Expansion of NK Cells and Reduction of NKG2D Expression in Chronic Lymphocytic Leukemia. Correlation with Progressive Disease

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.     

The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the repertoire of mature human natural killer cell receptors

Natural killer (NK) cells play a crucial role in the immune response to micro-organisms and tumours. Recent evidence suggests that NK cells also regulate the adaptive T-cell response and that it might be possible to exploit this ability to eliminate autoreactive T cells in autoimmune disease and alloreactive T cells in transplantation. Mature NK cells consist of a highly diverse population of cells that expresses different receptors to facilitate recognition of diseased cells and possibly pathogens themselves. Ex vivo culture of NK cells with cytokines such as IL-2 and IL-15 is an approach that permits significant expansion of the NK cell subpopulations, which are likely to have potent antitumour, antiviral, or immunomodulatory effects in autoimmunity. Our data indicate that the addition of IL-21 has a synergistic effect by increasing the numbers of NK cells on a large scale. IL-2 and IL-15 may induce the expression of killer cell immunoglobulin-like receptors (KIRs) in KIR-negative populations, the c-lectin receptor NKG2D and the natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15 or IL-2 can modify the pattern of the KIR receptors and inhibit NKp44 expression by reducing the expression of the adaptor DAP-12. IL-21 also preserved the production of interferon-γ and enhanced the cytotoxic properties of NK cells. Our findings indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21 can modify the peripheral repertoire of NK cells. These properties may be used to endow subpopulations of NK cells with specific phenotypes, which may be used in ex vivo cellular immunotherapy strategies.     

Roles for common cytokine receptor gamma-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo

NK cells differentiate in adult mice from bone marrow hemopoietic progenitors. Cytokines, including those that signal via receptors using the common cytokine receptor gamma-chain (gamma(c)), have been implicated at various stages of NK cell development. We have previously described committed NK cell precursors (NKPs), which have the capacity to generate NK cells, but not B, T, erythroid, or myeloid cells, after in vitro culture or transfer to a fetal thymic microenvironment. NKPs express the CD122 Ag (beta chain of the receptors for IL-2/IL-15), but lack other mature NK markers, including NK1.1, CD49b (DX5), or members of the Ly49 gene family. In this report, we have analyzed the roles for gamma(c)-dependent cytokines in the generation of bone marrow NKP and in their subsequent differentiation to mature NK cells in vivo. Normal numbers of NKPs are found in gamma(c)-deficient mice, suggesting that NK cell commitment is not dependent on IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. Although IL-2, IL-4, and IL-7 have been reported to influence NK cell differentiation, we find that mice deficient in any or all of these cytokines have normal NK cell numbers, phenotype, and effector functions. In contrast, IL-15 plays a dominant role in early NK cell differentiation by maintaining normal numbers of immature and mature NK cells in the bone marrow and spleen. Surprisingly, the few residual NK cells generated in absence of IL-15 appear relatively mature, expressing a variety of Ly49 receptors and demonstrating lytic and cytokine production capacity.     

Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

Interleukin-2-stimulated natural killer cells are less susceptible to mycophenolate mofetil than non-activated NK cells: possible consequences for immunotherapy

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Bacterial infection of human hematopoietic stem cells induces monocytic differentiation

Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34(+) hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response.     

Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice

The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the alpha-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.     

DAP10 deficiency breaks the immune tolerance against transplantable syngeneic melanoma

DAP10, an activating adaptor protein, associates with the NKG2D protein to form a multisubunit receptor complex that is expressed in lymphoid and myeloid cells. The ligands for NKG2D-DAP10 receptor are expressed in both normal and tumor cells, suggesting distinct roles for this receptor in autoimmunity and cancer. In this study, we report that constitutive DAP10 activating signaling is part of regulatory mechanisms that control immunity against tumors. Mice lacking DAP10 (DAP10KO), showed enhanced immunity against melanoma malignancies due to hyperactive functioning of NK1.1+CD3+ NKT cells. DAP10 deficiency resulted in substantially increased NKT cell functions, including cytokine production and cytotoxicity, leading to efficient killing of melanoma tumors. Moreover, the antitumor phenotype of DAP10KO mice correlated with impaired activation status of CD4+CD25+ T regulatory cells (Tregs). Upon activation, DAP10KO Tregs maintained higher levels of IL-2 and produced significantly lower amounts of IL-10 and IFN-gamma cytokines when compared with wild-type Tregs. Our data suggest that DAP10 signaling is involved in adjusting the activation threshold and generation of NKT cells and Tregs to avoid autoreactivity, but also modulates antitumor mechanisms.     

Multiple cytokines regulate the NK gene complex-encoded receptor repertoire of mature NK cells and T cells

Mature NK cells comprise a highly diverse population of lymphocytes that express different permutations of receptors to facilitate recognition of diseased cells and perhaps pathogens themselves. Many of these receptors, such as those belonging to the NKRP1, NKG2, and Ly49 families are encoded in the NK gene complex (NKC). It is generally thought that these NKC-encoded receptors are acquired by a poorly understood stochastic mechanism, which operates exclusively during NK cell development, and that following maturation the repertoire is fixed. However, we report a series of observations that demonstrates that the mature NK cell repertoire in mice can in fact be radically remodeled by multiple cytokines. Thus, both IL-2 and IL-15 selectively induce the de novo expression of Ly49E on the majority of mature NK cells. By contrast, IL-4 not only blocks this IL-2-induced acquisition of Ly49E, but reduces the proportion of mature NK cells that expresses pre-existing Ly49 receptors and abrogates the expression of NKG2 receptors while leaving the expression of several NKRP1 receptors unaltered. IL-21 also abrogates NKG2 expression on mature NK cells and selectively down-regulates Ly49F. IL-4 and IL-21 additionally cause dramatic and selective alterations in the NKC-encoded receptor repertoire of IL-2-activated T cells but these are quite different to the changes induced on NK cells. Collectively these findings reveal an unexpected aspect of NKC receptor expression that has important implications for our understanding of the function of these receptors and of the genetic mechanisms that control their expression.     

Interleukin-21 enhances NK cell activation in response to antibody-coated targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.     

Essential role of IL-21 in B cell activation, expansion, and plasma cell generation during CD4+ T cell-B cell collaboration

During T cell-B cell collaboration, plasma cell (PC) differentiation and Ig production are known to require T cell-derived soluble factors. However, the exact nature of the cytokines produced by activated T cells that costimulate PC differentiation is not clear. Previously, we reported that costimulation of purified human B cells with IL-21 and anti-CD40 resulted in efficient PC differentiation. In this study, we addressed whether de novo production of IL-21 was involved in direct T cell-induced B cell activation, proliferation, and PC differentiation. We found that activated human peripheral blood CD4(+) T cells expressed mRNA for a number of cytokines, including IL-21, which was confirmed at the protein level. Using a panel of reagents that specifically neutralize cytokine activity, we addressed which cytokines are essential for B cell activation and PC differentiation induced by anti-CD3-activated T cells. Strikingly, neutralization of IL-21 with an IL-21R fusion protein (IL-21R-Fc) significantly inhibited T cell-induced B cell activation, proliferation, PC differentiation, and Ig production. Inhibition of PC differentiation was observed even when the addition of IL-21R-Fc was delayed until after initial B cell activation and expansion had occurred. Importantly, IL-21 was found to be involved in PC differentiation from both naive and memory B cells. Finally, IL-21R-Fc did not inhibit anti-CD3-induced CD4(+) T cell activation, but rather directly blocked T cell-induced B cell activation and PC differentiation. These data are the first to document that B cell activation, expansion, and PC differentiation induced by direct interaction of B cells with activated T cells requires IL-21.     

Autocrine IL-15 mediates intestinal epithelial cell death via the activation of neighboring intraepithelial NK cells

Intestinal intraepithelial lymphocytes (IELs), which reside between the basolateral faces of intestinal epithelial cells (IECs), provide a first-line defense against pathogens via their cytotoxic activity. Although IEC-derived IL-7 and IL-15 are key regulatory cytokines for the development and activation of IELs, we report here that IL-15 but not IL-7 mediates the reciprocal interaction between IELs and IECs, an important interaction for the regulation of appropriate mucosal immunohomeostasis. IL-15-treated IELs induced cell death in IECs via the cytotoxic activity in vitro. Among the different subsets of IL-15-treated IELs, CD4(-)CD8(-)TCR(-) IELs, which express NK marker (DX5 or NK1.1), showed the most potent syngenic IEC killing activity. These intraepithelial NK cells expressed Ly-49 molecules, NKG2 receptors, and perforin. These results suggest the possibility that the cell death program of IECs could be regulated by self-produced IL-15 through the activation of intraepithelial NK cells.     

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.     

IL-21 induces the functional maturation of murine NK cells

IL-21 is a recently identified cytokine that stimulates mouse NK cell effector functions in vitro. In this study we demonstrate that IL-21 achieves its stimulatory effect by inducing the development of mature NK cells into a large granular lymphocyte phenotype with heightened effector function. IL-21 treatment results in increased cell size and granularity and a corresponding decrease in cell viability and proliferative potential. These cells up-regulate the expression of the inhibitory CD94-NKG2A receptor complex and the activation markers CD154 and killer cell, lectin-like-receptor G1. Surprisingly, IL-21 treatment also results in down-regulation of the pan-NK marker, NK1.1. Coinciding with these cellular changes IL-21 enhances cytolytic capacity across a spectrum of target sensitivities and induces IL-10 and IFN-gamma production. In vivo treatment with IL-21 results in a very similar activation and phenotypic maturation of NK cells as well as a potent increase in NK cell-mediated anti-tumor immunity that is perforin dependent. These developmental changes suggested that IL-21 functions to induce the terminal differentiation of mouse NK cells, resulting in heightened NK cell-mediated cytotoxicity and immune surveillance.     

Mesenchymal stromal cells of the bone marrow and natural killer cells: cell interactions and cross modulation

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are multipotent progenitor cells that have shown promise for several different therapeutic applications. As they are able to modulate the function of several types of immune cells, BM-MSCs are highly important in the field of cell-based immunotherapy. Understanding BM-MSC-natural killer (NK) cell interactions is crucial for improving their therapeutic efficiency. Here, we observed that the type of NK cell-activating cytokine (e.g., IL-2, IL-12, IL-15 and IL-21) strongly influenced the outcomes of their interactions with BM-MSCs. The expression patterns of the ligands (CD112, CD155, ULPB-3) and receptors (LAIR, NCR) mediating the cross-talk between BM-MSCs and NK cells were critically modulated following co-culture. BM-MSCs partially impaired NK cell proliferation but up-regulated their secretion of IFN-γ and TNF-α. As they are cytotoxic, activated NK cells induced the killing of BM-MSCs. Indeed, BM-MSCs triggered the degranulation of NK cells and increased their release of perforin and granzymes. Interestingly, activated NK cells induced ROS generation within BM-MSCs that caused their decreased viability and reduced expression of serpin B9. Collectively, our observations reveal that BM-MSC-NK cell interactions may impact the immunobiology of both cell types. The therapeutic potential of BM-MSCs will be significantly improved once these issues are well characterized.