MHC Class I-Dependent Shaping of the NK Cell Ly49 Receptor Repertoire Takes Place Early during Maturation in the Bone Marrow.

MHC class I (MHC I) expression in the host influences NK cells in a process termed education. The result of this education is reflected in the responsiveness of NK cells at the level of individual cells as well as in the repertoire of inhibitory MHC I-specific receptors at the NK cell system level. The presence of MHC I molecules in the host environment gives rise to a skewed receptor repertoire in spleen NK cells where subsets expressing few (one or two) inhibitory receptors are expanded whereas subsets with many (three or more) receptors are contracted. It is not known whether this MHC I-dependent skewing is imposed during development or after maturation of NK cells. In this study, we tested the hypothesis that the NK cell receptor repertoire is shaped already early during NK cell development in the bone marrow. We used mice with a repertoire imposed by a single MHC I allele, as well as a C57BL/6 mutant strain with exaggerated repertoire skewing, to investigate Ly49 receptor repertoires at different stages of NK cell differentiation. Our results show that NK cell inhibitory receptor repertoire skewing can indeed be observed in the bone marrow, even during the earliest developmental steps where Ly49 receptors are expressed. This may partly be accounted for by selective proliferation of certain NK cell subsets, but other mechanisms must also be involved. We propose a model for how repertoire skewing is established during a developmental phase in the bone marrow, based on sequential receptor expression as well as selective proliferation.

NK- and T-cell subsets in malignant mesothelioma patients: Baseline pattern and changes in the context of anti-CTLA-4 therapy.

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK-cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti-CTLA-4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL-15 stimulation improved NK cell-mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56bright and CD56dim NK subsets and increased serum concentrations of the cytokines IL-10, IL-8 and TNF-α. After tremelimumab treatment, the ratio between the CD56bright and CD56dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM-3+ CD8+ T-cell frequency, high DNAM-1+ CD56dim NK-cell frequency and high expression levels of NKp46 on the CD56dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.

Dynamic Regulation of NK Cell Responsiveness.

Natural killer (NK) cells deliver cytotoxic granules and immunomodulatory cytokines in response to transformed and virally infected cells. NK cell functions are under the control of a large number of germline-encoded receptors that recognize various ligands on target cells, but NK cells also respond to cytokines in the surrounding environment. The interaction between NK cell receptors and their ligands delivers either inhibitory or activating signals. The cytokine milieu further shapes NK cell responses, either directly or by influencing the way inhibitory or activating signals are perceived by NK cells. In this review, we discuss how NK cell function is controlled by inhibitory receptors and MHC-I molecules, how activating receptors contribute to NK cell education, and finally, how cytokines secreted by the surrounding cells affect NK cell responsiveness. Inputs at these three levels involve different cell types and are seamlessly integrated to form a functional NK cell population.

A genetic defect in mice that impairs missing self recognition despite evidence for normal maturation and MHC class I-dependent education of NK cells.

In studies of a CD1d1-deficient mouse strain, we unexpectedly observed a severely impaired capacity for NK cell-mediated rejection of MHC class I-deficient (spleen or tumor) cells. Studies of another CD1-defective strain, as well as intercrosses with C57BL/6 mice, indicated that the impaired missing self rejection (IMSR) NK cell defect was a recessive trait, independent from the targeted CD1 locus. Studies with mixed bone marrow chimeras indicated that the defect is intrinsic to NK cells. The IMSR mice had normal proportions of NK cells, displaying a typical cell surface phenotype, as evaluated using a panel of Abs to developmental markers and known receptors. The impaired missing self recognition could not be overcome through cytokine stimulation. There was also an impaired capacity with respect to NKG2D-dependent cytotoxicity, whereas the mice exhibited normal Ly49D/DAP12-dependent responses in vivo and in vitro. The NK cell system of IMSR mice showed two hallmarks of MHC-dependent education: skewing of the Ly49 receptor repertoire and differential in vitro responsiveness between NK cells with and without inhibitory receptors for self-MHC ("licensing"). We conclude that these mice have a recessive trait that perturbs the missing self reaction, as well as NKG2D-dependent responses, whereas other aspects of the NK system, such as development, capacity to sense MHC molecules during education, and Ly49D/DAP12-dependent responses, are largely intact.

Androgens Modulate the Immune Profile in a Mouse Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is associated with a low-grade inflammation, but it is unknown how hyperandrogenism, the hallmark of PCOS, affects the immune system. Using a PCOS-like mouse model, it is demonstrated that hyperandrogenism affects immune cell populations in reproductive, metabolic, and immunological tissues differently in a site-specific manner. Co-treatment with an androgen receptor antagonist prevents most of these alterations, demonstrating that these effects are mediated through androgen receptor activation. Dihydrotestosterone (DHT)-exposed mice displayed a drastically reduced eosinophil population in the uterus and visceral adipose tissue (VAT). A higher frequency of natural killer (NK) cells and elevated levels of IFN-γ and TNF-α are seen in uteri of androgen-exposed mice, while NK cells in VAT and spleen displayed a higher expression level of CD69, a marker of activation or tissue residency. Distinct alterations of macrophages in the uterus, ovaries, and VAT are also found in DHT-exposed mice and can potentially be linked to PCOS-like traits of the model. Indeed, androgen-exposed mice are insulin-resistant, albeit unaltered fat mass. Collectively, it is demonstrated that hyperandrogenism causes tissue-specific alterations of immune cells in reproductive organs and VAT, which can have considerable implications on tissue function and contribute to the reduced fertility and metabolic comorbidities associated with PCOS.

In vivo tumor cell rejection induced by NK cell inhibitory receptor blockade: maintained tolerance to normal cells even in the presence of IL-2.

Missing-self-reactivity can be mimicked by blocking self-specific inhibitory receptors on NK cells, leading to increased rejection of syngeneic tumor cells. Using a mouse model, we investigated whether Ab-mediated blocking of inhibitory receptors, to a degree where NK cells rejected syngeneic tumor cells, would still allow self-tolerance toward normal syngeneic cells. Ly49C/I inhibitory receptors on C57BL/6 (H-2(b)) NK cells were blocked with F(ab')(2) fragments of the mAb 5E6. Inhibitory receptor blockade in vivo caused rejection of i.v. inoculated fluorescence-labeled syngeneic lymphoma line cells but not of syngeneic spleen cells, BM cells or lymphoblasts. The selective rejection of tumor cells was NK cell-dependent and specifically induced by Ly49C/I blockade. Moreover, selective tumor rejection was maintained after treatment with 5E6 F(ab')(2) for 9 wk, arguing against the induction of NK cell anergy or autoreactivity during this time. Combination therapy using 5E6 F(ab')(2) together with high dose IL-2 treatment further increased lymphoma cell rejection. In addition, combination therapy reduced growth of melanoma cell line tumors established by s.c. inoculation 3 days before start of treatment. Our results demonstrate that inhibitory receptor blockade does not result in attack on normal cells, despite potent reactivity against MHC class I-expressing tumors.

Accumulation and activation of natural killer cells in local intraperitoneal HIV-1/MuLV infection results in early control of virus infected cells.

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo.

NK cell activation by KIR-binding antibody 1-7F9 and response to HIV-infected autologous cells in viremic and controller HIV-infected patients.

Natural killer (NK) cells may be protective in HIV infection and are inhibited by killer cell immunoglobulin-like receptors (KIRs) interacting with MHC class I molecules, including HLA-C. Retention of HLA-C despite downregulation of other MHC class I molecules on HIV infected cells might protect infected cells from NK cell recognition in vitro. To assess the role of inhibitory HLA-C ligands in the capacity of NK cells to recognize autologous infected T cells, we measured NK cell degranulation in vitro in viremic patients, controllers with low viremia, and healthy donors. No difference in NK cell response to uninfected compared to HIV-1(IIIB) infected targets was observed. Activation of NK cells was regulated by KIRs, because NK cell degranulation was increased by 1-7F9, a human antibody that binds KIR2DL1/L2/L3 and KIR2DS1/S2, and this effect was most pronounced in KIR haplotype B individuals.

NK cells control breast cancer and related cancer stem cell hematological spread.

The growth and recurrence of a number of cancers is driven by a scarce population of cancer stem cells (CSCs), which are resistant to most current therapies. It has been shown previously that natural killer (NK) cells recognize human glioma, melanoma, colon and prostate CSCs in vitro. We herein show that human and mouse breast CSCs are also susceptible to NK cytotoxic activity in vitro. Moreover, CSC induced autologous NK cell activation and expansion in vivo, which correlate with the inhibition of CSC metastatic spread. These data suggest that NK cells control CSC metastatic spread in vivo and that their use in breast cancer therapy may well be fruitful.

Computational modeling of human natural killer cell development suggests a selection process regulating coexpression of KIR with CD94/NKG2A.

Natural killer cells fail to lyse target cells expressing sufficient levels of self MHC class I molecules, providing one mechanism to secure self tolerance. Inhibition of lysis is mediated by inhibitory receptors expressed by NK cells, such as the murine Ly49 receptors, human KIR receptors and CD94/NKG2A, expressed by both species. To ensure that most, if not all, NK cells express at least one inhibitory receptor for self MHC class I, selection processes have been postulated for murine NK cells regulating the number and identity of inhibitory receptors expressed by each cell. The presence of similar selection processes in human NK cells has not been demonstrated. In previous studies using mathematical modeling we have shown that, in the Ly49 system, the sequential model (in which gene expression and selection operate simultaneously) is most likely to explain the observed expression frequencies. We also predicted the parameters (such as receptor-ligand binding affinity levels) under which the models fit with the observed frequencies. This study aims to evaluate whether these models may be valid in the human system. Our data suggest that if selection operates during human NK cell development, it affects the co-expression of CD94/NKG2A and KIR rather than KIR expression alone, and is more likely to be governed by the two-step selection model.

Natural killer cell tolerance persists despite significant reduction of self MHC class I on normal target cells in mice.

BACKGROUND: A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8(+) T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached. CONCLUSION: Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.

Probing natural killer cell education by Ly49 receptor expression analysis and computational modelling in single MHC class I mice.

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for "missing self" recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an "educating impact" on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.

The dynamics of natural killer cell tolerance.

Natural killer (NK) cells are important mediators of resistance against tumor growth and metastasis. NK cell reactivity is regulated by a balance of signals from activating and inhibitory receptors. While reactivity against tumor cells is beneficial, it is essential that NK cells do not attack normal tissue. The distinction between tumor cells and normal cells is partly made at the level of activating receptors: transformation often results in induction of ligands for such receptors. In addition, NK cells discriminate self from non-self using MHC class I-binding inhibitory receptors. Host MHC class I molecules regulate development of NK cell reactivity and tolerance, a process that is not well understood. Recent data suggest that functional maturation may not be a binary phenomenon: quantitative aspects, with regards to avidity and frequency in interactions between developing NK cells and normal cells, may be important for the generation of NK cells that are 'tuned' to optimally sensing the absence of self-MHC class I. In this article, we discuss models for development of NK cell reactivity and tolerance. Our understanding of this process may have significant implications for the use of NK cells in cancer therapy.

Mapping of quantitative trait loci determining NK cell-mediated resistance to MHC class I-deficient bone marrow grafts in perforin-deficient mice.

NK cells reject allogeneic and MHC class I-deficient bone marrow (BM) grafts in vivo. The mechanisms used by NK cells to mediate this rejection are not yet thoroughly characterized. Although perforin plays a major role, perforin-independent mechanisms are involved as well. C57BL/6 mice deficient in perforin (B6 perforin knockout (PKO)) reject class I-deficient TAP-1 KO BM cells as efficiently as normal B6 mice. In contrast, perforin-deficient 129S6/SvEvTac mice (129 PKO) cannot mediate this rejection while normal 129 mice efficiently reject. This suggests that in 129, but not in B6, mice, perforin is crucial for NK cell-mediated rejection of MHC class I-deficient BM grafts. To identify loci linked to BM rejection in perforin-deficient mice, we generated backcross 1 progeny by crossing (129 x B6)F(1) PKO mice to 129 PKO mice. In transplantation experiments, >350 backcross 1 progeny were analyzed and displayed a great variation in ability to reject TAP-1 KO BM grafts. PCR-based microsatellite mapping identified four quantitative trait loci (QTL) on chromosomes 2, 4, and 8, with the QTL on chromosome 8 showing the highest significance, as well as a fifth epistatic QTL on chromosome 3. This study describes the first important step toward identifying BM graft resistance gene(s).

Synthesis and characterization of molybdenum oxo complexes of two tripodal ligands: reactivity studies of a functional model for molybdenum oxotransferases.

Reaction of the tetradentate ligand N-(2-hydroxybenzyl)-N,N-bis(2-pyridylmethyl)amine (L-OH) with MoO2Cl2 in methanol in the presence of NaOMe and PF6- results in the formation of [MoO2(L-O)]PF6. Similarly, the reaction of N-(2-mercaptobenzyl)-N,N-bis(2-pyridylmethyl)amine (L-SH) with MoO2(acac)2 leads to the formation of [MoO2(L-S)]+. The dioxo-molybdenum complex [MoO2(L-O)]+ reacts with phosphines in methanol to afford phosphine oxides and an air-sensitive molybdenum complex, tentatively identified as [Mo(IV)O(L-O)(OCH3)]. The latter complex is capable of reducing biological oxygen donors such as DMSO or nitrate, thereby mimicking the activity of DMSO reductase and nitrate reductase. Reaction of [MoO2(L-O)]PF6 with PPh3 in other solvents than methanol leads to the formation of the Mo(V) dimer [(L-O)OMo(micro-O)MoO(L-O)](PF6)2. The crystal structures of [MoO2(L-O)]PF6 and the micro-oxo bridged dimer are presented.

Tetrakis(dimethyl sulfide)palladium(II) bis(tetrafluoroborate) and tetrakis(1,4-oxathiane-kappaS)palladium(II) bis(tetrafluoroborate).

Tetrakis(dimethyl sulfide)palladium(II) bis(tetrafluoroborate), [Pd(C(2)H(6)S)(4)](BF(4))(2), (I), and tetrakis(1,4-oxathiane-kappaS)palladium(II) bis(tetrafluoroborate), [Pd(C(4)H(8)OS)(4)](BF(4))(2), (II), both crystallize as mononuclear square-planar complexes with tetrafluoroborate as the counter-ions. The Pd atom accepts four S-donor atoms and is positioned at an inversion centre in both compounds. The two unique S atoms in the dimethyl sulfide complex, (I), are disordered. The Pd-S distances are in the range 2.3338(12)-2.3375(12)A in (I), and the corresponding distances in the thioxane complex, (II), are 2.3293(17) and 2.3406(17)A. The anions in both compounds interact weakly with the Pd atom.

Structures of trans-[PtCl(2)(PBz(3))(2)], trans-[PtI(2)(PBz(3))(2)], trans-[Pt(NCS)(2)(PBz(3))(2)].0.5C(6)H(6) and trans-[PdI(2)(PBz(3))(2)].

A series of structures of trans-[MX(2)(PBz(3))(2)] [M = Pt, X = Cl(-); PBz(3) = tribenzylphosphine (1), I(-), trans-diiodobis(tribenzylphosphine)platinum(II) (2), and NCS(-), trans-di(thiocyanate)bis(tribenzylphosphine)platinum(II) (3); M = Pd, X = I(-), trans-diiodobis(tribenzylphosphine)palladium(II) (4)] have been characterized by X-ray crystallography. In all compounds each tribenzylphosphine has one benzylcarbon close to the coordination plane. In (1), (2) and (4) those (in-plane) C atoms, from the two different PBz(3), exhibit an anti conformation along the P-P axis, while (3) has the gauche conformation. Root mean square (RMS) calculations and half-normal probability plots show that the complexes in (2) and (4) are very similar and the only significant differences between them are the M-P bonds, 2.354 (4) and 2.330 (5) A, and the M-I bond distances, 2.604 (1) and 2.611 (2) A, for Pd and Pt, respectively. Calculations of the steric demand of the PBz(3) ligands based on the Tolman model gave values ranging from 155 to 178 degrees for the effective and 156 to 179 degrees for the Tolman angles, respectively.

Structural and mechanistic look at the orthoplatination of aryl oximes by dichlorobis(sulfoxide or sulfide)platinum(II) complexes.

Structural and mechanistic aspects of orthoplatination of acetophenone and benzaldehyde oximes by the platinum(II) sulfoxide and sulfide complexes [PtCl(2)L(2)] (2, L = SOMe(2) (a), rac-SOMePh (b), R-SOMe(C(6)H(4)Me-4) (c), and SMe(2) (d)) to afford the corresponding platinacycles cis-(C,S)-[Pt(II)(C(6)H(3)-2-CR'=NOH-5-R)Cl(L)] (3, R, R' = H, Me) have been investigated. The reaction of acetophenone oxime with sulfoxide complex 2a in methanol solvent occurs noticeably faster than with sulfide complex 2d due to the fact that the sulfoxide is a much better platinum(II) leaving ligand than the sulfide. Evidence is presented that the orthoplatination is a multistep process. The formation of unreactive dichlorobis(N-oxime)platinum(II) cations accounts for the rate retardation by excess acetophenone oxime and suggests the importance of pseudocoordinatively unsaturated species for the C-H bond activation by Pt(II). A comparative X-ray structural study of dimethyl sulfoxide platinacycle 3b (R = R' = Me) and its sulfide analogue 3e (R = H, R' = Me), as well as of SOMePh complex 3c (R = H, R' = Me), indicated that they are structurally similar and a sulfur ligand is coordinated in the cis position with respect to the sigma-bound phenyl carbon. The differences concern the Pt-S bond distance, which is notably longer in the sulfide complex 3e (2.2677(11) A) as compared to that in sulfoxide complexes 3b (2.201(2)-2.215(2) A) and 3c (2.2196(12) A). Whereas the metal plane is practically a plane of symmetry in 3b due to the H-bonding between the sulfoxide oxygen and the proton at carbon ortho to the Pt-C bond, an S-bonded methyl of SOMePh and SMe(2) is basically in the platinum(II) plane in complexes 3c and 3e, respectively. There are intra- and intermolecular hydrogen bond networks in complex 3b. An interesting structural feature of complex 3c is that the two independent molecules in the asymmetric unit of the crystal reveal an extremely short Pt-Pt contact of 3.337 A.