhibitory and activating receptor expression by natural killer(NK), natural killer T-like(NKT-like) and CD8+ T lymphocytes in patients with chronic hepatitis C virus(HCV) infection with elevated and with persistently normal alanine aminotransferase(PNALT).METHODS: The percentage of peripheral blood Treg cells, KIR2DL3, ILT-2, KIR3DL1, CD160, NKG2 D, NKG2 C expressing NK, T and NKT-like cells, cytokine production and NK cytotoxicity were determined by flow cytometry. Twenty-one patients with chronic HCV infection with elevated alanine aminotransferase, 11 HCV carriers with persistently normal alanine aminotransferase and 15 healthy volunteers were enrolled. RESULTS: No significant differences were observed in the percentage of total T, NK or NKT-like cells between study groups. Comparing the activating and inhibitoryreceptor expression by NK cells obtained from HCV carriers with PNALT and chronic HCV hepatitis patients with elevated alanine aminotransferase, NKG2 D activating receptor expression was the only receptor showing a significant difference. NKG2 D expression of NK cells was significantly lower in patients with elevated alanine aminotransferase. The expression of CD160, NKG2 D and NKG2 C activating receptor by CD8+ T cells were significantly lower in patients with chronic HCV hepatitis than in healthy controls and in HCV carriers with PNALT. Plasma TGF-β1 levels inversely correlated with NKG2 D expression by NK cells. In vitro TGF-β1 treatment inhibited NK cells cytotoxic activity and downregulated NKG2 D expression. CD8+ T cells from HCV carriers with PNALT showed significantly elevated expression of CD160, NKG2 D and NKG2 C activating receptors compared to chronic HCV patients with elevated alanine aminotransferase. Enhanced expression of inhibitory KIR2DL3 receptor, and decreased ILT-2 expression on NK cells were also found in chronic hepatitis C patients compared to healthy controls.CONCLUSION: Our study demonstrated a complex dysregulation of activating and inhibitory receptor expression, such as decreased NKG2 D and CD160 activating receptor expression and increased KIR2DL3 inhibitory receptor expression by NK and cytotoxic T cells and may provide further mechanism contributing to defective cellular immune functions in chronic hepatitis C. Increased NKG2 D receptor expression in HCV patients with persistently normal ALT suggests an important pathway for sustaining NK and CD8 T cell function and a protective role against disease progression.     

Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that, in most transplantation patients, variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype, as well as through phenotypic and functional analyses of NK cells, both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether, these results may have important clinical implications for the selection of optimal donors for haplo-HSCT.     

Functional inhibitory human leucocyte antigen class I receptors on natural killer (NK) cells in patients with chronic NK lymphocytosis

Chronic natural killer (NK) lymphocytosis is a rare disorder characterized by an indolent clinical course. Despite high NK cell numbers, many patients present with only mild clinical symptoms, and are often asymptomatic. NK cells are equipped with a range of receptors that bind human leucocyte antigen (HLA)-class I molecules. The killer immunoglobulin-like receptors (KIR, CD158) bind groups of HLA alleles, the CD94/NKG2 receptors bind HLA-E, and the CD85j (ILT2, LIR-1) receptor binds to the relatively non-polymorphic alpha3 domain of HLA molecules. Inhibitory HLA class I receptors silence NK cells against cells expressing normal levels of HLA class I. Analysis of NK cells in six patients with chronic NK lymphocytosis revealed a high level of the inhibitory CD94/NKG2A receptor on all NK cells. In four patients, KIR were absent, in one patient a single KIR was expressed in the absence of self-ligand, and in one patient CD85j and multiple KIR were expressed. Cytotoxicity assays demonstrated that all HLA class I receptors were functional. The ability of monoclonal antibodies to block the receptors and allow killing of autologous target cells established that both receptor and ligand expression were adequate for inhibitory function. We propose that the silent behaviour of NK cells in patients with chronic NK lymphocytosis is due to effective inhibitory HLA class I receptors.     

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL)

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.     

HLA alleles determine differences in human natural killer cell responsiveness and potency

Epidemiological studies have associated certain human disease outcomes with particular killer cell Ig-like receptor (KIR) and HLA genotypes. However, the functional explanation for these associations is poorly understood, because the KIRs were initially described as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. Yet resolution of infections is often associated with genotypic pairing of inhibitory KIRs with their cognate HLA ligands. Recent studies in mice indicate a second role for MHC-specific inhibitory receptors, i.e., self-MHC recognition confers functional competence on the NK cell to be triggered through their activation receptors, a process termed licensing. As a result, licensed NK cells with self-MHC-specific receptors are more readily activated as compared with unlicensed NK cells without self-MHC-specific receptors. Such results predict that human NK cells may undergo a similar process. Here, we examined the human NK cell subset expressing KIR3DL1, the only known KIR specific for HLA-Bw4 alleles. The KIR3DL1(+) subset in normal donors with two HLA-B-Bw4 genes displayed increased responsiveness to tumor stimulation compared with the KIR3DL1(+) subset from individuals with only one or no Bw4 genes. By contrast, NK cells lacking KIR3DL1 showed no differences. Therefore, these data indicate that particular KIR and HLA alleles are associated with more responsive NK cells, strongly suggesting that human NK cells are also subjected to NK cell licensing, and providing a potential functional explanation for the influence of KIR and HLA genes in disease as well as interindividual differences in NK cell potency.