A global look into human T cell subsets before and after cryopreservation using multiparametric flow cytometry and two-dimensional visualization analysis

Lemieux, Jennifer; Jobin, Christine; Simard, Carl; Néron, Sonia

doi:10.1016/j.jim.2016.04.010

Cited by 44 publications

(75 citation statements)

References 37 publications

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“…To varying degrees cryopreservation has been implicated in both the stability of surface marker expression [14], [15] and in reduced T-cell functionality [16]. A recent paper from Lemieux [17] demonstrated that whilst the process of cryopreservation can reduce the ability to detect some T-cell phenotypes (predominantly CD3 + CD4 + ) by flow cytometry, this can be partly mitigated by allowing the thawed cells to rest before staining for phenotypic analysis. The effect of cryopreservation on B-cell function by ELISpot has also been studied [18] and the practice of cryopreservation largely endorsed for these purposes.…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

Ford

Wenden

Mbekeani

et al. 2017

Vaccine

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Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3–5-fold reduction of malaria antigen-specific IFNγ-producing CD3+CD4+ effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8+ T cells are relatively unaffected, as well as CD4+ T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines.

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Section: Introductionmentioning

confidence: 99%

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

Ford

Wenden

Mbekeani

et al. 2017

Vaccine

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“…LRSCs contain high numbers of peripheral blood mononuclear cells (PBMCs) and are a reliable source of human cells for research. As such, LRSCs are an alternative to peripheral blood collections or apheresis for the study of B cells, monocytes, dendritic cells (DCs), stem cells, and T cells . Regular platelet apheresis donors undergo extensive testing for transmissible diseases, are often well characterized in terms of human leukocyte antigen (HLA) typing as well as serostatus for common latent pathogens.…”

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confidence: 99%

Leukoreduction system chambers are a reliable cellular source for the manufacturing of T‐cell therapeutics

et al. 2018

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BACKGROUND Following solid organ or hematopoietic cell transplantation, refractory opportunistic viral reactivations are a significant cause of morbidity and mortality but can effectively be controlled by virus‐specific T‐cell transfer. Among effective and safe strategies is the use of “third‐party” (neither from the transplant donor nor recipient) virus‐specific T cells that can be manufactured from healthy donors and used as “off‐the‐shelf” therapies. Leukoreduction system chambers (LRSCs), recovered after routine plateletpheresis, were evaluated as a potential source of peripheral blood mononuclear cells (PBMCs) for the manufacturing of clinical‐scale virus‐specific T cell. STUDY DESIGN AND METHODS PBMCs from the same donors obtained either from LRSCs or peripheral blood were compared, focusing on T‐cell function and phenotype as well as the potential to generate cytomegalovirus (CMV)‐specific T‐cell lines from both CMV seropositive and seronegative donors. RESULTS PBMCs from both sources were comparable except for a transient downregulation of CD62L expression on freshly extracted PBMCs from LRSCs. Both nonspecific stimulation using anti‐CD3/CD28 antibodies and CMV peptides revealed that LRSCs or blood T cells were equivalent in terms of expansion, differentiation, and function. Moreover, PBMCs from LRSCs can be used to generate autologous monocyte‐derived dendritic cells to prime and expand CMV‐specific T cells from seronegative donors. CONCLUSION LRSCs are a reliable source of PBMCs for the generation of virus‐specific T cells for immunotherapy. These findings have implications for the development of third‐party therapeutic T‐cell products from well‐characterized blood product donors.

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“…For immuno-oncology research, sample sources primarily consist of blood, tumor tissue (liquid or solid), or bone marrow. It is important to consider, however, that cryopreservation may induce functional and phenotypic changes and may significantly affect the relative frequencies of viable peripheral blood mononuclear cell (PBMC) subpopulations (7)(8)(9)(10)(11)(12)(13). Bone marrow and peripheral blood samples often undergo standardized Ficoll gradient separation through which mononuclear cells are harvested.…”

Section: Sample Preparation and Storage: The Crucial First Stepmentioning

confidence: 99%

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

et al. 2019

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The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work.

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A global look into human T cell subsets before and after cryopreservation using multiparametric flow cytometry and two-dimensional visualization analysis

Cited by 44 publications

References 37 publications

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

Leukoreduction system chambers are a reliable cellular source for the manufacturing of T‐cell therapeutics

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

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