William E. Carson

A potential role for interleukin-15 in the regulation of human natural killer cell survival.

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.

Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro.

Natural killer (NK) cells are large granular lymphocytes that constitutively express functional IL-2 receptors. We have shown that recombinant human IL-15 uses the IL-2 receptor to activate human NK cells and can synergize with recombinant human IL-12 to stimulate NK cell production of IFN-gamma in vitro. IFN-gamma production by NK cells is critical in the prevention of overwhelming infection by obligate intracellular microbial pathogens in several experimental animal models. Herein, we demonstrate that human monocytes produce IL-15 protein within 5 h of activation with LPS. Using an IL-15-neutralizing antiserum in a coculture of LPS-activated monocytes and NK cells, we demonstrate that monocyte-derived IL-15 is critical for optimal NK cell production of IFN-gamma. Endogenous IL-15 activates NK cells through the IL-2 receptor, and with endogenous IL-12, regulates NK cell IFN-gamma after monocyte activation by LPS. These in vitro studies are the first to characterize a function for endogenous IL-15, and as such, suggest an important role for IL-15 during the innate immune response. IL-15 may be an important ligand for the NK cell IL-2 receptor in vivo.

Psychological, Behavioral, and Immune Changes After a Psychological Intervention: A Clinical Trial

PURPOSE This randomized clinical trial tests the hypothesis that a psychological intervention can reduce emotional distress, improve health behaviors and dose-intensity, and enhance immune responses. PATIENTS AND METHODS We studied 227 women who were surgically treated for regional breast cancer. Before adjuvant therapy, women completed interviews and questionnaires assessing emotional distress, social adjustment, and health behaviors. A 60-mL blood sample was drawn for immune assays. Patients were randomly assigned to either the intervention group or assessment only group. The intervention was conducted in small patient groups, with one session per week for 4 months. The sessions included strategies to reduce stress, improve mood, alter health behaviors, and maintain adherence to cancer treatment and care. Reassessment occurred after completion of the intervention. RESULTS As predicted, patients receiving the intervention showed significant lowering of anxiety, improvements in perceived social support, improved dietary habits, and reduction in smoking (all P <.05). Analyses of adjuvant chemotherapy dose-intensity revealed significantly more variability (ie, more dispersion in the dose-intensity values) for the assessment arm (P <.05). Immune responses for the intervention patients paralleled their psychological and behavioral improvements. T-cell proliferation in response to phytohemagglutinin and concanavalin A remained stable or increased for the Intervention patients, whereas both responses declined for Assessment patients; this effect was replicated across three concentrations for each assay (all P <.01). CONCLUSION These data show a convergence of significant psychological, health behavior, and biologic effects after a psychological intervention for cancer patients.

Psychologic Intervention Improves Survival for Breast Cancer Patients A Randomized Clinical Trial

The question of whether stress poses a risk for cancer progression has been difficult to answer. A randomized clinical trial tested the hypothesis that cancer patients coping with their recent diagnosis but receiving a psychologic intervention would have improved survival compared with patients who were only assessed.

Myeloid derived suppressor cells – a new therapeutic target in the treatment of cancer

Myeloid Derived Suppressor Cells (MDSC) are a heterogeneous population of immature myeloid cells that are increased in states of cancer, inflammation and infection. In malignant states, MDSC are induced by tumor secreted growth factors. MDSC play an important part in suppression of host immune responses through several mechanisms such as production of arginase 1, release of reactive oxygen species and nitric oxide and secretion of immune-suppressive cytokines. This leads to a permissive immune environment necessary for the growth of malignant cells. MDSC may also contribute to angiogenesis and tumor invasion. This review focuses on currently available strategies to inhibit MDSC in the treatment of cancer.

BEAM: A Randomized Phase II Study Evaluating the Activity of Bevacizumab in Combination With Carboplatin Plus Paclitaxel in Patients With Previously Untreated Advanced Melanoma

PURPOSE Metastatic melanoma, a highly vascularized tumor with strong expression of vascular endothelial growth factor, has an overall poor prognosis. We conducted a placebo-controlled, double-blind phase II study of carboplatin plus paclitaxel with or without bevacizumab in patients with previously untreated metastatic melanoma. PATIENTS AND METHODS Patients were randomly assigned in a two-to-one ratio to carboplatin (area under the curve, 5) plus paclitaxel (175 mg/m(2)) and bevacizumab (15 mg/kg; CPB) or placebo (CP) administered intravenously once every 3 weeks. Progression-free survival (PFS) was the primary end point. Secondary end points included overall survival (OS) and safety. RESULTS Two hundred fourteen patients (73% with M1c disease) were randomly assigned. With a median follow-up of 13 months, median PFS was 4.2 months for the CP arm (n = 71) and 5.6 months for the CPB arm (n = 143; hazard ratio [HR], 0.78; P = .1414). Overall response rates were 16.4% and 25.5%, respectively (P = .1577). With 13-month follow-up, median OS was 8.6 months in the CP arm versus 12.3 months in the CPB arm (HR, 0.67; P = .0366), whereas in an evaluation 4 months later, it was 9.2 versus 12.3 months, respectively (HR, 0.79; P = .1916). In patients with elevated serum lactate dehydrogenase (n = 84), median PFS and OS were longer in the CPB arm (PFS: 4.4 v 2.7 months; HR, 0.62; OS: 8.5 v 7.5 months; HR, 0.52). No new safety signals were observed. CONCLUSION The study did not meet the primary objective of statistically significant improvement in PFS with the addition of bevacizumab to carboplatin plus paclitaxel. A larger phase III study will be necessary to determine whether there is benefit to the addition of bevacizumab to carboplatin plus paclitaxel in this disease setting.

Interleukin‐2 enhances the natural killer cell response to Herceptin‐coated Her2 / neu‐positive breast cancer cells

The Her2 / neu (c‐erbB‐2) oncogene encodes a 185‐kDa protein tyrosine kinase which is overexpressed in 20 % of breast adenocarcinomas and is recognized by a humanized anti‐Her2 / neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody‐dependent cell cytotoxicity (ADCC) against antibody‐coated targets via their expression of a low‐affinity receptor for IgG (FcγRIII or CD16). NK cells can be expanded in cancer patients via the administration of low‐dose interleukin‐2 (IL‐2) and become potent cytotoxic effectors following exposure to high doses of IL‐2. We tested IL‐2‐activated NK cells against Her2 / neu+ (MCF‐7Her2 / neu) and Her2 / neu– (MDA‐468) breast cancer cell lines in a 4‐h 51Cr‐release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5‐coated MCF‐7Her2 / neu and MDA‐468 target cells by IL‐2‐activated NK cells was 35 % and 3 %, respectively (p < 0.05). Lysis was less than 5 % when targets were treated with either the non‐humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5‐coated MCF‐7Her2 / neu cells was inhibited by 80 % when NK cells were pre‐treated with an anti‐Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF‐7Her2 / neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low‐dose IL‐2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5‐coated tumor cells in the presence of IL‐2 also produced large amounts of IFN‐γ with concomitant up‐regulation of cell‐surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL‐2 therapy in patients with cancers that express the Her2 / neu oncogene.

Myeloid-derived suppressor cell inhibition of the IFN response in tumor-bearing mice.

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid-derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to IFNs. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26-bearing mice had significantly elevated levels of GR1(+)CD11b(+) MDSC as compared with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr(701) in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4(+) and CD8(+) T cells as well as natural killer cells. In vitro coculture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26-bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in nitric oxide in C26-bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN response as compared with control C26 tumor-bearing mice. These data suggest that nitric oxide produced by MDSC can lead to reduced IFN responsiveness in immune cells.

IL-12 enhances the natural killer cell cytokine response to Ab-coated tumor cells

The anti-tumor activity of recombinant mAbs directed against tumor cell growth receptors has generally been considered to result from direct antiproliferative effects, the induction of apoptosis, or possibly Ab-dependent cellular cytotoxicity mediated against tumor targets. However, it remains unclear to what degree these mechanisms actually aid in the clearance of Ab-coated tumor cells in vivo. We show here that NK cells secrete a distinct profile of potent immunostimulatory cytokines in response to dual stimulation with Ab-coated tumor cells and IL-12. This response could not be duplicated by costimulation with other ILs and was significantly enhanced in the presence of monocytes. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIII) and the IL-12 receptor expressed on NK cells. Coadministration of Ab-coated tumor cells and IL-12 to BALB/c mice resulted in enhanced circulating levels of NK cell-derived cytokines with the capacity to augment anti-tumor immunity. These findings suggest that, in addition to mediating cellular cytotoxicity and apoptosis, the anti-tumor activity of mAbs might also result from activation of a potent cytokine secretion program within immune effectors capable of recognizing mAb-coated targets.

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 (FcγRIIIa) 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-γ 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-γ production than was observed with either agent alone. NK cell–derived IFN-γ significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1α, 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.