Separation, banking, and quality control of peripheral blood mononuclear cells from whole blood of melanoma patients

Holland, Martha; Cunningham, Rachel; Seymour, Lake J.; Kleinsteuber, Katja; Cunningham, Amy; Patel, Tejas; Manos, Michael P.; Brennick, Ryan; Zhou, Jun; Hodi, F. Stephen; Severgnini, Mariano

doi:10.1007/s10561-018-9734-x

Cited by 19 publications

(10 citation statements)

References 18 publications

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“…Analyte concentration for each patient was calculated using standard curves. Fold changes were calculated as a ratio relative to the patient’s baseline (C0D1) ( 47 – 49 ).…”

Section: Methodsmentioning

confidence: 99%

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse

Shapiro¹,

Birch²,

Hu³

et al. 2022

Journal of Clinical Investigation

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Background Responses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell–based therapy is a promising modality to treat post-HCT relapse. Methods We initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion. Results In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. Conclusion Given their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies. Trial Registration ClinicalTrials.gov NCT04024761. Funding Dunkin’ Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

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“…Analyte concentration for each patient was calculated using standard curves. Fold changes were calculated as a ratio relative to the patient’s baseline (C0D1) ( 47 – 49 ).…”

Section: Methodsmentioning

confidence: 99%

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse

Shapiro¹,

Birch²,

Hu³

et al. 2022

Journal of Clinical Investigation

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“…Patient plasma was isolated from whole blood within 6 hours of the collection via centrifugation (3000× g , 10 min, 4°C) and stored at −80°C. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved following the protocol outlined by Holland et al 8 Plasma samples were thawed at room temperature and soluble analyte assessment was performed following the manufacturer’s protocol, as previously described. 9 A custom Magnetic Luminex Kit (Bio-Techne, Minneapolis, Minnesota) was used to detect interleukin (IL)-1a, IL-1b, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-12p70, IL-15, IL-17a, chemokine ligand (CCL)2, CCL3, CCL4, CCL7, chemokine ligand (CXCL)2, CXCL5, CXCL6, CXCL8, CXCL10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ.…”

Section: Methodsmentioning

confidence: 99%

Combining CTLA-4 and angiopoietin-2 blockade in patients with advanced melanoma: a phase I trial

Ott

Nazzaro

Pfaff

et al. 2021

J Immunother Cancer

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BackgroundAngiogenic factors promote the growth of tumor vasculature, modulate lymphocyte trafficking into tumors, and inhibit maturation of dendritic cells. We hypothesized that MEDI3617, a human IgG1 kappa monoclonal antibody directed against human angiopoietin-2, in combination with tremelimumab (treme), an IgG2 monoclonal antibody blocking cytotoxic T-lymphocyte-associated protein- (CTLA-4), is safe in patients with advanced melanoma.MethodsIn a phase I, 3+3 dose escalation trial, patients with metastatic or unresectable melanoma received treme in combination with MEDI3617. The primary objectives of the study were safety and determination of recommended phase II dose (RP2D). The secondary objectives included determination of 6-month and 1-year overall survival and best overall response rate. Immune cell populations and soluble factors were assessed in peripheral blood and metastatic tumors using Fluorescence activated cell sorting (FACS), Luminex, and multiplexed immunofluorescence.ResultsFifteen patients (median age: 62) were enrolled in the study (3 patients in cohort 1: treme at 10 mg/kg and MEDI3617 at 200 mg; and 12 patients in cohort 2: treme at 10 mg/kg and MEDI3617 at 600 mg). The most common all-grade treatment-related adverse events were rash, pruritus, fatigue, and extremity edema. No dose-limiting toxicities were observed. Cohort 2 was determined to be the RP2D. There were no patients with confirmed immune-related complete response or immune-related partial response. Six of 15 patients had immune-related stable disease, resulting in a disease control rate of 0.40 (95% CI 0.16 to 0.68). An increase in frequencies of circulating inducible T-cell costimulator (ICOS)+ and human leukocyte antigen (HLA)-DR+ CD4+ and CD8+ T cells and production of Interleukin-2 and Interleukin-10 was observed post therapy.ConclusionsTremelimumab in combination with MEDI3617 is safe in patients with advanced melanoma. Angiopoietin-2 inhibition in combination with immune checkpoint inhibition warrants further exploration.Trial registration numberNCT02141542.

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“…PBMCs were isolated from whole blood within 6 h of collection and processed according to the methods described by Holland et al . [24].…”

Section: Methodsmentioning

confidence: 99%

Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry

et al. 2019

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As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4 + and CD8 + T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.

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Separation, banking, and quality control of peripheral blood mononuclear cells from whole blood of melanoma patients

Cited by 19 publications

References 18 publications

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse

Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse

Combining CTLA-4 and angiopoietin-2 blockade in patients with advanced melanoma: a phase I trial

Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry

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