MetaGate: Interactive Analysis of High-Dimensional Cytometry Data with Meta Data Integration.

Flow cytometry is a powerful technology for high-throughput protein quantification at the single-cell level, widely used in basic research and routine clinical diagnostics. Traditionally, data analysis is carried out using manual gating, in which cut-offs are defined manually for each marker. Recent technical advances, including the introduction of mass cytometry, have increased the number of proteins that can be simultaneously assessed in each cell. To tackle the resulting escalation in data complexity, numerous new analysis algorithms have been developed. However, many of these show limitations in terms of providing statistical testing, data sharing, cross-experiment comparability integration with clinical data. We developed MetaGate as a platform for interactive statistical analysis and visualization of manually gated high-dimensional cytometry data with integration of clinical meta data. MetaGate allows manual gating to take place in traditional cytometry analysis software, while providing a combinatorial gating system for simple and transparent definition of biologically relevant cell populations. We demonstrate the utility of MetaGate through a comprehensive analysis of peripheral blood immune cells from 28 patients with diffuse large B-cell lymphoma (DLBCL) along with 17 age- and sex-matched healthy controls using two mass cytometry panels made of a total of 55 phenotypic markers. In a two-step process, raw data from 143 FCS files is first condensed through a data reduction algorithm and combined with information from manual gates, user-defined cellular populations and clinical meta data. This results in one single small project file containing all relevant information to allow rapid statistical calculation and visualization of any desired comparison, including box plots, heatmaps and volcano plots. Our detailed characterization of the peripheral blood immune cell repertoire in patients with DLBCL corroborate previous reports showing expansion of monocytic myeloid-derived suppressor cells, as well as an inverse correlation between NK cell numbers and disease progression.

Perturbed NK-cell homeostasis associated with disease severity in chronic neutropenia.

Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

A Systemic Protein Deviation Score Linked to PD-1+ CD8+ T Cell Expansion That Predicts Overall Survival in Diffuse Large B Cell Lymphoma.

BACKGROUND: Current prognostic variables can only partly explain the large outcome heterogeneity in diffuse large B cell lymphoma (DLBCL). We aimed to investigate the utility of systems-level protein and immune repertoire profiling for outcome prognostication in DLBCL. METHODS: In this retrospective study, we used proximity extension assay technology to quantify 81 immune-related proteins in serum or plasma in 2 independent cohorts in a total 111 DLBCL patients. Protein levels were assessed before and after treatment with rituximab and chemotherapy, and the patients were compared with 19 age- and sex-matched healthy blood donors. In a subset of the patients, we performed a broad mass cytometric characterization of immune cell repertoires in peripheral blood. FINDINGS: Patients displayed large deviations in protein profiles compared with healthy controls. Development of a systemic protein deviation (SPD) score provided a 4-protein-based metric that reflected the overall degree of protein deviations compared with age- and sex-matched healthy blood donors. The SPD score identified patients with very poor overall survival in both cohorts and correlated with increased frequencies of peripheral blood PD-1+ CD8+ T cells, and expansion of myeloid-derived suppressor cells. CONCLUSIONS: Our results show that a simple metric based on measurement of a small set of serum or plasma proteins can be used to probe systemic immune changes associated with poor survival in DLBCL. This finding warrants further investigation in larger, prospective studies to establish a clinical prognostic biomarker.

Cellular immunotherapy with multiple infusions of in vitro-expanded haploidentical natural killer cells after autologous transplantation for patients with plasma cell myeloma.

BACKGROUND AIMS: To investigate the feasibility and safety of haploidentical natural killer (NK) cell infusions as consolidation immunotherapy after autologous stem cell transplant (ASCT) in patients with plasma cell myeloma. METHODS: Ten patients (median age, 59 years) received induction treatment followed by high-dose melphalan (200 mg/m2) at day -1, ASCT at day 0 and increasing NK cell doses (1.5 × 106, 1.5 × 107 and multiple doses of 1.0 × 108 cells/kg body weight) from day +1 to day +30 after ASCT. NK cells were harvested and purified from peripheral blood of haploidentical donors and expanded for 19 days with interleukin (IL)-2 and IL-15 under Good Manufacturing Practice conditions. RESULTS: NK cell numbers increased 56.0-fold (37.4- to 75.5-fold). Patients received a median of 3.8 × 108 (0.9-5.7 × 108) NK cells/kg body weight in six (three to eight) infusions. Multiparametric mass cytometry analysis demonstrated an altered surface receptor repertoire of expanded NK cells with increased degranulation and cytokine production activities but diminished expression of perforin. Donor NK cells were detectable in the peripheral blood, peaking 1 h after each dose (up to 90% donor NK cells). The treatment was safe and well tolerated, without evidence of graft-versus-host disease. Comparison with a control patient population receiving ASCT without NK cell infusions showed no significant difference in relapse, progression-free survival and overall survival. CONCLUSIONS: This study demonstrates reliable manufacturing of high numbers of activated NK cells for multiple-dose infusions and safe administration of these cellular products. The trial was registered at ClinicalTrials.gov (identifier no. NCT01040026).

Biological markers of hemostasis and endothelial activation in patients with a hematological malignancy with or without stem cell transplants.

INTRODUCTION: In this study, we analyzed the changes of thrombin generation as marker of coagulation activation and von Willebrand factor (vWF) levels as a marker of endothelial activation in patients undergoing chemotherapy, autologous, or allogeneic HSCT. We studied possible associations to triggering factors, including acute GVHD, thrombosis, time to engraftment, and bleeding complications. METHODS: Seventy-six patients treated for hematologic malignancies at the University Hospital Basel between 2005 and 2008 took part in this study. Blood samples were collected before the start of chemotherapy or conditioning regime (median day -2), in an early phase (median day + 12), and at a later point in time (median day + 24). RESULTS: Thrombin generation decreased in all three groups to about 50% of the initial value. Patients undergoing autologous or allogeneic HSCT showed significantly (P = .026 and P = .01) higher vWF levels than patients undergoing chemotherapy. Eighteen patients (42%) receiving allogeneic HSCT developed GVHD, vWF levels in patients with GVHD were significantly (P = .008) higher than in patients without GVHD. DISCUSSION: Patients receiving autologous or allogeneic HSCT had significantly higher vWF levels in the acute phase after the transplant than patients receiving chemotherapy alone, implicating a persistent stimulation of the endothelium, possibly within the context of GVHD.

[Cellular therapies]. / Zelluläre Therapien.

Cellular therapies Abstract. Transfusion medicine and allogeneic stem cell transplantation are well known and established cellular therapies in hematology. Since decades many efforts have been made, in order to re-program the patient's own immune system in order to clear malignancies. A breakthrough was achieved with the manufacturing and optimizing of so-called chimeric antigen receptor (CAR) T-cells, genetically engineered cells, specifically directed against tumor antigens. In this review we discuss the structure of CAR T-cells, their manufacturing and the different steps of a CAR T-cell treatment according to the current licensing. Furthermore, we give an outlook on future prospects of cellular therapies including the major issues in the field.

Human Cytomegalovirus Infection Enhances NK Cell Activity In Vitro.

BACKGROUND: Occurring frequently after solid organ and hematopoietic stem cell transplantation, cytomegalovirus (CMV) replication remains a relevant cause of mortality and morbidity in affected patients. Despite these adverse effects, an increased alloreactivity of natural killer (NK) cells after CMV infection has been assumed, but the underlying physiopathological mechanisms have remained elusive. METHODS: We used serial analyses of NK cells before and after CMV infection in kidney transplant recipients as an in vivo model for CMV primary infection to explore the imprint of CMV infection using every patient as their own control: We analyzed NK cell phenotype and function in 47 CMV seronegative recipients of CMV seropositive kidney grafts, who developed CMV primary infection posttransplant. Seronegative recipients of seronegative kidney grafts served as controls. RESULTS: We observed a significant increase of NKG2C expressing NK cells after CMV infection (mean increase, 17.5%; 95% confidence interval [95% CI], 10.2-24.9, P < 0.001), whereas cluster of differentiation (CD)57 expressing cells decreased (mean decrease, 14.1%; 95% CI, 8.0-20.2; P < 0.001). Analysis of killer immunoglobulin-like receptor (KIR) expression showed an increase of cells expressing KIR2DL1 as their only inhibitory KIR in patients carrying the cognate ligand HLA-C2 (mean increase, 10.0%; 95% CI, 1.7-18.3; P = 0.018). In C2-negative individuals, KIR2DL1 expression decreased (mean decrease, 3.9%; 95% CI, 1.6-6.2; P = 0.001). As for activating KIR, there was no conclusive change pattern. Most importantly, we observed a significantly higher NK cell degranulation and IFNγ production in response to different target cells (target K562, CD107a: mean increase, 9.9%; 95% CI, 4.8-15.0; P < 0.001; IFNγ: mean increase, 6.6%; 95% CI, 1.6-11.1; P < 0.001; target MRC-5, CD107a: mean increase, 6.9%; 95% CI, 0.7-13.1; P = 0.03; IFNγ: mean increase, 4.8%; 95% CI, 1.7-7.8; P = 0.002). CONCLUSIONS: We report evidence for an increased function of NK cells induced by CMV infection. This increased in vitro functionality was seen in NKG2C-positive and NKG2C-negative subsets, arguing for an NKG2C independent mechanism of action.