Increased Susceptibility of the CD57- NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57-NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57- NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients.

Regional lymph nodes (LN)s represent important immunological barriers in spreading of malignant tumors. However, they are the most frequent early metastatic site in melanoma. Immunomodulatory agents including cytokines have been included in therapy of melanoma and have shown severe side effects and toxicity. In this sense, there is a growing need for bringing these agents to further in vitro testing that may enlighten aspects of their regional application. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, the two cytokines with similar immune-enhancing effects, on the expression of activating NKG2D, inhibitory CD158a and CD158b receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients. In this study, we showed significant effects of IL-2 and IL-15 cytokine treatments on the expression of activating NKG2D and on inhibitory CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cell lymphocyte subsets originating from regional LNs of melanoma patients. Furthermore, IL-2 and IL-15 by inducing the expression of NKG2D activating receptor on innate and on adaptive lymphocyte subsets and by augmenting NK cell antitumor cytotoxicity that correlated with the cytokine-induced NKG2D expression, increased antitumor potential of immune cells in regional LNs of melanoma patients irrespective of LN involvement. These findings indicate the importance of immune cell population from regional LNs of melanoma patients in the development of immune intervention strategies that may if applied locally increase antitumor potential to the level that controls tumor progressions.

Tissue-Specific Education of Decidual NK Cells.

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self-HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK.

Amplified NKG2C+ NK cells in cytomegalovirus (CMV) infection preferentially express killer cell Ig-like receptor 2DL: functional impact in controlling CMV-infected dendritic cells.

CMV infection represents a major complication in hematopoietic stem cell transplantation, which compromises graft outcome. Downregulation of HLA class I expression is one mechanism by which CMV evades T cell-mediated immune detection, rendering infected cells vulnerable to killer cell Ig-like receptor (KIR)(+) NK cells. In this study, we observed that the amplified NKG2C(+) NK cell population observed specifically in CMV seropositive individuals mainly expressed KIR2DL receptors. We have shown that HLA class I expression was downregulated on CMV-infected immature dendritic cells (iDCs), which escape to HLA-A2-pp65-specific T lymphocytes but strongly trigger the degranulation of KIR2D(+) NK cells. CMV infection conferred a vulnerability of C2C2(+) iDCs to educated KIR2DL1(+) and KIR2DL3(+) NK cell subsets. Alloreactivity of KIR2DL1(+) NK cell subsets against C1C1(+) iDCs was maintained independently of CMV infection. Unexpectedly, CMV-infected C1C1(+) iDCs did not activate KIR2DL3(+) NK cell reactivity, suggesting a potential CMV evasion to KIR2DL3 NK cell recognition. Altogether, the coexpression of KIR and NKG2C on expanded NK cell subsets could be related to a functional contribution of KIR in CMV infection and should be investigated in hematopoietic stem cell transplantation, in which the beneficial impact of CMV infection has been reported on the graft-versus-leukemia effect.

Investigation of NK cell function and their modulation in different malignancies.

NK cells have become a subject of investigation not only in the field of tumor immunology and infectious diseases, but also within all aspects of immunology, such as transplantation, autoimmunity, and hypersensitivity. Our early studies aside from investigating NK cell activity in experimental animals and humans included studies of perforin expression and modulation in this lymphocyte subset. As NK cell activity is modified by their environment, we showed clinical stage-dependent impairment of their activity and in vitro effect of different sera, Th1 cytokines, and their combination in breast cancer, Hodgkin's disease, and non-Hodgkin's lymphoma patients, especially with respect to metabolic and cell membrane changes of peripheral blood lymphocytes evaluated by spontaneous release of the enzyme lactate dehydrogenase (LDH) that led to the correction of the LDH enzyme release assay for natural cytotoxicity. By long-term immuno-monitoring of patients with malignancies, we also showed the kinetics of NK cell modulation during chemo-immunotherapy. In our more recent studies, we give data of NK function and novel families of NK cell receptor expression in healthy individuals that may be of help in NK cell profiling, by giving referent values of basic and cytokine-induced expression of some NK cell receptors either in evaluation of disease or in immuno-monitoring during cytokine therapy of patients with malignancies. Moreover, we give novel aspects of modulation of NK cell activity by cytokines approved for immunotherapy, IFN and IL-2, in melanoma and other malignancies with respect to alterations in new activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptor characteristics and signaling molecules in CD16- and CD56-defined NK cells and their small immunoregulatory and large cytotoxic subsets in peripheral blood and lymph nodes, as NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling.

Increased killer immunoglobulin-like receptor expression and functional defects in natural killer cells in lung cancer.

Frequencies of natural killer (NK) cells from patients with non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) did not differ from healthy controls. A higher proportion of NK cells from NSCLC patients expressed the killer immunoglobulin-like receptor (KIR) CD158b than in controls (P = 0.0004), in the presence or absence of its ligand, HLA-C1. A similar result was obtained for CD158e in the presence of its ligand HLA-Bw4 in NSCLC patients (P = 0.003); this was entirely attributable to the Bw4I group of alleles in the presence of which a fivefold higher percentage of CD158e(+) NK cells was found in NSCLC patients than controls. Proportions of CD158b(+) NK cells declined with advancing disease in NSCLC patients. Expression of NKp46, CD25 and perforin A, and production of interferon-γ following stimulation with interleukin-12 and interleukin-18, were all significantly lower in NK cells from NSCLC patients than in controls. Both NK cell cytotoxicity and granzyme B expression were also reduced in lung cancer patients. Increased inhibitory KIR expression would decrease NK cell cytotoxic function against tumour cells retaining class I HLA expression. Furthermore, the reduced ability to produce interferon-γ would restrict the ability of NK cells to stimulate T-cell responses in patients with lung cancer.

Epigenetic regulation of killer immunoglobulin-like receptor expression in T cells.

With increasing age, T cells gain expression of killer immunoglobulin-like receptors (KIRs) that transmit negative signals and dampen the immune response. KIR expression is induced in CD4 and CD8 T cells by CpG DNA demethylation suggesting epigenetic control. To define the mechanisms that underlie the age-associated preferential KIR expression in CD8 T cells, we examined KIR2DL3 promoter methylation patterns. With age, CD8 T cells developed a patchy and stochastic promoter demethylation even in cells that did not express the KIR2DL3-encoded CD158b protein; complete demethylation of the minimal KIR2DL3 promoter was characteristic for CD158b-expressing cells. In contrast, the promoter in CD4 T cells was fully methylated irrespective of age. The selectivity for CD8 T cells correlated with lower DNMT1 recruitment to the KIR2DL3 promoter which further diminished with age. In contrast, binding of the polycomb protein EZH2 known to be involved in DNMT1 recruitment was not different. Our data suggest that CD8 T cells endure increasing displacement of DNMT1 from the KIR promoter with age, possibly because of an active histone signature. The ensuing partial demethylation lowers the threshold for transcriptional activation and renders CD8 T cells more susceptible to express KIR, thereby contributing to the immune defect in the elderly.

Evolution and survival of marine carnivores did not require a diversity of killer cell Ig-like receptors or Ly49 NK cell receptors.

Ly49 lectin-like receptors and killer cell Ig-like receptors (KIR) are structurally unrelated cell surface glycoproteins that evolved independently to function as diverse NK cell receptors for MHC class I molecules. Comparison of primates and various domesticated animals has shown that species have either a diverse Ly49 or KIR gene family, but not both. In four pinniped species of wild marine carnivore, three seals and one sea lion, we find that Ly49 and KIR are each represented by single, orthologous genes that exhibit little polymorphism and are transcribed to express cell surface protein. Pinnipeds are therefore species in which neither Ly49 nor KIR are polygenic, but retain the ancestral single-copy state. Whereas pinniped Ly49 has been subject to purifying selection, we find evidence for positive selection on KIR3DL during pinniped evolution. This selection, which focused on the D0 domain and the stem, points to the functionality of the KIR and most likely led to the sea lion's loss of D0. In contrast to the dynamic and rapid evolution of the KIR and Ly49 genes in other species, the pinniped KIR and Ly49 have been remarkably stable during the >33 million years since the last common ancestor of seals and sea lions. These results demonstrate that long-term survival of placental mammal species need not require a diverse system of either Ly49 or KIR NK cell receptors.

Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3.

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the "cross-reactions" were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor's interactions with HLA-C.

Cutting edge: selective expression of inhibitory or activating killer cell Ig-like receptors in circulating CD4+ T lymphocytes.

Apart from NK cells, TCRgammadelta and CD8+ T cells, killer cell Ig-like receptor (KIR) expression was described on a minor subset of CD4+ T cells. However, their functions remain to be elucidated in this latter lymphocyte population. We demonstrated that KIR2DL2/L3 (CD158b) and KIR2DS2 (CD158j) transcripts were synthesized by sorted CD4+CD158b/j+ T cells obtained from healthy individuals. In contrast, we observed that only the inhibitory or activating receptor was expressed at the cell surface according to the donor tested. In CD158b-expressing cells, KIR triggering leads to an inhibition of the CD3-induced cell proliferation and Erk activation, and the receptor exhibits an activation-dependent tyrosine phosphorylation and association with the Src homology 2-containing phosphatase 1. In CD158j-positive cells, KIR-engagement results in an enhanced CD3-mediated cell growth and Erk phosphorylation. Our results suggested that, in contrast to NK cells, the functions of KIR in CD4+ T lymphocytes might derive from a selective expression of their activating or inhibiting forms.