BackgroundAdoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair NK cell and T cell function. Auranofin, a gold (I)-containing phosphine compound, is a strong activator of the transcription factor Nrf2. Nrf2 controls a wide range of downstream targets important for the cells to obtain increased resistance to ROS. In this study, we present a strategy using auranofin to render human cytotoxic lymphocytes resistant toward oxidative stress.MethodsMelanoma patient-derived tumor infiltrating lymphocytes (TIL) and healthy donor-derived NK cells and CD19-directed CAR T cells were pretreated with a low dose of auranofin. Their resistance toward oxidative stress was assessed by measuring antitumoral responses (killing-assay, degranulation/CD107a, cytokine production) and intracellular ROS levels (flow cytometry) in conditions of oxidative stress. To confirm that the effects were Nrf2 dependent, the transcription level of Nrf2-driven target genes was analyzed by qPCR.ResultsPretreatment of human TIL and NK cells ex vivo with a low-dose auranofin significantly lowered their accumulation of intracellular ROS and preserved their antitumoral activity despite high H2O2 levels or monocyte-derived ROS. Furthermore, auranofin pretreatment of CD19 CAR-T cells or TIL increased their elimination of CD19 +tumor cells or autologous tumor spheroids, respectively, especially during ROS exposure. Analysis of Nrf2-driven target genes revealed that the increased resistance against ROS was Nrf2 dependent.ConclusionThese novel findings suggest that Nrf2 activation in human cytotoxic lymphocytes could be used to enhance the efficacy of adoptive cell therapy.

Successful translation of chimeric antigen receptor (CAR) T cell therapy for the treatment of solid tumors has proved to be troublesome, mainly due to the complex tumor microenvironment promoting T cell dysfunction and antigen heterogeneity. Mesothelin (MSLN) has emerged as an attractive target for CAR T cell therapy of several solid malignancies, including ovarian cancer. To improve clinical response rates with MSLN-CAR T cells, a better understanding of the mechanisms impacting CAR T cell functionality in vitro is crucial. Here, we demonstrated superior cytolytic capacity of CD28-costimulated MSLN-CAR T cells (M28z) relative to 4–1BB-costimulated MSLN-CAR T cells (MBBz). Furthermore, CD28-costimulated MSLN CAR T cells displayed enhanced cytolytic capacity against tumor spheroids with heterogeneous MSLN expression compared to MBBz CAR T cells. In this study, we identified CAR-mediated trogocytosis as a potential impeding factor for successful MSLN-CAR T cell therapy due to fratricide killing and contributing to tumor antigen heterogeneity. Moreover, we link antigen-dependent upregulation of LAG-3 with reduced CAR T cell functionality. Taken together, our study highlights the therapeutic potential and bottlenecks of MSLN-CAR T cells, providing a rationale for combinatorial treatment strategies.

For the past decade, adoptive cell therapy including tumor-infiltrating lymphocytes, genetically modified cytotoxic lymphocytes expressing a chimeric antigen receptor, or a novel T-cell receptor has revolutionized the treatment of many cancers. Progress within exome sequencing and neoantigen prediction technologies provides opportunities for further development of personalized immunotherapies. In this study, we present a novel strategy to deliver in silico predicted neoantigens to autologous dendritic cells (DCs) using paramagnetic beads (EpiTCer beads). DCs pulsed with EpiTCer beads are superior in enriching for healthy donor and patient blood-derived tumor-specific CD8+ T cells compared to DC loaded with whole-tumor lysate or 9mer neoantigen peptides. A dose-dependent effect was observed, with higher EpiTCer bead per DC being favorable. We concluded that CD8+ T cells enriched by DC loaded with EpiTCer beads are tumor specific with limited tumor cross-reactivity and low recognition of autologous non-activated monocytes or CD8+ T cells. Furthermore, tumor specificity and recognition were improved and preserved after additional expansion using our Good Manufacturing Process (GMP)-compatible rapid expansion protocol. Phenotypic analysis of patient-derived EpiTCer DC expanded CD8+ T cells revealed efficient maturation, with high frequencies of central memory and effector memory T cells, similar to those observed in autologous expanded tumor-infiltrating lymphocytes. These results indicate that DC pulsed with EpiTCer beads enrich for a T-cell population with high capacity of tumor recognition and elimination, which are features needed for a T-cell product to be used for personalized adoptive cell therapy.

Cancer cells in Hodgkin's lymphoma [HL] account for only a few % of the tumor mass which mainly consists of normal cells of the immune system including macrophages. Macrophages may perform different functions depending on the signals they receive. Tumor-associated macrophages (TAM) are often polarized towards the M2-like phenotype -immunosuppressive phenotype, preferring oxygen metabolism, and their presence in HL is associated with a worse prognosis. HL is highly avid in positron emission tomography, which suggests that glucose (Glc) metabolism is increased in non-cancerous microenvironmental cells. Here we asked if and what type of glucose metabolism is increased in TAMs and monocytes/Cd14+ cells. The project was conducted on the in vitro and the ex vivo model. In the in vitro model, the studied populations were THP-1 monocytic macrophages stimulated by Reed-Sternberg [RS] cells, and control macrophages that were not subjected to stimulation. In the ex vivo model, CD14+ cells were isolated, using the immunomagnetic method, from: fine-needle biopsy lymph node aspirate, peripheral blood of patients diagnosed with HL and from peripheral blood of a healthy donor. The cell's phenotype was determined by flow cytometry using CD14, CD16, CD163, CD206, CD68, CD11b, HLA-DR, PDL1 antibodies. Glc metabolism of the studied cell populations was analyzed using the Seahorse XFp analyser [by directly measuring the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR)]. The in vitro model experiment showed the intensification of Glc metabolism, in both glycolysis and oxidative phosphorylation pathways, in cells that have been stimulated by RS cells compared to control macrophages. Comparable results were obtained in the ex vivo model, which corroborates the reliability of the in vitro model. Glucose metabolism increases not only the population of cells obtained from lymph node aspirate but also from peripheral blood of patients diagnosed with HL, which may indicate both paracrine and endocrine neoplastic cells impact. Our data indicate that RS cells stimulate Glc metabolism of tumor microenvironment and peripheral blood CD14+ cells through both glycolysis and oxidative phosphorylation pathways.