Mesenchymal Stromal Cells Induce Peculiar Alternatively Activated Macrophages Capable of Dampening Both Innate and Adaptive Immune Responses.

Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (M(MSC) ), through a prostaglandin E2-dependent mechanism. M(MSC) were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-ß. These cytokines contributed to the immunoregulatory properties of M(MSC) , which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, M(MSC) inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNγ, while M2 affected only IFNγ production. Moreover, M(MSC) inhibited the proliferation of CD8(+) T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of M(MSC) to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches. Stem Cells 2016;34:1909-1921.

Cellular and molecular interactions of mesenchymal stem cells in innate immunity.

In recent years, human mesenchymal stem/stromal cells (MSC) have attracted major attention for their possible clinical applications. In addition to their tissue regenerative capacity, they display immune-modulatory properties for which they have been used in the treatment of acute graft-versus-host disease and autoimmune diseases. Various studies have analyzed the inhibitory effect exerted by MSC on cells belonging to acquired or to innate immunity. In this context, MSC have been shown to inhibit proliferation and function of natural killer (NK) cells and to hinder the generation of dendritic cells and macrophages, thus interfering with inflammatory processes and with the generation of type I immune responses. In addition, MSC promote the differentiation of regulatory cells and participate in the regeneration of tissues damaged as a consequence of the inflammatory process. Different molecular mechanisms are involved in the immunosuppressive effect. Further investigation on the biology of MSC and on the regulatory events involved in their functional activities can help to optimize their use in clinical practice.

miR-24-3p down-regulates the expression of the apoptotic factors FasL and BIM in human natural killer cells.

MicroRNAs are involved in the regulation of different functions in immune and non-immune cells. Here we show that miR-24-3p functionally interacts with FASLG mRNA and down-regulates its expression. This interaction occurs in human natural killer cells (NK), leading to the modulation of FasL surface expression. Moreover, miR-24-3p also modulates the mRNA and protein expression of BIM in NK cells. Thus, it likely contributes to the control of both the extrinsic and intrinsic apoptotic pathways. In line with this hypothesis, inhibition of miR-24-3p improves both initiator caspase-8 and effector caspase-3 and -7 activities, increases cell apoptosis, and reduces cell viability. Our data suggest that miR-24-3p can act as a survival factor in NK cells, affecting the FasL-mediated killing of Fas expressing cells and the BIM-dependent cell death. More generally, miR-24-3p may condition the level of cell apoptosis, which increases at the contraction phase of the immune response when the clearance of various expanded effector cells is needed.

Cord Blood-Derived Natural Killer Cell Exploitation in Immunotherapy Protocols: More Than a Promise?

In the last 20 years, Natural Killer (NK) cell-based immunotherapy has become a promising approach to target various types of cancer. Indeed, NK cells play a pivotal role in the first-line defense against tumors through major histocompatibility complex-independent immunosurveillance. Their role in the control of leukemia relapse has been clearly established and, moreover, the presence of NK cells in the tumor microenvironment (TME) generally correlates with good prognosis. However, it has also been observed that, often, NK cells poorly infiltrate the tumor tissue, and, in TME, their functions may be compromised by immunosuppressive factors that contribute to the failure of anti-cancer immune response. Currently, studies are focused on the design of effective strategies to expand NK cells and enhance their cytotoxic activity, exploiting different cell sources, such as peripheral blood (PB), umbilical cord blood (UCB) and NK cell lines. Among them, UCB represents an important source of mature NK cells and CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs), as precursors of NK cells. In this review, we summarize the UCB-derived NK cell activity in the tumor context, review the different in-vitro models to expand NK cells from UCB, and discuss the importance of their exploitation in anti-tumor immunotherapy protocols.

Neuroblastoma Tumor-Associated Mesenchymal Stromal Cells Regulate the Cytolytic Functions of NK Cells.

Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been thoroughly investigated. Herein, we study the immune-regulatory potential of six primary young and senescent NB-TA-MSC on NK cell function. Young cells display a phenotype (CD105+/CD90+/CD73+/CD29+/CD146+) typical of MSC cells and, in addition, express high levels of immunomodulatory molecules (MHC-I, PDL-1 and PDL-2 and transcriptional-co-activator WWTR1), able to hinder NK cell activity. Notably, four of them express the neuroblastoma marker GD2, the most common target for NB immunotherapy. From a functional point of view, young NB-TA-MSC, contrary to the senescent ones, are resistant to activated NK cell-mediated lysis, but this behavior is overcome using anti-CD105 antibody TRC105 that activates antibody-dependent cell-mediated cytotoxicity. In addition, proliferating NB-TA-MSC, but not the senescent ones, after six days of co-culture, inhibit proliferation, expression of activating receptors and cytolytic activity of freshly isolated NK. Inhibitors of the soluble immunosuppressive factors L-kynurenine and prostaglandin E2 efficiently counteract this latter effect. Our data highlight the presence of phenotypically heterogeneous NB-TA-MSC displaying potent immunoregulatory properties towards NK cells, whose inhibition could be mandatory to improve the antitumor efficacy of targeted immunotherapy.

MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2.

Various studies analyzed the inhibitory effect exerted by mesenchymal stem cells (MSCs) on cells of the innate or acquired immunity. Myeloid dendritic cells (DCs) are also susceptible to such inhibition. In this study, we show that MSCs strongly inhibit DC generation from peripheral blood monocytes. In the presence of MSCs, monocytes supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) did not acquire the surface phenotype typical of immature (CD14(-), CD1a(+)) or mature (CD80(+), CD86(+), CD83(+)) DCs, failed to produce IL-12, and did not induce T-cell activation or proliferation. Analysis of the molecular mechanism(s) responsible for the inhibitory effect revealed a major role of prostaglandin E(2) (PGE(2)). Thus, addition of the PGE(2) inhibitor NS-398 restored DC differentiation and function. Moreover, PGE(2) directly added to cultures of monocytes blocked their differentiation toward DCs in a manner similar to MSCs. Although IL-6 has been proposed to play a role in MSC-mediated inhibition of DC differentiation, our data indicate that PGE(2) and not IL-6 represents the key inhibitory mediator. Indeed, NS-398 inhibited PGE(2) production and restored DC differentiation with no effect on IL-6 production. These data emphasize the role of MSCs in inhibiting early DC maturation and identifying the molecular mechanisms responsible for the inhibitory effect.

Stromal-like Wilms tumor cells induce human Natural Killer cell degranulation and display immunomodulatory properties towards NK cells.

The similarity of stromal-like Wilms tumor (str-WT) cells with mesenchymal stem cells (MSC), suggests their relevant role in the interplay with immune cells in the tumor microenvironment. We investigated the interaction between str-WT cells and NK cells. We observed that str-WT cells expressed some major ligands for activating and inhibitory NK cell receptors. Moreover, they expressed inhibitory checkpoint molecules involved in the negative regulation of anti-tumor immune response. The analysis of the interaction between str-WT cells and NK lymphocytes revealed that activated NK cells could efficiently degranulate upon interaction with str-WT cells. On the other hand, str-WT cells could exert potent inhibitory effects on cytokine-induced activation of NK cell proliferation and phenotype, which were mediated by the production of IDO and PGE2 inhibitory factors. Our data provide insight into the molecular interactions between str-WT cells and NK lymphocytes that may result in different outcomes possibly occurring in the WT microenvironment.

Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants.

BACKGROUND: High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3. METHOD: Here, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 106 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel. RESULTS: No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system. CONCLUSIONS: Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.

Recent Advances in the Role of Natural Killer Cells in Acute Kidney Injury.

Growing evidence is revealing a central role for natural killer (NK) cells, cytotoxic cells belonging to the broad family of innate lymphoid cells (ILCs), in acute and chronic forms of renal disease. NK cell effector functions include both the recognition and elimination of virus-infected and tumor cells and the capability of sensing pathogens through Toll-like receptor (TLR) engagement. Notably, they also display immune regulatory properties, exerted thanks to their ability to secrete cytokines/chemokines and to establish interactions with different innate and adaptive immune cells. Therefore, because of their multiple functions, NK cells may have a major pathogenic role in acute kidney injury (AKI), and a better understanding of the molecular mechanisms driving NK cell activation in AKI and their downstream interactions with intrinsic renal cells and infiltrating immune cells could help to identify new potential biomarkers and to select clinically valuable novel therapeutic targets. In this review, we discuss the current literature regarding the potential involvement of NK cells in AKI.

Isolation and characterization of renal cancer stem cells from patient-derived xenografts.

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133- over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.

Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

BACKGROUND: Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. METHODS AND RESULTS: Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15-4.8) vs 0.3% (0.032-2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18-4.8) and 0.36% (0.15-3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). CONCLUSIONS: We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.

NCR(+)ILC3 concentrate in human lung cancer and associate with intratumoral lymphoid structures.

Tertiary lymphoid structures (TLSs) are a common finding in non-small cell lung cancer (NSCLC) and are predictors of favourable clinical outcome. Here we show that NCR(+) innate lymphoid cell (ILC)-3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumour-associated TLSs. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, on activation, produces IL-22, TNF-α, IL-8 and IL-2, and activates endothelial cells. Tumour NCR(+)ILC3 may interact with both lung tumour cells and tumour-associated fibroblasts, resulting in the release of cytokines primarily on engagement of the NKp44-activating receptor. In patients, NCR(+)ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumour stages and their presence correlate with the density of intratumoral TLSs. Our results indicate that NCR(+)ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumour-associated TLSs.

Interactions between mesenchymal stem cells and dendritic cells.

Mesenchymal stem or stromal cells (MSC) are considered a promising new therapeutic strategy for the treatment of several pathological conditions. Due to their immunomodulatory properties, they are currently employed in clinical trials aimed at preventing or treating steroid-resistant acute graft-versus-host disease (GvHD), a frequent complication of allogeneic hematopoietic stem cell transplantation (HSCT). In addition, the use of MSC has been proposed for the treatment of autoimmune diseases. A number of recent studies have focused on the influence of MSC on dendritic cell (DC) function. DCs play a critical role in initiating and regulating immune responses by promoting antigen-specific T cell activation. Moreover, they are involved in efficient cross-talk with different cells of the innate immune system. DC are the most effective antigen-presenting cells and prime naïve T cells to initiate adaptive immune responses including those against allogeneic cells or self-antigens. Thus, alteration of DC generation or function may greatly contribute to the inhibition of T cell responses. In this context, MSC were shown to interfere with DC maturation from monocytes or CD34(+) hemopoietic precursors thus further confirming their role in immune regulation and their usefulness in cell-based therapies.

Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2.

Recently, a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT, we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs, whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study, we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions, such as cytotoxic activity and cytokine production. Moreover, we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30, NKp44, and NKG2D. Finally, we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells.

Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation.

In recent years, mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However, no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells, whereas they only partially affect the proliferation of activated NK cells. In addition, we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30, NKG2D, and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly, MSCs expressed the known ligands for these activating NK receptors-ULBPs, PVR, and Nectin-2. Moreover, NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation.

Expression of the DNAM-1 ligands, Nectin-2 (CD112) and poliovirus receptor (CD155), on dendritic cells: relevance for natural killer-dendritic cell interaction.

In this study, we demonstrate the involvement of DNAM-1-triggering receptor and its ligands, poliovirus receptor (PVR) and Nectin-2, in natural killer (NK) cell-mediated lysis of dendritic cells (DCs). The surface expression of both ligands was up-regulated in DCs as compared to monocytes. It reached maximal densities after DC maturation induced by different stimuli including lipopolysaccharide (LPS), poly I:C, flagellin, and CD40L. Both immunohistochemical analysis and confocal microscopy revealed expression of DNAM-1 ligands by DCs in lymph nodes in which they were localized in the parafollicular T-cell region and surrounded the high endothelial venules. Remarkably, in cytolytic assays, DNAM-1 cooperated with NKp30 in the NK-mediated killing of both immature and mature DCs and the degree of contribution of DNAM-1 appeared to correlate with the surface densities of its specific ligands PVR and Nectin-2.

Analysis of the receptor-ligand interactions in the natural killer-mediated lysis of freshly isolated myeloid or lymphoblastic leukemias: evidence for the involvement of the Poliovirus receptor (CD155) and Nectin-2 (CD112).

On the basis of recent clinical and experimental data, natural killer (NK) cells appear to play a crucial role in eradication of acute myeloid leukemias. In the present study, by exploiting our current knowledge on NK receptors and their ligands on target cells, we investigated the interactions between NK and leukemic cells. We show that the size of the NK cell subset expressing the killer immunoglobulin-like receptor (KIR) not engaged by the HLA-class I alleles of the patient parallels the degree of NK cytotoxicity against leukemic cells. A sharp down-regulation of HLA-class I molecules has been detected in various leukemias and it was more frequent in myeloid than in lymphoblastic leukemias. Analysis of the ligands for triggering NK receptors revealed the consistent expression of Poliovirus receptor (PVR) and Nectin-2 in myeloid leukemias. In contrast, major histocompatibility complex class I-related chain molecules A/B (MICA/B) and UL1b-binding protein (ULBPs) were either absent or weakly expressed. Accordingly, NK-mediated lysis of these leukemias was dependent on DNAM-1 but not NKG2D. The major role of NKp46 and NKp30 was also confirmed. The expression of PVR and/or Nectin-2 was less frequent in lymphoblastic leukemias. In most leukemias, both CD48 and NTBA were down-regulated. The correlation found between marker expression and susceptibility to lysis may reveal useful information for NK-based immunotherapy.

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction.

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.

IFN-gamma production in human NK cells through the engagement of CD8 by soluble or surface HLA class I molecules.

The engagement of CD8 on NK cell surface by either surface or soluble HLA class I (sHLA-I) molecules induces synthesis and secretion of IFN-gamma. HLA-I-mediated effects were inhibited by the covering of CD8 with specific anti-CD8 monoclonal antibodies, indicating a direct interaction of HLA-I and CD8. That CD8 ligation induces IFN-gamma production was further supported by the finding that cross-linking of CD8 led to release of IFN-gamma at similar levels to those obtained with HLA-I. The sHLA-I-induced IFN-gamma production via CD8 was strongly down-regulated by the engagement of the inhibitory isoforms of either CD94/NKG2 complex by sHLA-I-non-(A,B,C,G) (putative sHLA-E) or CD158b by sHLA-I-Cw3 allele. Ligation of CD8 did not elicit, different from other activating NK cell surface molecules such as CD16 or CD69, triggering of NK cell-mediated cytolysis. Cyclosporin A, but not concanamycin A, an H+-ATPase vacuolar inhibitor which affects perforin and granzyme release, strongly reduced the sHLA-I-mediated CD8-dependent IFN-gamma production but did not affect cytolytic activity of NK cells, suggesting that different biochemical pathways are involved. Altogether, these findings indicate that CD8 engagement by sHLA-I activates a cyclosporin A-dependent pathway leading to production and secretion of IFN-gamma which may play a role in the regulation of innate immune responses in humans.

NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium-calmodulin kinase II: inhibition by HIV-1 Tat C-terminal domain.

In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin and granzymes, and IFN-gamma secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-gamma release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.