Peripheral Blood Mononuclear Cell Preparations Research Articles

Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

High-resolution respirometry (HRR) can assess peripheral blood mononuclear cell (PBMC) bioenergetics, but no standardized medium for PBMC preparation and HRR analysis exist. Here, we study the effect of four different media (MiR05, PBS, RPMI, Plasmax) on the count, size, and HRR (Oxygraph-O2k) of intact PBMCs. Remarkably, the cell count was 21 % higher when PBMCs were resuspended in MiR05 than in PBS or Plasmax, causing O2 flux underestimation during HRR due to inherent adjustments. Moreover, smaller cell size and cell aggregation was observed in MiR05. Based on our findings, we propose that Plasmax, PBS or RPMI is more suitable than MiR05 for HRR of intact PBMCs. We provide oxygen solubility factors for Plasmax and PBS and encourage further optimization of a standardized HRR protocol for intact PBMCs.

Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 μM, 1 μM, and 2 μM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 μM) as a substrate (18.1, 14.9, and 6.43 μM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

Abstract A005: Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models

Abstract Background The preclinical evaluation of novel immune therapies requires humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By xenotransplantation of cell-line-derived (CDX) or patient-derived (PDX) tumors on humanized mice, we successfully generated a full human tumor-immune-cell model for different disease entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies, and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient 1st and 2nd generation NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities were s.c. (i.e. lymphoma) and/or i.v. (leukemia) transplanted on those humanized mice. Tumor growth of orthotopic models was followed up by BLI measurement. Blood and tumor samples were analysed by for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. The 2nd generation NOG mice are showing an increased total engraftment and lineage specific differentiation of cells. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Check point inhibitor treatments (Ipi, Nivo, or Pembro) induced tumor growth delay in selected models. BLI measurement is a suitable method to follow up systemic disease models and treatment effects over time. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on different immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been successfully used for preclinical evaluation of novel check point inhibitors, cell therapies and immune cell engagers. Our human tumor-immune-cell models allow preclinical translational research on tumor immune biology as well as evaluation of new therapies, drug combinations, as well as biomarker identification and validation in subcutaneous and orthotopic models. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Joshua Alcaniz, Glenn Smits, Wolfgang Walther, Jens Hoffmann. Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A005.

Optimization of Peripheral Blood Mononuclear Cell Processing for Improved Clinical ELISpot Assay Performance.

Cell and gene therapies have demonstrated impressive therapeutic efficacy in various human diseases. Nevertheless, cellular immune response directed against these therapeutic agents is an obstacle for achieving long-lasting clinical efficacy. Therefore, it is crucial to develop robust assays to accurately monitor cellular immunogenicity towards these therapies. Enzyme-linked immunospot (ELISpot) assay is one of the primarily used methods for measuring cellular immune response in clinical programs, which requires isolation of the peripheral blood mononuclear cells (PBMCs). The quality of this clinical material is one of the most critical factors that impact the robust assessment of cellular immune responses. The optimal blood sample processing conditions, however, remain poorly understood. In this study, we examined the impact of blood sample processing time on the performance characteristics of ELISpot to measure antigen-specific cellular responses. Blood samples that were processed after overnight delay resulted in a loss of ELISpot signals. We subsequently optimized several parameters of sample processing, and successfully recovered ELISpot signals for the blood samples that are processed within 32h. Furthermore, several mitigation strategies were employed that would potentially address the impact of granulocyte contamination on detection of antigen-specific cellular responses. Our investigation provides an extension of sample processing window for clinical studies and is significant for resolving the logistical challenge of whole blood sample shipment for timely PBMC preparation in cell/gene therapy clinical studies.

Impact of delayed PBMC processing on functional and genomic assays

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results.Whole blood samples from 20 healthy adults were collected and incubated at 20–25 °C for 2–48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2–4 h) and longer (8–48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs.PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased.PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.

Upgrading Monocytes Therapy for Critical Limb Ischemia Patient Treatment: Pre-Clinical and GMP-Validation Aspects.

Advanced cell therapy medicinal products (ATMP) are at the forefront of a new range of biopharmaceuticals. The use of ATMP has evolved and increased in the last decades, representing a new approach to treating diseases that are not effectively managed with conventional treatments. The standard worldwide recognized for drug production is the Good Manufacturing Practices (GMP), widely used in the pharma production of synthesized drugs but applying also to ATMP. GMP guidelines are worldwide recognized standards to manufacture medicinal products to guarantee high quality, safety, and efficacy. In this report, we describe the pre-clinical and the GMP upgrade of peripheral blood mononuclear cell (PBMC) preparation, starting from peripheral blood and ending up with a GMP-grade clinical product ready to be used in patients with critical limb ischemia (CLI). We also evaluated production in hypoxic conditions to increase PBMC functional activity and angiogenic potential. Furthermore, we extensively analyzed the storage and transport conditions of the final product as required by the regulatory body for ATMPs. Altogether, results suggest that the whole manufacturing process can be performed for clinical application. Peripheral blood collected by a physician should be transported at room temperature, and PBMCs should be isolated in a clean room within 8 h of venipuncture. Frozen cells can be stored in nitrogen vapors and thawed for up to 12 months. PBMCs resuspended in 5% human albumin solution should be stored and transported at 4 °C before injection in patients within 24 h to thawing. Hypoxic conditioning of PBMCs should be implemented for clinical application, as it showed a significant enhancement of PBMC functional activity, in particular with increased adhesion, migration, and oxidative stress resistance. We demonstrated the feasibility and the quality of a GMP-enriched suspension of monocytes as an ATMP, tested in a clean room facility for all aspects related to production in respect of all the GMP criteria that allow its use as an ATMP. We think that these results could ease the way to the clinical application of ATMPs.

32 (PB022) - Humanized mouse models for preclinical evaluation of novel immune therapies, checkpoint inhibitors and immune cell engagers

Background: The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have established a humanized immune system with functional T- B- and NK cells, as well as monocytes in immunodeficient mice by transfer of hematopoietic stem cells (HSCs) or peripheral blood mononuclear cells (PBMC). By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors or immune cell engagers. Methods: HSC-humanized mice were generated by single i.v. transplantation of CD34+ stem cells to immunodeficient NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. CDX and PDX from different entities were transplanted on the humanized mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections into tumor-bearing mice or with simultaneous tumor cell transplantation. These models were used to evaluate immune checkpoint inhibitors. Blood and tumor samples were analysed by FACS and immunohistochemistry for immune cell infiltration and activation. Results: The transplanted HSCs engrafted in mice and established a functional human differentiated immune system with proliferating immune cells. Up to 20% of the human immune cells in the blood were functional T cells, characterized by a high PD-1 expression 14 weeks after HSC inoculation. Selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of xenograft tumors revealed an increased percentage of tumor-infiltrating T cells. In addition, we identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other immune therapeutics. Conclusions: We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSCs on immunodeficient mouse strains generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors and immune cell engagers. Our human tumor-immune-cell models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. No conflict of interest.

The peripheral blood mononuclear cells preparation and the hematology of Varanus salvator.

The objectives of this study were to evaluate the proper anticoagulants coated in blood-collecting tube for the peripheral blood mononuclear cells (PBMCs) isolation and to evaluate the proper culture temperature for the Varanus salvator's PBMCs, in addition, the hematological characteristics also reported. The heparin treated blood (n = 10) and EDTA treated blood (n = 10) from Varanus salvator were obtained for PBMCs evaluation. The PBMCs obtained from the heparin treated blood was significantly higher than that of EDTA treated blood during the culture period (P < 0.05) indicated heparin would be more appropriated anticoagulant for blood collection. The PBMCs cultured under 37°C and 27°C were not significantly difference on first three days but 37°C showed significantly higher effect in the following days (P < 0.05) indicated both temperatures can be used which 37°C should be an optimal for PBMCs preparation. The peripheral blood cells of Varanus salvator (n = 49) were analyzed for hematological profiles and characteristics which the number of erythrocytes 1.19 ± 0.04 x 1012/L (1.17-1.35 x 1012/L) and WBC 2.41 ± 0.13 x 109/L (2.29-2.81 x 109/L) with absolute differential count of heterophils 0.92 ± 0.02 x 109/L (0.87-0.95 x 109/L), lymphocytes 1.17 ± 0.01 x 109/L (1.15-1.23 x 109/L), azurophils 0.40 ± 0.01 x 109/L (0.37-0.42 x 109/L), basophils 0.000 ± 0.001 x 109/L (0.004-0.011 x 109/L) and monocytes 0.027 ± 0.002 x 109/L (0.028-0.039 x 109/L). These results would play an important role on the cell immunological studies of the Varanus salvator in the future.

Abstract 1650: Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers

Abstract Background The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+stem cells to immunodeficient NOG mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted on those humanized mice. These models were used to evaluate novel immune therapies. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were functional T-cells, characterized by a high PD-1 expression. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of tumors revealed an increased percentage of tumor infiltrating T-cells. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors, cell therapies and immune cell engagers. Our models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1650.

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

Long non-coding RNAs as novel therapeutic targets in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50–60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.

PolyQ-expanded ataxin-3 protein levels in peripheral blood mononuclear cells correlate with clinical parameters in SCA3: a pilot study

In view of upcoming clinical trials, quantitative molecular markers accessible in peripheral blood are of critical importance as prognostic or pharmacodynamic markers in genetic neurodegenerative diseases such as Spinocerebellar Ataxia Type 3 (SCA3), in particular for signaling target engagement. In this pilot study, we focused on the quantification of ataxin-3, the protein altered in SCA3, in human peripheral blood mononuclear cells (PBMCs) acquired from preataxic and ataxic SCA3 mutation carriers as well as healthy controls, as a molecular marker directly related to SCA3 pathophysiology. We established two different highly sensitive TR-FRET-based immunoassays to measure the protein levels of either total full-length, non-expanded and expanded, ataxin-3 or specifically polyQ-expanded ataxin-3. In PBMCs, a clear discrimination between SCA3 mutation carrier and controls were seen measuring polyQ-expanded ataxin-3 protein level. Additionally, polyQ-expanded ataxin-3 protein levels correlated with disease progression and clinical severity as assessed by the Scale for the Assessment and Rating of Ataxia. Total full-length ataxin-3 protein levels were directly influenced by the expression levels of the polyQ-expanded ataxin-3 protein, but were not correlated with clinical parameters. Assessment of ataxin-3 levels in fibroblasts or induced pluripotent stem cells allowed to distinguish mutation carriers from controls, thus providing proof-of-principle validation of our PBMC findings across cell lines. Total full-length or polyQ-expanded ataxin-3 protein was not detectable by TR-FRET assays in other biofluids like plasma or cerebrospinal fluid, indicating the need for ultra-sensitive assays for these biofluids. Standardization studies revealed that tube systems, blood sampling, and PBMC preparation may influence ataxin-3 protein levels indicating a high demand for standardized protocols in biomarker studies. In conclusion, the polyQ-expanded ataxin-3 protein is a promising candidate as a molecular target engagement marker in SCA3 in future clinical trials, determinable even in—easily accessible—peripheral blood biomaterials. These results, however, require validation in a larger cohort and further standardization of modifying conditions.

Multi-site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system.

High-dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30-marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2-week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay-specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter-site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

Phenotype and Function of Activated Natural Killer Cells From Patients With Prostate Cancer: Patient-Dependent Responses to Priming and IL-2 Activation.

Background: Although immunotherapy has emerged as the “next generation” of cancer treatments, it has not yet been shown to be successful in the treatment of patients with prostate cancer, for whom therapeutic options remain limited to radiotherapy and androgen (hormone) deprivation therapy. Previous studies have shown that priming natural killer (NK) cells isolated from healthy individuals via co-incubation with CTV-1 cells derived from an acute lymphoblastic leukemia (ALL) enhances their cytotoxicity against human DU145 (metastatic) prostate cancer cells, but it remains unknown to what extent NK cells from patients with prostate cancer can be triggered to kill. Herein, we explore the phenotype of peripheral blood NK cells in patients with prostate cancer and compare the capacity of CTV-1 cell-mediated priming and IL-2 stimulation to trigger NK cell-mediated killing of the human PC3 (metastatic) prostate cancer cell line.Methods: The phenotype of resting, primed (co-incubation with CTV-1 cells for 17 h) and IL-2 activated (100 IU/ml IL-2 for 17 h) NK cells isolated from frozen-thawed peripheral blood mononuclear cell (PBMC) preparations from patients with benign disease (n = 6) and prostate cancer (n = 18) and their cytotoxicity against PC3 and K562 cells was determined by flow cytometry. Relationship(s) between NK cell phenotypic features and cytotoxic potential were interrogated using Spearman Rank correlation matrices.Results and Conclusions: NK cell priming and IL-2 activation of patient-derived NK cells resulted in similar levels of cytotoxicity, but distinct NK cell phenotypes. Importantly, the capacity of priming and IL-2 stimulation to trigger cytotoxicity was patient-dependent and mutually exclusive, in that NK cells from ~50% of patients preferentially responded to priming whereas NK cells from the remaining patients preferentially responded to cytokine stimulation. In addition to providing more insight into the biology of primed and cytokine-stimulated NK cells, this study supports the use of autologous NK cell-based immunotherapies for the treatment of prostate cancer. However, our findings also indicate that patients will need to be stratified according to their potential responsiveness to individual therapeutic approaches.

Phenotypic and functional stability of leukocytes from human peripheral blood samples: considerations for the design of immunological studies

BackgroundHuman peripheral blood mononuclear cells (PBMCs) are extensively used for research of immune cell functions, identification of biomarkers and development of diagnostics and therapeutics for human diseases, among others. The assumption that “old blood samples” are not appropriate for isolation of PBMCs for functional assays has been a dogma in the scientific community. However, partial data on the impact of time after phlebotomy on the quality and stability of human PBMCs preparations impairs the design of studies in which time-controlled blood sampling is challenging such as field studies involving multiple sampling centers/sites. In this study, we evaluated the effect of time after phlebotomy over a 24 h time course, on the stability of human blood leukocytes used for immunological analyses. Blood samples from eight healthy adult volunteers were obtained and divided into four aliquots, each of which was left in gentle agitation at room temperature (24 °C) for 2 h (control), 7 h, 12 h and 24 h post phlebotomy. All samples at each time point were independently processed for quantification of mononuclear cell subpopulations, cellular viability, gene expression and cytokine secretion.ResultsA 24 h time delay in blood sample processing did not affect the viability of PBMCs. However, a significantly lower frequency of CD3+ T cells (p < 0.05) and increased LPS-induced CXCL10 secretion were observed at 12 h post-phlebotomy. Alterations in TNFα, CCL8, CCR2 and CXCL10 gene expression were found as early as 7 h after blood sample procurement.ConclusionsThese data reveal previously unrecognized early time-points for sample processing control, and provide an assay-specific time reference for the design of studies that involve immunological analyses of human blood samples.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

Studies of Isolated Peripheral Blood Cells as a Model of Immune Dysfunction.

Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of immune function in studies of multiple medical areas, such as metabolic diseases and immune dysfunction. This chapter describes a standardized technique for blood draw and preparation of PBMCs from whole blood using density gradient centrifugation, followed by cell culture. The main focus is on collection of the PBMC culture media and extraction of cellular proteins in order to provide the materials for biomarker studies.

Preparation of Peripheral Blood Mononuclear Cells (PBMCs) as a Model for Proteomic Studies of Psychiatric Disorders.

Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of brain function in studies of psychiatric disorders, such as schizophrenia. This chapter describes the preparation of PBMCs from whole blood using a density gradient/cell culture protocol. This includes collection of the PBMC culture media and preparation of cell extracts in order to provide the raw materials for proteomic analyses.

Kinetics of human myeloid-derived suppressor cells after blood draw.

BackgroundHuman myeloid-derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes. While there is a huge scientific interest to study these cells in multiple human diseases, the methodological approach varies substantially between published studies. This is problematic as human MDSC seem to be a sensible cell type concerning not only cryopreservation but also time point after blood draw. To date data on delayed blood processing influencing cell numbers and phenotype is missing. We therefore evaluated the kinetics of granulocytic MDSC (gMDSC) and monocytic MDSC (mMDSC) frequencies after blood draw in order to determine the best time point for analysis of this recently defined cell type.MethodsIn this study, we isolated peripheral blood mononuclear cells (PBMC) of patients with HIV infection or solid tumors directly after blood draw. We then analyzed the frequencies of gMDSC and mMDSC 2, 4 and 6 h after blood draw and after an overnight rest by FACS analysis using the standard phenotypic markers. In addition, part of the cells was frozen directly after PBMC preparation and was measured after thawing.ResultsgMDSC levels showed no significant difference using fresh PBMC over time with a limitation for the overnight sample. However they were massively diminished after freezing (p = 0.0001 for all subjects). In contrast, frequencies of fresh mMDSC varied over time with no difference between time point 2 and 4 h but a significantly reduction after 6 h and overnight rest (p = 0.0005 and p = 0.005 respectively). Freezing of PBMC decreased the yield of mMDSC reaching statistical significance (p = 0.04). For both MDSC subgroups, FACS analysis became more difficult over time due to less sharp divisions between populations.ConclusionsAccording to our data human MDSC need to be studied on fresh PBMC. gMDSC can be studied with delay, mMDSC however should be studied no later than 4 h after blood draw. These results are crucial as an increasing number of clinical trials aim at analyzing MDSC nowadays and the logistics of blood processing implies delayed sample processing in some cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0755-y) contains supplementary material, which is available to authorized users.

Laser Ablation Inductively Coupled Plasma Mass Spectrometry: An Emerging Technology for Multiparametric Analysis of Tissue Antigens.

New analytical techniques for multiparametric characterisation of individual cells are likely to reveal important information about the heterogeneity of immunological responses at the single-cell level. In this proof-of-principle study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was applied to the problem of concurrently detecting 24 lineage and activation markers expressed by human leucocytes. This approach was sufficiently sensitive and specific to identify subpopulations of isolated T, B, and natural killer cells. Leucocyte subsets were also accurately detected within unfractionated peripheral blood mononuclear cells preparations. Accordingly, we judge LA-ICP-MS to be a suitable method for assessing expression of multiple tissue antigens in solid-phase biological specimens, such as tissue sections, cytospins, or cells grown on slides. These results augur well for future development of LA-ICP-MS-based bioimaging instruments for general users.