Intact interferon signaling in peripheral blood leukocytes of high-grade osteosarcoma patients

High-grade osteosarcoma has a poor prognosis with an overall survival rate of about 60 percent. The recently closed European and American Osteosarcoma Study Group (EURAMOS)-1 trial investigates the efficacy of adjuvant chemotherapy with or without interferon-α. It is however unknown whether the interferon-signaling pathways in immune cells of osteosarcoma patients are functional. We studied the molecular and functional effects of interferon treatment on peripheral blood lymphocytes and monocytes of osteosarcoma patients, both in vivo and ex vivo. In contrast to other tumor types, in osteosarcoma, interferon signaling as determined by the phosphorylation of signal transducer and activator of transcription (STAT)1 at residue 701 was intact in immune cell subsets of 33 osteosarcoma patients as compared to 19 healthy controls. Also, cytolytic activity of interferon-α stimulated natural killer cells against allogeneic (n = 7 patients) and autologous target cells (n = 3 patients) was not impaired. Longitudinal monitoring of three osteosarcoma patients on interferon-α monotherapy revealed a relative increase in the CD16-positive subpopulation of monocytes during treatment. Since interferon signaling is intact in immune cells of osteosarcoma patients, there is a potential for indirect immunological effects of interferon-α treatment in osteosarcoma

Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell–mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell–mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell–mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient–derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell–activating agents in patients with high-grade osteosarcoma

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2(+) γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2(+) T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2(+) T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2(+) T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2(+) T cell cytotoxicity. Vδ2(+) T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2(+) T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts

<p>Abstract</p> <p>Background</p> <p>Conventional high-grade osteosarcoma is a primary malignant bone tumor, which is most prevalent in adolescence. Survival rates of osteosarcoma patients have not improved significantly in the last 25 years. Aiming to increase this survival rate, a variety of model systems are used to study osteosarcomagenesis and to test new therapeutic agents. Such model systems are typically generated from an osteosarcoma primary tumor, but undergo many changes due to culturing or interactions with a different host species, which may result in differences in gene expression between primary tumor cells, and tumor cells from the model system. We aimed to investigate whether gene expression profiles of osteosarcoma cell lines and xenografts are still comparable to those of the primary tumor.</p> <p>Methods</p> <p>We performed genome-wide mRNA expression profiling on osteosarcoma biopsies (n = 76), cell lines (n = 13), and xenografts (n = 18). Osteosarcoma can be subdivided into several histological subtypes, of which osteoblastic, chondroblastic, and fibroblastic osteosarcoma are the most frequent ones. Using nearest shrunken centroids classification, we generated an expression signature that can predict the histological subtype of osteosarcoma biopsies.</p> <p>Results</p> <p>The expression signature, which consisted of 24 probes encoding for 22 genes, predicted the histological subtype of osteosarcoma biopsies with a misclassification error of 15%. Histological subtypes of the two osteosarcoma model systems, <it>i.e</it>. osteosarcoma cell lines and xenografts, were predicted with similar misclassification error rates (15% and 11%, respectively).</p> <p>Conclusions</p> <p>Based on the preservation of mRNA expression profiles that are characteristic for the histological subtype we propose that these model systems are representative for the primary tumor from which they are derived.</p