Anti-tumor activity of a novel LAIR1 antagonist in combination with anti-PD-1 to treat collagen-rich solid tumors.

We recently reported that resistance to PD-1-blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and thus we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. Here, we report LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist monoclonal antibody, elicits myeloid inflammation and allogeneic T cell responses by binding to LAIR1 and blocking collagen engagement. Further, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

Hypoxia-Inducible Factor α Subunits Regulate Tie2-Expressing Macrophages That Influence Tumor Oxygen and Perfusion in Murine Breast Cancer.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

Hypoxia inducible factors-mediated inhibition of cancer by GM-CSF: a mathematical model.

Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.

Stabilization of HIF-2α induces sVEGFR-1 production from tumor-associated macrophages and decreases tumor growth in a murine melanoma model.

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.

Hypoxia-inducible factor-2α regulates GM-CSF-derived soluble vascular endothelial growth factor receptor 1 production from macrophages and inhibits tumor growth and angiogenesis.

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O(2) secrete high levels of a soluble form of the VEGF receptor 1 (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using small interfering RNA targeting to deplete hypoxia-inducible factor (HIF)-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O(2) was dependent on HIF-2α, whereas HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1α(flox/flox)- or HIF-2α(flox/+)/LysMcre mice). GM-CSF treatment increased intratumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1α(flox/flox)/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared with control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1α(flox/flox)/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2α(flox/+)/LysMcre mice, and, correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production.

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

Opposing roles for HIF-1α and HIF-2α in the regulation of angiogenesis by mononuclear phagocytes.

Macrophages contribute to tumor growth through the secretion of the proangiogenic molecule vascular endothelial growth factor (VEGF). We previously observed that monocytes treated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produce a soluble form of the VEGF receptor-1 (sVEGFR-1), which neutralizes VEGF biologic activity. The VEGF and VEGFR-1 promoters both contain a hypoxia regulatory element, which binds the hypoxia-inducible factor (HIF) transcription factors under hypoxic conditions. Based on this observation, we examined VEGF and sVEGFR-1 production from monocytes cultured at various O(2) concentrations. The amount of sVEGFR-1 production observed from GM-CSF-treated monocytes increased with decreasing levels of O(2). This sVEGFR-1 was biologically active and sequestered VEGF. To evaluate the role of the HIFs in sVEGFR-1 production, we used macrophages with a genetic deletion of HIF-1α. HIF-1α(-/-) macrophages cultured with GM-CSF at hypoxia secreted diminished amounts of VEGF compared with HIF-1α(+/+) macrophages, whereas sVEGFR-1 secretion was unaffected. In contrast, siRNA-mediated knockdown of HIF-2α inhibited the production of sVEGFR-1 in response to GM-CSF and low O(2), whereas VEGF production was unaffected. These studies suggest that hypoxia, generally thought to promote angiogenesis, can induce antiangiogenic behavior from macrophages within a GM-CSF-rich environment. Furthermore, these results suggest specific and independent roles for HIF-1α and HIF-2α in hypoxic macrophages.

CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain-engineered monoclonal antibody.

CD19 is a B cell-specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified constant fragment (Fc)-domain designed to enhance binding of FcgammaRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity, and antibody-dependent cellular phagocytosis but not complement-mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC compared with both the humanized anti-CD19 nonengineered antibody it is derived from and also rituximab, a therapeutic antibody widely used in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a granzyme B-dependent mechanism. The NK cell-mediated cytolytic and secretory function with XmAb5574 compared with the nonengineered antibody is associated with enhanced NK-cell activation, interferon production, extracellular signal-regulated kinase phosphorylation downstream of Fcgamma receptor, and no increased NK-cell apoptosis. Notably, enhanced NK cell-mediated ADCC with XmAb5574 was enhanced further by lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with lenalidomide for the therapy of CLL and related CD19(+) B-cell malignancies.

A phase I trial of paclitaxel and trastuzumab in combination with interleukin-12 in patients with HER2/neu-expressing malignancies.

Our preclinical work showed a dramatic synergy between interleukin-12 (IL-12) and trastuzumab for stimulation of natural killer cell cytokine secretion. We aimed to determine the safety profile of IL-12 when given in combination with trastuzumab and paclitaxel to patients with metastatic HER2-overexpressing cancers. Paclitaxel was given i.v. at 175 mg/m(2) every 3 weeks. Trastuzumab was given on day 1 each week (4 mg/kg initially and 2 mg/kg thereafter) in combination with injections of IL-12 on days 2 and 5 starting in cycle 2. This trial accrued 21 patients with metastatic HER2-positive tumors (breast, 7; colon, 6; esophagus, 4; stomach, 2; pancreas, 1; thyroid, 1). The IL-12 component was dose-escalated in cohorts of three patients. The dose-limiting toxicity was grade 3 fatigue at the 300 ng/kg dose level in two patients. The recommended phase II dose was 200 ng/kg administered s.c. There was one complete response in a patient with breast cancer, partial responses in 4 patients (breast, 2; esophageal, 2), and stabilization of disease lasting 3 months or greater (SD) in 6 other patients. All but one response occurred in patients with HER2 3+ disease. Two SD patients completed 1 year of therapy. Ten patients had progressive disease. There was increased activation of extracellular signal-regulated kinase in peripheral blood mononuclear cells and increased levels of IFN-gamma and several chemokines in patients with clinical benefit (complete response, partial response, or SD), but not in patients with progressive disease. IL-12 in combination with trastuzumab and paclitaxel therefore exhibits an acceptable toxicity profile and has activity in patients with HER2-overexpressing cancers.

Colocalization of the IL-12 receptor and FcgammaRIIIa to natural killer cell lipid rafts leads to activation of ERK and enhanced production of interferon-gamma.

Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcgammaRIIIa) that mediates interferon (IFN)-gamma production in response to antibody (Ab)-coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-gamma as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-gamma production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal-regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-gamma production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-gamma production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-gamma production. Confocal microscopy and cellular fractionation experiments revealed that FcgammaRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-gamma production. These data suggest that dual recruitment of FcgammaRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-gamma production.

The activation of natural killer cell effector functions by cetuximab-coated, epidermal growth factor receptor positive tumor cells is enhanced by cytokines.

PURPOSE: Natural killer (NK) cells express an activating Fc receptor (FcgammaRIIIa) that mediates antibody-dependent cellular cytotoxicity (ADCC) and production of immune modulatory cytokines in response to antibody-coated targets. Cetuximab is a therapeutic monoclonal antibody directed against the HER1 antigen. We hypothesized that the NK cell response to cetuximab-coated tumor cells could be enhanced by the administration of NK cell-stimulatory cytokines. EXPERIMENTAL DESIGN: Human NK cells stimulated with cetuximab-coated tumor cells and interleukin-2 (IL-2), IL-12, or IL-21 were assessed for ADCC and secretion of IFN-gamma and T cell-recruiting chemokines. IL-21 and cetuximab were given to nude mice bearing HER1-positive xenografts. RESULTS: Stimulation of human NK cells with cetuximab-coated tumor cells and IL-2, IL-12, or IL-21 resulted in 3-fold to 10-fold higher IFN-gamma production than was observed with either agent alone. NK cell-derived IFN-gamma significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1alpha, and RANTES) that could direct the migration of naive and activated T cells. IL-2, IL-12, and IL-21 enhanced NK cell ADCC against tumor cells treated with cetuximab. The combination of cetuximab, trastuzumab (an anti-HER2 monoclonal antibody), and IL-21 mediated greater NK cell cytokine secretion and ADCC than any agent alone. Furthermore, administration of IL-21 enhanced the effects of cetuximab in a murine tumor model. CONCLUSIONS: These results show that cetuximab-mediated NK cell activity can be significantly enhanced in the presence of NK cell-stimulatory cytokines. These factors, therefore, may be effective adjuvants to administer, in combination with cetuximab, to patients with HER1-positive malignancies.

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.

Natural killer cells produce T cell-recruiting chemokines in response to antibody-coated tumor cells.

In the current report, we have examined the ability of natural killer (NK) cells to produce T cell-recruiting chemokines following dual stimulation with interleukin (IL)-2 or IL-12 and human breast cancer cells coated with an antitumor antibody (trastuzumab). NK cells stimulated in this manner secreted an array of T cell-recruiting chemotactic factors, including IL-8, macrophage-derived chemokine, macrophage inflammatory protein 1alpha (MIP-1alpha), monocyte chemoattractant protein 1, and regulated on activation, normal T-cell expressed and secreted (RANTES), whereas stimulation of NK cells with either agent alone had minimal effect. Furthermore, these factors were functional for T-cell chemotaxis as culture supernatants derived from costimulated NK cells induced migration of both naïve and activated T cells in an in vitro chemotaxis assay. T-cell migration was significantly reduced when neutralizing antibodies to IL-8, MIP-1alpha, or RANTES were added to culture supernatants before their use in the chemotaxis assay. In addition, coadministration of trastuzumab-coated tumor cells and IL-12 to mice led to enhanced serum MIP-1alpha. As a clinical correlate, we examined the chemokine content of serum samples from breast cancer patients enrolled on a phase I trial of trastuzumab and IL-12, and found elevated levels of IL-8, RANTES, IFN-gamma inducible protein 10, monokine induced by IFN-gamma, and MIP-1alpha, specifically in those patients that experienced a clinical benefit. Sera from these patients exhibited the ability to direct T-cell migration in a chemotaxis assay, and neutralization of chemokines abrogated this effect. These data are the first to show chemokine production by NK cells, specifically in response to stimulation with antibody-coated tumor cells, and suggest a potential role for NK cell-derived chemokines in patients receiving therapeutic monoclonal antibodies.

Src homology 2-containing inositol 5'-phosphatase 1 negatively regulates IFN-gamma production by natural killer cells stimulated with antibody-coated tumor cells and interleukin-12.

We have previously shown that natural killer (NK) cells secrete a distinct profile of immunomodulatory cytokines in response to dual stimulation with antibody-coated tumor cells and interleukin-12 (IL-12). This NK cell cytokine response is dependent on synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIIIa) and the IL-12 receptor (IL-12R), both constitutively expressed on NK cells. The phosphatase Src homology 2-containing inositol 5'-phosphatase 1 (SHIP1) is known to exert inhibitory effects on Fc receptor (FcR) signaling via its enzymatic activity on phosphatidylinositol 3-kinase (PI3-K) products within many cells of the immune system, most notably mast cells, B cells, and monocytes. However, its activity in the context of FcR activation on NK cells has not been fully explored. The current study focused on the regulation of FcgammaRIIIa-induced NK cell cytokine production by SHIP1. Inhibitor studies showed that NK cell IFN-gamma production following FcR stimulation in the presence of IL-12 depended, in part, on the downstream products of PI3-K. Overexpression of wild-type (WT) SHIP1, but not a catalytic-deficient mutant, via retroviral transfection of primary human NK cells, resulted in a >70% reduction of NK cell IFN-gamma production in response to costimulation. In addition, NK cells from SHIP1-/- mice produced 10-fold greater amounts of IFN-gamma following culture with antibody-coated tumor cells plus IL-12 compared with NK cells from WT mice. Further, activation of the mitogen-activated protein kinase (MAPK) family member extracellular signal-regulated kinase (Erk; a downstream target of PI3-K) was significantly enhanced within SHIP1-/- NK cells compared with WT NK cells following costimulation. Pharmacologic inhibition of Erk activity, but not Jnk MAPK activity, led to significantly decreased IFN-gamma production from both SHIP1-/- and WT NK cells under these conditions. These results are the first to show a physiologic role for SHIP1 in the regulation of NK cell cytokine production and implicate PI3-K in the induction of MAPK signal transduction following costimulation of NK cells via the FcR and the IL-12R.

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells.

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.