Immunogenic cell death induced by chemotherapy and immune tolerance mechanisms in acute myeloid leukemia

Sia nei tumori solidi che nelle neoplasie ematologiche, è stato dimostrato che alcuni agenti chemioterapici, come le antracicline, sono altamente immunogenici e determinano, attraverso le cellule dendritiche (DCs), un’efficiente attivazione della risposta antitumorale mediata dalle cellule T. Tale processo, caratterizzato dalla traslocazione della calreticulina e delle HSPs 70/90 sulla superficie cellulare delle cellule tumorali e dal rilascio di HMGB1 e di ATP, è detto morte cellulare immunogenica (ICD). Oltra alla ICD, però, la chemioterapia genera fenomeni infiammatori nel microambiente tumorale e attiva pathway immunosoppressivi a carico delle DCs, che potrebbero alterare la risposta immunitaria. Scopo del presente studio è stato caratterizzare la ICD indotta da antracicline nella leucemia acuta mieloide (LAM) e valutare l’induzione di pathway inibitori, come l’enzima indoleamina-2,3-diossigenasi (IDO1). In vitro, ex vivo ed in vivo abbiamo dimostrato che il trattamento con antracicline induce ICD anche nella LAM. Nei pazienti sottoposti a chemioterapia è stata riscontrata una risposta anti-leucemica caratterizzata dalla produzione di IFN- nei linfociti CD4+ e CD8+, capaci di uccidere i blasti autologhi pur mostrando un fenotipo exhausted, ma anche l’espansione di una popolazione di linfociti T regolatori. La ICD infatti determina la maturazione delle DCs, che attivano efficacemente linfociti T antigene-specifici ma, allo stesso tempo, esprimendo elevati livelli di IDO1, inducono linfociti T regolatori che limitano la risposta anti-leucemica. Questi risultati sono stati confermati nel modello murino leucemico dove, a seguito della chemioterapia, l’infiltrato tumorale è arricchito di DCs, mature esprimenti IDO1, ma anche di linfociti T caratterizzati da fenotipo exhausted. I nostri dati confermano che la ICD, attiva anche nella LAM, determina una risposta immunitaria efficace nei confronti della malattia ma anche l’attivazione di pathway inibitori nelle DCs che alterano tale risposta. La combinazione della chemioterapia con inibitori di IDO1 rappresenterebbe un approccio interessante per potenziare l'effetto immunogenico della chemioterapia e la risposta anti-leucemica.Recently, both in solid tumors and haematological malignancies, it has been shown that some chemotherapeutic agents, such as daunorubicin, are highly immunogenic and activate the immune response via cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by tumor cells modifications, such as cell surface translocation of calreticulin and heat shock proteins, extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately hamper anti-tumor T-cells. Aim of this study is to characterize ICD in acute myeloid leukemia (AML) and to evaluate the involvement of some DC-related inhibitory pathways, such as indoleamine-2,3-dioxygenase 1 (IDO1) expression. In vitro, ex vivo and in vivo we demonstrated that daunorubicin treatment induced ICD in AML. Ex vivo, T-cell monitoring of daunorubicin-treated AML patients displayed an increase in leukemia-specific IFN--producing CD4+ and CD8+ T cells, which were capable to kill autologous blasts but showed an exhausted phenotype. We also observed the expansion of regulatory T cells (Tregs). ICD, in fact, induced maturation of DCs which efficiently activate antigen-specific T lymphocytes but simultaneously express high levels of IDO1, inducing a population of Tregs which inhibit the anti-leukemia response. These results were confirmed in leukemic murine model where daunorubicin treatment resulted in increased maturation status of tumor infiltrating DCs expressing IDO1, but also in an increased infiltration of exhausted T lymphocytes. Our data confirm that ICD is active in AML and results in an immunological response against AML but also in the induction of inhibitory pathway. The combination of chemotherapy with IDO1 inhibitors represents an interesting approach to enhance the immunogenic effect of chemotherapy and anti-leukemic immune response

Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia

In solid tumors and hematological malignancies, including acute myeloid leukemia, some chemotherapeutic agents, such as anthracyclines, have proven to activate an immune response via dendritic cell-based cross-priming of anti-tumor T lymphocytes. This process, known as immunogenic cell death, is characterized by a variety of tumor cell modifications, i.e., cell surface translocation of calreticulin, extracellular release of adenosine triphosphate and pro-inflammatory factors, such as high mobility group box 1 proteins. However, in addition to with immunogenic cell death, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, such as the overexpression of indoleamine 2,3-dioxygensase 1, which may ultimately hamper anti-tumor T-cells via the induction of T regulatory cells. The aim of this review is to summarize the current knowledge about the mechanisms and effects by which chemotherapy results in both activation and suppression of anti-tumor immune response. Indeed, a better understanding of the whole process underlying chemotherapy-induced alterations of the immunological tumor microenvironment has important clinical implications to fully exploit the immunogenic potential of anti-leukemia agents and tune their application

Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

Background: High-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD 1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma. Methods: The human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-g production, before and after nivolumab exposure, were analyzed. Results: Along with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-g. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-g-producing effector T cells. Conclusions: Taken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-g-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.This study was supported by the Associazione Onlus ‘il Pensatore: Matteo Amitrano’, by Bologna AIL (Associazione Italiana contro le Leucemie), Section of Bologna; FATRO, Foundation Corrado and Bruno Maria Zaini-BolognaYe

An evaluation of genotoxicity in human neuronal-type cells subjected to oxidative stress under an extremely low frequency pulsed magnetic field.

The possible genotoxicity of extremely low frequency magnetic field (ELF-MF) exposure is still a controversial topic. The most of the reported data suggests that it alone does not affect DNA integrity, but several recent reports have suggested that sinusoidal ELF-MF may increase the effect of known genotoxic agents. Only a few studies deal with non sinusoidal ELF-MF, including pulsed magnetic field (PMF), which are produced by several devices. The aim of this study is to investigate whether PMF exposure can interfere with DNA damage and repair in the presence of a genotoxic oxidative agent in neuronal type cells. To this purpose gamma-H2AX foci formation, which is a sensitive marker of DNA double strand breaks (DSB), was investigated at different points of time (1, 24, 48, 72. h) after the H2O2 treatment (300. μM for 1. h) under PMF exposure (1. mT, 50. Hz) in human neuroblastoma BE(2)C cells. Moreover, cytotoxicity evaluation, by MTT assay and cell cycle analysis, was performed at various points of time after the treatment. Taken together, results suggest that PMF exposure does not interfere with genotoxicity and cytotoxicity induced by oxidative stress

Flow cytometry analysis of T-cell subsets in cerebrospinal fluid of narcolepsy type 1 patients with long-lasting disease

Background Type 1 narcolepsy (NT1) is a central hypersomnia linked to the destruction of hypocretin-producing neurons. A great body of genetic and epidemiological data points to likely autoimmune disease aetiology. Recent reports have characterized peripheral blood T-cell subsets in NT1, whereas data regarding the cerebrospinal fluid (CSF) immune cell composition are lacking. The current study aimed to characterize the T-cell and natural killer (NK) cell subsets in NT1 patients with long disease course. Methods Immune cell subsets from CSF and peripheral blood mononuclear cell (PBMC) samples were analysed by flow cytometry in two age-balanced and sex-balanced groups of 14 NT1 patients versus 14 healthy controls. The frequency of CSF cell groups was compared with PBMCs. Non-parametric tests were used for statistical analyses. Results The NT1 patients did not show significant differences of CSF immune cell subsets compared to controls, despite a trend towards higher CD4+ terminally differentiated effector memory T cells. T cells preferentially displayed a memory phenotype in the CSF compared to PBMCs. Furthermore, a reduced frequency of CD4+ terminally differentiated effector memory T cells and an increased frequency of NK CD56bright cells was observed in PBMCs from patients compared to controls. Finally, the ratio between CSF and peripheral CD4+ terminally differentiated effector memory T cells was two-fold increased in NT1 patients versus controls. Conclusions Significant differences in PBMCs and in CSF/PBMC ratios of immune cell profile were found in NT1 patients compared to healthy controls. These differences might have arisen from the different HLA status, or be primary or secondary to hypocretin deficiency. Further functional studies in patients close to disease onset are required to understand NT1 pathophysiology

Immunosenescence and immunotherapy in elderly acute myeloid leukemia patients: Time for a biology-driven approach

Acute myeloid leukemia (AML) is a disease, which mainly affects the elderly population. Unfortunately, the prognosis of patients aged >65 years is dismal, with 1-year overall survival approaching 10% with conventional therapies. The hypothesis of harnessing the immune system against cancer, including leukemia, has been postulated for a long time, and several clinical attempts have been made in this field. In the last years, we increased our knowledge about the interplay between AML and immune cells, but no major improvement has been translated, up to now, from bench to bedside. However, the outstanding results coming from the modern immuno-oncology trials with new drugs have granted a new interest for immunotherapy in AML. Accordingly, the elderly population represents an ideal target, given the low percentage of patients eligible for allogeneic stem cell transplant. With that in mind, in the era of immunotherapy, we consider immunosenescence as the optimal background to start investigating a biology-driven approach to AML therapy in the elderly. By taking into account the physiological age-related changes of immune response, more personalized and tailored use of the new drugs and strategies harnessing the immune system against AML, has the potential to increase their efficacy and impact on clinical outcomes

Tumour-derived PGD2 and NKp30-B7H6 engagement drives an immunosuppressive ILC2-MDSC axis

Group 2 innate lymphoid cells (ILC2s) are involved in human diseases, such as allergy, atopic dermatitis and nasal polyposis, but their function in human cancer remains unclear. Here we show that, in acute promyelocytic leukaemia (APL), ILC2s are increased and hyper-activated through the interaction of CRTH2 and NKp30 with elevated tumour-derived PGD2 and B7H6, respectively. ILC2s, in turn, activate monocytic myeloid-derived suppressor cells (M-MDSCs) via IL-13 secretion. Upon treating APL with all-trans retinoic acid and achieving complete remission, the levels of PGD2, NKp30, ILC2s, IL-13 and M-MDSCs are restored. Similarly, disruption of this tumour immunosuppressive axis by specifically blocking PGD2, IL-13 and NKp30 partially restores ILC2 and M-MDSC levels and results in increased survival. Thus, using APL as a model, we uncover a tolerogenic pathway that may represent a relevant immunosuppressive, therapeutic targetable, mechanism operating in various human tumour types, as supported by our observations in prostate cancer.Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth

Larger size of donor alloreactive NK cell repertoire correlates with better response to NK cell immunotherapy in elderly acute myeloid leukemia patients

In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy.Purpose: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Experimental Design: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53-73) received NK cells from haploidentical KIR-ligand-mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. Results: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6-68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3-51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively; P = 0.03). Conclusions: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells