Functional Assays Evaluating Immunosuppression Mediated by Myeloid‐Derived Suppressor Cells

Reuven, Or; Mikula, I.; Ashkenazi‐Preiser, Hadas; Twaik, Nira; Ben‐Meir, Kerem; Meirow, Yaron; Daniel, Leonor; Kariv, Guy; Kurd, Mahdi; Baniyash, Michal

doi:10.1002/cpz1.557

Cited by 5 publications

(4 citation statements)

References 18 publications

(32 reference statements)

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“…Eluted purified cells are now ready for further use, such as flow cytometry as described in Basic Protocol 2. In addition, the obtained cells can be further used for evaluating MDSC suppressive features and suppressive function, following protocols described in a Current Protocols article by Reuven et al (2022).…”

Section: Preparation Of Single-cell Suspension For Cell Separationmentioning

confidence: 99%

“…Cells are now ready for further use, such as for flow cytometry staining following Basic Protocol 2. Functional assays are described in a companion Current Protocols article by Reuven et al (2022). a Fluorophore panels can be modified as desired, according to the experiment and the equipment available.…”

Section: Resuspend Tumor Pellet In Elbmentioning

confidence: 99%

“…The gating strategy for flow cytometry of MDSCs allows for distinguishing total MDSCs and MDSC subpopulations. The isolated MDSCs obtained following these protocols are suitable for evaluating their suppressive features, functions, and differentiation state (described in detail in a companion Current Protocols article by Reuven et al., 2022).…”

Section: Introductionmentioning

confidence: 99%

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Phenotypic Characterization and Isolation of Myeloid‐Derived Suppressor Cells

Reuven¹,

Mikula²,

Ashkenazi‐Preiser³

et al. 2022

Current Protocols

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Myeloid‐derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood‐derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single‐cell suspension generation from different tissue Alternate Protocol 1: Single‐cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan‐MDSCs or PMN‐MDSC and M‐MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN‐MDSC and M‐MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples

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Section: Preparation Of Single-cell Suspension For Cell Separationmentioning

confidence: 99%

Section: Resuspend Tumor Pellet In Elbmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Phenotypic Characterization and Isolation of Myeloid‐Derived Suppressor Cells

Reuven¹,

Mikula²,

Ashkenazi‐Preiser³

et al. 2022

Current Protocols

Self Cite

View full text Add to dashboard Cite

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“…To further quantify cell subpopulations and characterize MDSC levels and phenotype, refer to the Current Protocols article by Reuven et al (2022a). To further characterize suppressive features and functions, refer to the parallel Current Protocols article by Reuven et al (2022b).…”

Section: Sacrifice On Day 16mentioning

confidence: 99%

An In Vivo Mouse Model for Chronic Inflammation–Induced Immune Suppression: A “Factory” for Myeloid‐Derived Suppressor Cells (MDSCs)

Ben‐Meir¹,

Twaik²,

Meirow³

et al. 2022

Current Protocols

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Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells known to play a role in perpetuating a wide range of pathologies, such as chronic infections, autoimmune diseases, and cancer. MDSCs were first identified in mice by the markers CD11b + Gr1 + , and later, based on their morphology, they were classified into two subsets: polymorphonuclear MDSCs, identified by the markers CD11b + Ly6G + Ly6C Low , and monocytic MDSCs, detected as being CD11b + Ly6G -Ly6C Hi . MDSCs are studied as immunosuppressive cells in various diseases characterized by chronic inflammation and are associated with disease causes/triggers such as pathogens, autoantigens, and cancer. Therefore, different diseases may diversely affect MDSC metabolism, migration, and differentiation, thus influencing the generated MDSC functional features and ensuing suppressive environment. In order to study MDSCs in a pathology-free environment, we established and calibrated a highly reproducible mouse model that results in the development of chronic inflammation, which is the major cause of MDSC accumulation and immune suppression. The model presented can be used to study MDSC phenotypes, functional diversity, and plasticity. It also permits study of MDSC migration from the bone marrow to peripheral lymphatic and non-lymphatic organs and MDSC crosstalk with extrinsic factors, both in vivo and ex vivo. Furthermore, this model can serve as a platform to assess the effects of anti-MDSC modalities.

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The current state and future of T-cell exhaustion research

Jenkins

Whitehead

Fellermeyer

et al. 2023

Oxford Open Immunology

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“Exhaustion” is a term used to describe a state of native and redirected T-cell hypo-responsiveness resulting from persistent antigen exposure during chronic viral infections or cancer. Although a well-established phenotype across mice and humans, exhaustion at the molecular level remains poorly defined and inconsistent across the literature. This is, in part, due to an overreliance on surface receptors to define these cells and explain exhaustive behaviours, an incomplete understanding of how exhaustion arises, and a lack of clarity over whether exhaustion is the same across contexts, e.g., chronic viral infections versus cancer. With the development of systems-based genetic approaches such as single-cell RNA-seq and CRISPR screens applied to in vivo data, we are moving closer to a consensus view of exhaustion, although understanding how it arises remains challenging given the difficulty in manipulating the in vivo setting. Accordingly, producing and studying exhausted T-cells ex vivo is burgeoning, allowing experiments to be conducted at scale up and with high throughput. Here, we first review what is currently known about T-cell exhaustion and how it’s being studied. We then discuss how improvements in their method of isolation/production and examining the impact of different microenvironmental signals and cell interactions has now become an active area of research. Finally, we discuss what the future holds for the analysis of this physiological condition and, given the diversity of ways in which exhausted cells are now being generated, propose the adoption of a unified approach to clearly defining exhaustion using a set of metabolic-, epigenetic-, transcriptional-, and activation-based phenotypic markers, that we call ‘M.E.T.A.’.

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Functional Assays Evaluating Immunosuppression Mediated by Myeloid‐Derived Suppressor Cells

Cited by 5 publications

References 18 publications

Phenotypic Characterization and Isolation of Myeloid‐Derived Suppressor Cells

Phenotypic Characterization and Isolation of Myeloid‐Derived Suppressor Cells

An In Vivo Mouse Model for Chronic Inflammation–Induced Immune Suppression: A “Factory” for Myeloid‐Derived Suppressor Cells (MDSCs)

The current state and future of T-cell exhaustion research

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