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-γ 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-γ. 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-γ-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-γ-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

Long-Term Outcome After Adoptive Immunotherapy With Natural Killer Cells: Alloreactive NK Cell Dose Still Matters

open26noRecently, many reports were published supporting the clinical use of adoptively transferred natural killer (NK) cells as a therapeutic tool against cancer, including acute myeloid leukemia (AML). Our group demonstrated promising clinical response using adoptive immunotherapy with donor-derived alloreactive KIR-ligand-mismatched NK cells in AML patients. Moreover, the antileukemic effect was correlated with the dose of infused alloreactive NK cells (“functional NK cell dose”). Herein, we update the results of our previous study on a cohort of adult AML patients (median age at enrollment 64) in first morphological complete remission (CR), not eligible for allogeneic stem cell transplantation. After an extended median follow-up of 55.5 months, 8/16 evaluable patients (50%) are still off-therapy and alive disease-free. Overall survival (OS) and disease-free survival (DFS) are related with the dose of infused alloreactive NK cells (≥2 × 105/kg).openParisi S.; Ruggeri L.; Dan E.; Rizzi S.; Sinigaglia B.; Ocadlikova D.; Bontadini A.; Giudice V.; Urbani E.; Ciardelli S.; Sartor C.; Cristiano G.; Nanni J.; Zannoni L.; Chirumbolo G.; Arpinati M.; Lewis R.E.; Bonifazi F.; Marconi G.; Martinelli G.; Papayannidis C.; Paolini S.; Velardi A.; Cavo M.; Lemoli R.M.; Curti A.Parisi, S.; Ruggeri, L.; Dan, E.; Rizzi, S.; Sinigaglia, B.; Ocadlikova, D.; Bontadini, A.; Giudice, V.; Urbani, E.; Ciardelli, S.; Sartor, C.; Cristiano, G.; Nanni, J.; Zannoni, L.; Chirumbolo, G.; Arpinati, M.; Lewis, R. E.; Bonifazi, F.; Marconi, G.; Martinelli, G.; Papayannidis, C.; Paolini, S.; Velardi, A.; Cavo, M.; Lemoli, R. M.; Curti, A

An IDO1-related immune gene signature predicts overall survival in acute myeloid leukemia

The contribution of the bone marrow (BM) immune microenvironment (TME) to acute myeloid leukemia (AML) development is well-known, but its prognostic significance is still elusive. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is an interferon (IFN)-γ-inducible mediator of immune tolerance. With the aim to develop a prognostic IDO1-based immune gene signature, biological and clinical data of 732 patients with newly diagnosed, non-promyelocytic AML were retrieved from public datasets and analyzed using established computational pipelines. Targeted transcriptomic profiles of 24 diagnostic BM samples were analyzed using the NanoString's nCounter platform. BIN1 and IDO1 were inversely correlated and individually predicted overall survival. PLXNC1, a semaphorin receptor involved in inflammation and immune response, was the IDO1-interacting gene retaining the strongest prognostic value. The incorporation of PLXNC1 into the 2-gene IDO1-BIN1 score gave rise to a powerful immune gene signature predicting survival, especially in patients receiving chemotherapy. The top differentially expressed genes between IDO1low and IDO-1high and between PLXNC1low and PLXNC1 high cases further improved the prognostic value of IDO1 providing a 7 and 10-gene immune signature, highly predictive of survival and correlating with AML mutational status at diagnosis. Taken together, our data indicate that IDO1 is pivotal for the construction of an immune gene signature predictive of survival in AML patients. Given the emerging role of immunotherapies for AML, our findings support the incorporation of immune biomarkers into current AML classification and prognostication algorithms

Humoral and Cellular CMV Responses in Healthy Donors; Identification of a Frequent Population of CMV-Specific, CD4+ T Cells in Seronegative Donors

CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i.e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors

Blinatumomab differentially modulates peripheral blood and bone marrow immune cell repertoire: a Campus ALL study

Blinatumomab is the first bi-specific T-cell engager approved for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (B-ALL). Despite remarkable clinical results, the effects of blinatumomab on the host immune cell repertoire are not fully elucidated. In the present study, we characterized the peripheral blood (PB) and, for the first time, the bone marrow (BM) immune cell repertoire upon blinatumomab treatment. Twenty-nine patients with B-ALL received blinatumomab according to clinical practice. Deep multiparametric flow cytometry was used to characterize lymphoid subsets during the first treatment cycle. Blinatumomab induced a transient redistribution of PB effector T-cell subsets and Treg cells with a persistent increase in cytotoxic NK cells, which was associated with a transient upregulation of immune checkpoint receptors on PB CD4 and CD8 T-cell subpopulations and of CD39 expression on suppressive Treg cells. Of note, BM immune T-cell subsets showed a broader post-treatment subversion, including the modulation of markers associated with a T-cell-exhausted phenotype. In conclusion, our study indicates that blinatumomab differentially modulates the PB and BM immune cell repertoire, which may have relevant clinical implications in the therapeutic setting

A screening of antineoplastic drugs for acute myeloid leukemia reveals contrasting immunogenic effects of etoposide and fludarabine

Background: Recent evidence demonstrated that the treatment of acute myeloid leukemia (AML) cells with daunorubicin (DNR) but not cytarabine (Ara-C) results in immunogenic cell death (ICD). In the clinical setting, chemotherapy including anthracyclines and Ara-C remains a gold standard for AML treatment. In the last decade, etoposide (Eto) and fludarabine (Flu) have been added to the standard treatment for AML to potentiate its therapeutic effect and have been tested in many trials. Very little data are available about the ability of these drugs to induce ICD. Methods: AML cells were treated with all four drugs. Calreticulin and heat shock protein 70/90 translocation, non-histone chromatin-binding protein high mobility group box 1 and adenosine triphosphate release were evaluated. The treated cells were pulsed into dendritic cells (DCs) and used for in vitro immunological tests. Results: Flu and Ara-C had no capacity to induce ICD-related events. Interestingly, Eto was comparable to DNR in inducing all ICD events, resulting in DC maturation. Moreover, Flu was significantly more potent in inducing suppressive T regulatory cells compared to other drugs. Conclusions: Our results indicate a novel and until now poorly investigated feature of antineoplastic drugs commonly used for AML treatment, based on their different immunogenic potential

Flow cytometry T cell profiling in a recent-onset narcoleptic type 1 child: a case report

Background: Recent studies have disclosed the involvement of T cells in narcolepsy type 1 pathogenesis. We characterized the T cell subsets distribution in a recent-onset child at two different time points, two months from disease onset (T0) and 10 months later (follow-up), respectively. Methods: Peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) lymphocytes were characterized by flow cytometry at both evaluations. The distribution of T cell subsets was compared between the two time points, and the fold change was calculated as the CSF/PBMC frequencies ratio. Results: The patient showed a 2-fold increase in the frequency of CD4+ EMRA T cells, and an increase of more than one and a half in the frequency of CD4+ CM T cells in CSF at follow-up compared to T0. Moreover, the distribution of CD4+ EM T cells was slightly increased at T0 compared to follow-up. In PBMCs, the CD4+ and CD8+ T cell subsets showed a slight decrease in CM cells at the follow-up. Finally, the CSF/PBMC fold-change ratio showed a 3-fold increase of CD4+ and CD8+ CM T cells at the follow-up, a rise up to 1.5-fold of CD4+ EMRA subsets and a slight decrease in CD4+ EM T cells. Conclusions: Our data suggest that variations in the frequency of EM vs. CM of CD4+ and CD8+ T cell subsets in the CSF and in the CSF/PBMC fold change may represent a biological marker of disease progression

Flow cytometry T cell profiling in a recent-onset narcoleptic type 1 child: a case report

Background: Recent studies have disclosed the involvement of T cells in narcolepsy type 1 pathogenesis. We characterized the T cell subsets distribution in a recent-onset child at two different time points, two months from disease onset (T0) and 10 months later (follow-up), respectively. Methods: Peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) lymphocytes were characterized by flow cytometry at both evaluations. The distribution of T cell subsets was compared between the two time points, and the fold change was calculated as the CSF/PBMC frequencies ratio. Results: The patient showed a 2-fold increase in the frequency of CD4+ EMRA T cells, and an increase of more than one and a half in the frequency of CD4+ CM T cells in CSF at follow-up compared to T0. Moreover, the distribution of CD4+ EM T cells was slightly increased at T0 compared to follow-up. In PBMCs, the CD4+ and CD8+ T cell subsets showed a slight decrease in CM cells at the follow-up. Finally, the CSF/PBMC fold-change ratio showed a 3-fold increase of CD4+ and CD8+ CM T cells at the follow-up, a rise up to 1.5-fold of CD4+ EMRA subsets and a slight decrease in CD4+ EM T cells. Conclusions: Our data suggest that variations in the frequency of EM vs. CM of CD4+ and CD8+ T cell subsets in the CSF and in the CSF/PBMC fold change may represent a biological marker of disease progression

Isolation and expansion of allogeneic myeloma-specific interferon-gamma producing T cells for adoptive immunotherapy.

Adoptive immunotherapy is a promising approach in the treatment of multiple myeloma. We have tested the identification, separation, and expansion of allogeneic myeloma-specific T cells in vitro. Irradiated myeloma cell line ARH 77 has been used to stimulate allogeneic CD4(+) and CD8(+) T lymphocytes. Activated myeloma-specific T cells that produced interferon-gamma were isolated using immunomagnetic beads and further expanded in vitro to numbers of up to 400 x 106 T cells. Specificity of the T lymphocytes was tested using a 5-(6-)carboxyfluoresceine diacetate succinimidyl ester (CFSE)-based cytotoxicity test. This study demonstrates the feasibility of identification and isolation of tumor-specific T cells from allogeneic donors that can be expanded in vitro to numbers useful for clinical applications