Vincenzo BrontepersName, Sven BrandaupersName, Shu‐Hsia ChenpersName, Mario P. ColombopersName, Alan B. FreypersName, Tim F. GretenpersName, Susanna MandruzzatopersName, Peter J. MurraypersName, Augusto C. OchoapersName, Suzanne Ostrand‐RosenbergpersName, Paulo C. Rodrı́guezpersName, Antonio SicapersName, Viktor UmanskypersName, Robert H. VonderheidepersName, Dmitry I. GabrilovichpersName (2016date). “Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.” Nature Communicationstitle·venue.
Object of study — A CELL CLASS THAT MAY NOT BE A CELL TYPE - myeloid-derived suppressor cells, named nine years earlier to capture a function rather than a lineage, and now given minimal phenotypic, molecular and functional reporting criteria because the field had drifted from the original intent
Research question — what a paper must report before calling a cell an MDSCterm·def, when no marker combination is unique to the class and the defining function is often unmeasurable
Contribution — minimal reporting standards for mouse and human MDSCterm·def: named subsets (PMN-MDSCterm, M-MDSC, eMDSCterm·def) with gating criteria, required functional demonstration of T-cell suppression, and a molecular/biochemical table
phenomenon · claim-type · cross-domain-transfer · mechanism · claim-relation · replication-status · limitation — grounded in the full text| phenomenon | a term coined to organise a research programme, adopted by a field publishing 500 papers a year, and drifting away from what it was coined to mean — “The marked growth of publications over the last years has led to deviation from the original intent of the MDSC nomenclature. The objective of this review article is not to provide a comprehensive analysis of the MDSC biology, which has been done in many recent publications, but to suggest minimal phenotypic, molecular and functional criteria for reporting and classifying MDSCs. We also propose to integrate the increasing observations, in which expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression.” | |
| claim-type | method — “MDSC are the subject of intensive research with >500 papers published in 2015 alone. However, the phenotypic, morphological and functional heterogeneity of these cells generates confusion in investigation and analysis of their roles in inflammatory responses. The purpose of this communication is to suggest characterization standards in the burgeoning field of MDSC research.” | |
| method — evidence | — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | |
| object of study — evidence | — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | |
| research question — evidence | — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” | |
| contribution — evidence | — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” | |
| mechanism | A NAME INTRODUCED DELIBERATELY AS A RESEARCH FRAMEWORK, NOT AS A NATURAL KIND: `The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies.` The corpus has definitions built to classify, to diagnose and to enrol. This one was built to ORGANISE A LITERATURE, and its authors say so — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | mechanisms[0] |
| mechanism | AND THE USAGE HAS DRIFTED FROM THE INTENT, MEASURABLY: `The marked growth of publications over the last years has led to deviation from the original intent of the MDSC nomenclature ... We also propose to integrate the increasing observations, in which expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression.` Cells that look like the class and lack the function the class is named for - and the response is to widen the term rather than to exclude them — “The marked growth of publications over the last years has led to deviation from the original intent of the MDSC nomenclature. The objective of this review article is not to provide a comprehensive analysis of the MDSC biology, which has been done in many recent publications, but to suggest minimal phenotypic, molecular and functional criteria for reporting and classifying MDSCs. We also propose to integrate the increasing observations, in which expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression.” | mechanisms[1] |
| mechanism | AND A SUBOPTIMAL TERM KEPT FOR CONTINUITY - THE THIRD SUCH DECISION IN THIS CORPUS: `despite realization that the term `MDSC` may not be optimal, we feel that it is purposeful and should be retained to assure consistency as the field continues to develop.` After EWGSOP keeping `sarcopenia` against a term it concedes is better suited, and buck2011's `pragmatic compromise ... legacy usage` — “Therefore, despite realization that the term ‘MDSC' may not be optimal, we feel that it is purposeful and should be retained to assure consistency as the field continues to develop.” | mechanisms[2] |
| mechanism | AND THE CLASS CANNOT BE APPLIED BY THE INSTRUMENT THAT IS SUPPOSED TO APPLY IT: `These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation.` The definition is by surface markers and the surface markers do not separate the class - the separation is by DENSITY — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” | mechanisms[3] |
| mechanism | AND IN TISSUE IT IS NOT HARD BUT IMPOSSIBLE: `In mice, Gr-1 or Ly6G staining is used to identify tumour-associated MDSC. However, the distinction between neutrophils or monocytes and MDSC in frozen or paraffin-embedded tissues is impossible.` The word is the authors'. Most of the world's archived tumour material cannot be classified by this vocabulary at all — “In mice, Gr-1 or Ly6G staining is used to identify tumour-associated MDSC. However, the distinction between neutrophils or monocytes and MDSC in frozen or paraffin-embedded tissues is impossible.” | mechanisms[4] |
| mechanism | AND THE PRECISE NATURE OF A NAMED SUBSET IS UNKNOWN BECAUSE THE ASSAY IS POPULATION-LEVEL: `Since functional, biochemical and genomic characterization is performed on entire population of cells, and single-cell functional analysis is not yet feasible, the precise nature of PMN-MDSC remains unclear.` The class is defined per cell and measured per population — “Since functional, biochemical and genomic characterization is performed on entire population of cells, and single-cell functional analysis is not yet feasible, the precise nature of PMN-MDSC remains unclear.” | mechanisms[5] |
| mechanism | AND THE OBJECTION TO THE CATEGORY IS STATED BY THE PEOPLE DEFENDING IT: `One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different.` The counter-argument is written into the paper that answers it — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | mechanisms[6] |
| mechanism | AND TWO RENAMINGS, EACH WITH ITS REASON: `Initially we and others used the term `GRANULOCYTIC MDSC` to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils` - and `since some immaturity is a common trait of all MDSC subsets, we propose to define them more properly as `EARLY-STAGE MDSC` (eMDSC)`. The second is the sharper move: a modifier dropped because it does not DISCRIMINATE — “These cells have been defined as immature MDSC and the mouse equivalent is yet to be identified. However, since some immaturity is a common trait of all MDSC subsets, we propose to define them more properly as ‘early-stage MDSC' (eMDSC).” | mechanisms[7] |
| mechanism | AND THE CONFUSION HAS A NAMED PRE-HISTORY: `It became apparent that CD11b+ Gr-1+ cells WERE HETEROGENEOUS, which generated uncertainty in description of these cells among different groups of investigators, an ambiguity amplified by the use of different acronyms to define these cells.` The term was coined to end an acronym proliferation - buck2011's problem, nine years earlier and one field over — “It became apparent that CD11b + Gr-1 + cells were heterogeneous, which generated uncertainty in description of these cells among different groups of investigators, an ambiguity amplified by the use of different acronyms to define these cells.” | mechanisms[8] |
| mechanism | AND THE ACTIVATION STATE THAT JUSTIFIES THE CATEGORY IS DEFINED BY CONTRAST: `Classical activation of neutrophils and monocytes has evolved to protect the host ... In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals ... these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.` A class defined by a history of stimulation rather than by a marker — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” | mechanisms[9] |
| extends | the 2007 proposal that introduced the term 'myeloid-derived suppressor cells', whose intent was 'not to define a novel population' but to give the field a framework - and from which usage has since deviated — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | claim_relations[0] |
| critiques | the practice of designating cells MDSC on phenotype alone, when 'expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression' — “The marked growth of publications over the last years has led to deviation from the original intent of the MDSC nomenclature. The objective of this review article is not to provide a comprehensive analysis of the MDSC biology, which has been done in many recent publications, but to suggest minimal phenotypic, molecular and functional criteria for reporting and classifying MDSCs. We also propose to integrate the increasing observations, in which expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression.” | claim_relations[1] |
| sense | `MDSC` (2007 coinage) - a term chosen for `the myeloid origin, the immune-suppressive function and the systemic expansion`, explicitly NOT a claim about a distinct lineage — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | senses[0] |
| sense | `PMN-MDSC` - replaces `granulocytic MDSC` because the cells are `phenotypically distinct from steady-state neutrophils` in six named ways — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” | senses[1] |
| sense | `eMDSC` (early-stage MDSC) - replaces `immature MDSC`, because `some immaturity is a common trait of all MDSC subsets` and the modifier therefore discriminated nothing — “These cells have been defined as immature MDSC and the mouse equivalent is yet to be identified. However, since some immaturity is a common trait of all MDSC subsets, we propose to define them more properly as ‘early-stage MDSC' (eMDSC).” | senses[2] |
| sense | `pathological activation` - the state that separates MDSC from monocytes and neutrophils: persistent low-strength stimulation, poor phagocytosis, high ROS/RNS and anti-inflammatory cytokines, against short-lived CLASSICAL activation — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” | senses[3] |
| control | requiring a functional demonstration of T-cell suppression, with the input MDSC number TITRATED and the loss of suppression shown as the cells are titrated down — held — “A number of molecules produced by MDSCs have been implicated in suppression (discussed below) including: arginases, NO, ROS, IDO, TGFβ and PGE 2 , among others. Although important for a thorough understanding of MDSC suppressive mechanism(s), evaluation of their expression cannot substitute for functional assays. In different settings MDSCs utilize different mechanisms of suppression and it is difficult to predict which will be more prevalent. It is also challenging to ascertain what level of production of any given effector molecule is sufficient for the MDSC suppressive activity.” — rules out artefacts due to MDSC viability, and designation of cells as MDSC on phenotype alone — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” | controls[0] |
| control | controls with the SAME PHENOTYPE and preferably the SAME TISSUE, from healthy donors or naive mice — partial — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” — rules out attributing to MDSC what any cell of that phenotype would do - and the paper names the exception, that neutrophils are rarely in the PBMC fraction of healthy donors — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” | controls[1] |
| control | density-gradient centrifugation at 1.077 g/l to separate PMN-MDSC from neutrophils — failed — “Since functional, biochemical and genomic characterization is performed on entire population of cells, and single-cell functional analysis is not yet feasible, the precise nature of PMN-MDSC remains unclear.” — rules out nothing cleanly - 'The limitations of this approach are unavoidable'; the low-density fraction contains activated neutrophils too — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” | controls[2] |
| weight | the 2007 proposal that introduced the term 'myeloid-derived suppressor cells', whose intent was 'not to define a novel population' but to give the field a framework - and from which usage has since deviated — 0.95 — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | claim_relations[0] |
| weight | the practice of designating cells MDSC on phenotype alone, when 'expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression' — 0.85 — “The marked growth of publications over the last years has led to deviation from the original intent of the MDSC nomenclature. The objective of this review article is not to provide a comprehensive analysis of the MDSC biology, which has been done in many recent publications, but to suggest minimal phenotypic, molecular and functional criteria for reporting and classifying MDSCs. We also propose to integrate the increasing observations, in which expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression.” | claim_relations[1] |
| note | PMN-MDSC —distinct-from→ neutrophils — `Initially we and others used the term `GRANULOCYTIC MDSC` to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b).` The rename is justified BY the distinction - and elsewhere the paper says the two cannot be separated except by density gradient — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” | concept_edges[0] |
| relation | PMN-MDSC —distinct-from→ neutrophils — phenotypically distinct in six named ways - and that distinctness is the reason the subset was RENAMED from 'granulocytic MDSC' — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” — “but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils” | concept_edges[0] |
| weight | PMN-MDSC —distinct-from→ neutrophils — 0.95 — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” | concept_edges[0] |
| note | pathological activation —distinct-from→ Classical activation — 'Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals.' A class distinguished by the HISTORY of its stimulation rather than by anything measurable at one moment - which is why no marker combination is unique to it — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” | concept_edges[1] |
| relation | pathological activation —distinct-from→ Classical activation — classical activation is short-lived and ends with the stimulus; pathological activation is the product of PERSISTENT low-strength stimulation — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” — “Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals” | concept_edges[1] |
| weight | pathological activation —distinct-from→ Classical activation — 0.95 — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” | concept_edges[1] |
| note | MDSCs —distinct-from→ monocytes — 'The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils.' The edge records a distinction that is real by function and invisible by phenotype - 'there are no combinations of markers unique to MDSC', and in fixed tissue the distinction is 'IMPOSSIBLE' — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | concept_edges[2] |
| relation | MDSCs —distinct-from→ monocytes — FUNCTIONALLY different - immune-suppressive activity - where the morphology and phenotype are similar — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” — “The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils” | concept_edges[2] |
| weight | MDSCs —distinct-from→ monocytes — 0.95 — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | concept_edges[2] |
| outcome | control — held — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” — “A number of molecules produced by MDSCs have been implicated in suppression (discussed below) including: arginases, NO, ROS, IDO, TGFβ and PGE 2 , among others. Although important for a thorough understanding of MDSC suppressive mechanism(s), evaluation of their expression cannot substitute for functional assays. In different settings MDSCs utilize different mechanisms of suppression and it is difficult to predict which will be more prevalent. It is also challenging to ascertain what level of production of any given effector molecule is sufficient for the MDSC suppressive activity.” | controls[0] |
| outcome | control — partial — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” | controls[1] |
| outcome | control — failed — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” — “Since functional, biochemical and genomic characterization is performed on entire population of cells, and single-cell functional analysis is not yet feasible, the precise nature of PMN-MDSC remains unclear.” | controls[2] |
| limitation | THE DEFINING FUNCTION HAS A GOLD STANDARD IN SCARE QUOTES AND NO THRESHOLD: `inhibition of T cells is THE `GOLD` standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria` - while elsewhere `it is challenging to ascertain what level of production of any given effector molecule IS SUFFICIENT for the MDSC suppressive activity`. A criterion that is sufficient and unquantified at the same time — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” | limitations[0] |
| limitation | AND THE MECHANISM CANNOT STAND IN FOR THE FUNCTION: `A number of molecules produced by MDSCs have been implicated in suppression ... Although important for a thorough understanding of MDSC suppressive mechanism(s), evaluation of their expression cannot substitute for functional assays. In different settings MDSCs utilize different mechanisms of suppression and it is difficult to predict which will be more prevalent.` No marker, no molecule, and no fixed mechanism - only the assay — “A number of molecules produced by MDSCs have been implicated in suppression (discussed below) including: arginases, NO, ROS, IDO, TGFβ and PGE 2 , among others. Although important for a thorough understanding of MDSC suppressive mechanism(s), evaluation of their expression cannot substitute for functional assays. In different settings MDSCs utilize different mechanisms of suppression and it is difficult to predict which will be more prevalent. It is also challenging to ascertain what level of production of any given effector molecule is sufficient for the MDSC suppressive activity.” | limitations[1] |
| limitation | AND THE CLASS IS CUT FROM A CONTINUUM: `Since MDSC are a continuum of cells in different stages of differentiation, assignment of biochemical and molecular MDSC markers will depend on presently incompletely defined processes that govern differentiation stages of altered myelopoiesis, and some markers might thus be shared by cells with different phenotypes or with mature activated monocytes or neutrophils.` The boundary is drawn across a gradient whose governing processes are not yet described — “Since MDSC are a continuum of cells in different stages of differentiation, assignment of biochemical and molecular MDSC markers will depend on presently incompletely defined processes that govern differentiation stages of altered myelopoiesis, and some markers might thus be shared by cells with different phenotypes or with mature activated monocytes or neutrophils.” | limitations[2] |
| limitation | and one named subset has no counterpart in the model organism: `These cells have been defined as immature MDSC and the mouse equivalent is yet to be identified` - a class defined in humans, renamed to eMDSC in the same paragraph, with nothing on the other side of the translational bridge to compare it to — “These cells have been defined as immature MDSC and the mouse equivalent is yet to be identified. However, since some immaturity is a common trait of all MDSC subsets, we propose to define them more properly as ‘early-stage MDSC' (eMDSC).” | limitations[3] |
finding · quantity · variable · definition · key-term — grounded in the full text| finding | — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | content_tags.findings[0] |
| finding | — “The marked growth of publications over the last years has led to deviation from the original intent of the MDSC nomenclature. The objective of this review article is not to provide a comprehensive analysis of the MDSC biology, which has been done in many recent publications, but to suggest minimal phenotypic, molecular and functional criteria for reporting and classifying MDSCs. We also propose to integrate the increasing observations, in which expansion of cells with MDSC morphology, phenotype and main biochemical features is not accompanied by immune suppression.” | content_tags.findings[1] |
| finding | — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” | content_tags.findings[2] |
| finding | — “In mice, Gr-1 or Ly6G staining is used to identify tumour-associated MDSC. However, the distinction between neutrophils or monocytes and MDSC in frozen or paraffin-embedded tissues is impossible.” | content_tags.findings[3] |
| finding | — “Therefore, despite realization that the term ‘MDSC' may not be optimal, we feel that it is purposeful and should be retained to assure consistency as the field continues to develop.” | content_tags.findings[4] |
| finding | — “These cells have been defined as immature MDSC and the mouse equivalent is yet to be identified. However, since some immaturity is a common trait of all MDSC subsets, we propose to define them more properly as ‘early-stage MDSC' (eMDSC).” | content_tags.findings[5] |
| quantity | more than 500 papers in 2015 alone — — “MDSC are the subject of intensive research with >500 papers published in 2015 alone. However, the phenotypic, morphological and functional heterogeneity of these cells generates confusion in investigation and analysis of their roles in inflammatory responses. The purpose of this communication is to suggest characterization standards in the burgeoning field of MDSC research.” | content_tags.quantities[0] |
| quantity | none — — “These gating criteria cannot discriminate monocytes from M-MDSCs and neutrophils from PMN-MDSC since at present there are no combinations of markers unique to MDSC. Presently, the only method allowing for separation of neutrophils from PMN-MDSC is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSC are enriched in low density (mononuclear cell fraction), whereas neutrophils are high-density cells” | content_tags.quantities[1] |
| quantity | six: fewer granules, altered buoyancy, reduced CD16 and CD62L, increased arginase 1, peroxynitrite, CD11b and CD66b — — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” | content_tags.quantities[2] |
| variable | immune-suppressive activity — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | content_tags.variables[0] |
| definition | MDSC — “In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSC), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSC in a cancer-related context 6 . The initial intent to introduce MDSC nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSC are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.” | content_tags.definitions[0] |
| definition | eMDSC — “These cells have been defined as immature MDSC and the mouse equivalent is yet to be identified. However, since some immaturity is a common trait of all MDSC subsets, we propose to define them more properly as ‘early-stage MDSC' (eMDSC).” | content_tags.definitions[1] |
| concept | PMN-MDSC — not defined in this paper's own words; the polymorphonuclear MDSC subset, renamed from 'granulocytic MDSC' — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” | concepts[0] |
| concept | neutrophils — not defined in this paper's own words; the cells PMN-MDSC are phenotypically distinct from and cannot be separated from by surface markers alone — “Initially we and others used the term ‘granulocytic MDSC' to describe PMN-MDSC, but now we believe that the latter term better defines this MDSC subset, since PMN-MDSC are phenotypically distinct from steady-state neutrophils (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b)” | concepts[1] |
| concept | MDSCs — not defined in this paper's own words; the class as a whole, defined by function where the phenotype is shared — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | concepts[2] |
| concept | monocytes — not defined in this paper's own words; the mature myeloid cells M-MDSC share a phenotype with — “One of the most controversial aspects of MDSC biology is why cells with the morphology and phenotype similar to neutrophils and monocytes should require a designation of PMN-MDSC and M-MDSC. The main consideration reflects the potent immune-suppressive activity of MDSCs, the basis to define them as functionally different from monocytes and neutrophils. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSC, which demonstrate that they are pathologically activated.” | concepts[3] |
| concept | pathological activation — not defined in this paper's own words; persistent low-strength stimulation producing poorly phagocytic, immune-suppressive myeloid cells — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” | concepts[4] |
| concept | Classical activation — not defined in this paper's own words; the short-lived protective activation state that terminates when the stimulus does — “Classical activation of neutrophils and monocytes has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activation is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential.” | concepts[5] |
| sufficiency without a level | — “The ability to suppress immune cells is an important characteristic of MDSC. Although MDSC were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSC function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSC, provided that they meet the phenotypic criteria described above.” | content_tags.statistics[0] |
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Nat Commun Nat Commun 2873 ncomms Nature Communications 2041-1723 Nature Publishing Group PMC4935811 PMC4935811.1 4935811 4935811 27381735 10.1038/ncomms12150 ncomms12150 1 Review Article Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards BrontepersName Vincenzo a 1 * BrandaupersName Sven 2 ChenpersName Shu-Hsia 3 ColombopersName Mario P. 4 FreypersName Alan B. 5 GretenpersName Tim F. 6 MandruzzatopersName Susanna 7 8 MurraypersName Peter J. 9 OchoapersName Augusto 10 Ostrand-RosenbergpersName Suzanne 11 Rodriguez Paulo C. 12 SicapersName Antonio 13 14 UmanskypersName Viktor 15 16 VonderheidepersName Robert H. 17 GabrilovichpersName Dmitry I. b 18 * 1 Department of Medicine, University Hospital, University of Verona , Verona 37134, Italy 2 Department of Otorhinolaryngology, University Hospital Essen , Essen D-45122, Germany 3 Department of Oncological Sciences, Tisch Cancer Institute, Immunology Institute, Icahn School of Medicine at Mount Sinai , New York, New York 10029, USA 4 Department of Experimental Oncology and Molecular Medicine, Molecular Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori , Milano 20133, Italy 5 New York University School of Medicine , New York, New York 10029, USA 6 GI-Malignancy Section, Thoracic and GI Oncology Branch, NCI , Bethesda, Maryland 20892, USA 7 Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padova , Padova 35128, Italy 8 Veneto Institute of Oncology IOV-IRCCS , Padova 35128, Italy 9 Departments of Infectious Diseases and Immunology, St Jude Children's Research Hospital , Memphis, Tennessee 38105, USA 10 Stanley S. Scott Cancer Center, Louisiana State University , New Orleans, Louisiana 70112, USA 11 University of Maryland Baltimore County , Baltimore, Maryland 21250, USA 12 Georgia Regents University Cancer Center , Augusta, Georgia 30912, USA 13 Humanitas Clinical and Research Center , Via Manzoni 56, Rozzano, Milan 20089, Italy 14 Department of Pharmaceutical Sciences, Università del Piemonte Orientale ‘Amedeo Avogadro' , via Bovio 6, Novara 20089, Italy 15 Skin Cancer Unit, German Cancer Research Center (DKFZ) , Heidelberg 69120, Germany 16 Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg , Mannheim 69120, Germany 17 Abramson Cancer Center, University of Pennsylvania School of Medicine , Philadelphia, Pennsylvania 19104, USA 18 Translational Tumor Immunology, The Wistar Institute , Philadelphia, Pennsylvania 19104, USA a vincenzo.brontepersName@univr.it b dgabrilovich@wistar.org * These authors contributed equally to this work.
06 07 2016 2016 7 264076 12150 04 11 2015 02 06 2016 06 07 2016 14 07 2016 24 03 2024 Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Myeloid-derived suppressor cells (MDSCterm·def) are a heterogeneous population expanded in cancer and other chronic inflammatory conditions. Here the authors identify the challenges and propose a set of minimal reporting guidelines for mouse and human MDSCterm·def.
Myeloid-derived suppressor cells (MDSCsterm) have emerged as major regulators of immune responses in cancer and other pathological conditions. In recent years, ample evidence supports key contributions of MDSCterm·def to tumour progression through both immune-mediated mechanisms and those not directly associated with immune suppression. MDSCterm·def are the subject of intensive research with >500 papers published in 2015 alone. However, the phenotypic, morphological and functional heterogeneity of these cells generates confusion in investigation and analysis of their roles in inflammatory responses. The purpose of this communication is to suggest characterization standards in the burgeoning field of MDSCterm·def research.
pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement no pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes pmc-license-ref CC BY Definition of MDSCterm·def At steady-state, myelopoiesis is a structured process where progeny of common precursors acquire specific markers and functions of circulating leucocytes, and in doing so progressively lose the ability to self-renew. The turnover of mature leucocytes is substantial, with billions of cells generated and replaced daily. A variety of pathological conditions can perturb the steady supply of leucocytes resulting in emergency myelopoiesis 1 , which serves to provide cells to eliminate potential threats, either abnormal cellular growth (cancer), infectious agents or tissue damage. If these conditions resolve quickly, the balance of myeloid cells is restored without negative consequences for the host. However, a number of conditions associated with various types of chronic inflammation, autoimmune diseases and cancer results in aberrant, sustained myelopoiesis characterized by the accumulation of immature myeloid cells that deviate from the standard path of differentiation. These cells are distinct from mature, terminally differentiated myeloid cells (macrophage, dendritic cells or neutrophilsterm) and have an activation programme (pathologic activation), which is different from that of mature myeloid cells.
Early studies on inflammation in the mouse highlighted a shared, systemic expansion of myeloid cells bearing the markers CD11b (CR3A or integrin αM) and Gr-1 (anti-Gr-1 mAbs recognize epitopes common to Ly6C and Ly6G) (refs 2 , 3 , 4 , 5 ). It became apparent that CD11b + Gr-1 + cells were heterogeneous, which generated uncertainty in description of these cells among different groups of investigators, an ambiguity amplified by the use of different acronyms to define these cells. In an attempt to codify analysis of the nature and clinical significance of these cells, 9 years ago a group of investigators suggested to utilize the term of ‘myeloid-derived suppressor cells' (MDSCterm·def), informed by the myeloid origin, the immune-suppressive function and the systemic expansion of MDSCterm·def in a cancer-related context 6 . The initial intent to introduce MDSCterm·def nomenclature was not to define a novel population of myeloid cells (at that point being clear that MDSCterm·def are not a distinct myeloid lineage), but to provide a term that captured the function, origin and heterogeneity of the cells and offered a framework to guide studies on these remarkable players in tumour-dependent immune dysfunction.
Since the inception of this term, interest in MDSCterm·def has blossomed. MDSCsterm are implicated in various aspects of immune regulation in diseases that involve chronic inflammation, especially cancer, but also infection, autoimmune diseases, trauma, graft versus host disease and so on. Recently, evidence of the clinical significance of MDSCterm·def in cancer has emerged. Therefore, despite realization that the term ‘MDSCterm·def' may not be optimal, we feel that it is purposeful and should be retained to assure consistency as the field continues to develop.
However, several notions about MDSCterm·def now require reassessment. First, the cellular nature of MDSCterm·def is now better defined and includes two major subsets based on their phenotypic and morphological features: polymorphonuclear (PMN) and monocytic (M)-MDSC, and to reflect those discoveries, the terms PMN-MDSCterm and M-MDSC were introduced 7 8 . Initially we and others used the term ‘granulocytic MDSCterm·def' to describe PMN-MDSCterm, but now we believe that the latter term better defines this MDSCterm·def subset, since PMN-MDSCterm are phenotypically distinct from steady-state neutrophilsterm (having less granules, altered buoyancy, reduced CD16 and CD62L, and increased arginase 1, peroxynitrite, CD11b and CD66b) (refs 9 , 10 ). It is apparent now that PMN-MDSCterm and M-MDSC are not only phenotypically and morphologically distinct, but also have unique (although partially overlapping) functional characteristics and biochemical traits, which reflect their different roles under various pathological conditions. Therefore, we believe that the definition of MDSCterm·def in scientific reporting needs to include the specific subset of MDSCsterm under investigation.
Second, even though functional analyses of immunoregulatory activity are often lacking for practical reasons (typically relating to the paucity of MDSCterm·def in human samples), the systemic expansion of circulating myeloid cells and the correlation with clinical outcomes have been reported for both solid and hematologic human malignancies, confirming the concept that tumours can influence, at different stages, the ‘distant' hematopoietic compartment 11 12 . The recent identification of specialized molecular programs that orchestrate MDSCterm·def differentiation, joined with high-throughput technologies (see below), has provided new insight in the multifaceted and unique myeloid cell development leading to MDSCterm·def generation.
The marked growth of publications over the last years has led to deviation from the original intent of the MDSCterm·def nomenclature. The objective of this review article is not to provide a comprehensive analysis of the MDSCterm·def biology, which has been done in many recent publications, but to suggest minimal phenotypic, molecular and functional criteria for reporting and classifying MDSCsterm. We also propose to integrate the increasing observations, in which expansion of cells with MDSCterm·def morphology, phenotype and main biochemical features is not accompanied by immune suppression. The criteria for defining cells as MDSCsterm should include phenotypic and functional, and, if possible, molecular characteristics described below.
Phenotypic markers to define MDSCterm·def In mice, MDSCsterm historically were defined as cells expressing both Gr-1 and CD11b markers. Although initially useful in identifying MDSCterm·def, the use of this original criterion is no longer sufficient since subpopulations have been shown to exist: PMN-MDSCterm (CD11b + Ly6G + Ly6C lo ) and M-MDSC (CD11b + Ly6G − Ly6C hi ). Example of staining is provided in Fig. 1 . These cell-surface markers provide only an initial framework for characterization and can be complemented by various other markers (described in detail elsewhere) 13 14 .
In human peripheral blood mononuclear cell (PBMC), the equivalent to PMN-MDSCterm are defined as CD11b + CD14 − CD15 + or CD11b + CD14 − CD66b + and M-MDSC as CD11b + CD14 + HLA-DR −/lo CD15 − . CD33 myeloid marker can be used instead of CD11b since very few CD15 + cells are CD11b − . While M-MDSC express the myeloid marker CD33, PMN-MDSCterm display CD33 dim staining 15 . Lin − (including CD3, CD14, CD15, CD19, CD56) HLA-DR − CD33 + cells contain mixed groups of MDSCterm·def comprising more immature progenitors. These cells have been defined as immature MDSCterm·def and the mouse equivalent is yet to be identified. However, since some immaturity is a common trait of all MDSCterm·def subsets, we propose to define them more properly as ‘early-stage MDSCterm·def' (eMDSCterm·def). At this point, it appears that any characterization of MDSCterm·def needs to include each of these cell populations ( Table 1 ). Example of staining is provided in Fig. 2 . A recent report based on the experience of many laboratories around the world, suggested specific gating parameters for harmonized phenotyping of human MDSCterm·def by flow cytometry 16 . Although various additional populations of myeloid cells were described in that analysis, their morphological, biochemical or functional distinctions from each other and from PMN-MDSCterm, M-MDSC or eMDSCterm·def are not yet clear. In contrast to the three main populations, which are found (at various frequencies) in all types of cancer and every pathological condition, some of those cells may exist in one type of cancer but absent in the other. It is likely that there is a substantial overlap between cells belonging to different subsets. Therefore, more detailed analysis of MDSCterm·def populations is needed for better characterization. However, the minimum (and currently sufficient) requirements for definition and characterization of human MDSCsterm (PMN-MDSCsterm, M-MDSCs or eMDSCsterm·def) according to the phenotypic criteria are described in Table 1 . Detailed discussion of the specific gating criteria for phenotyping of mouse and human MDSCterm·def can be also found elsewhere 17 . These gating criteria cannot discriminate monocytesterm from M-MDSCs and neutrophilsterm from PMN-MDSCterm since at present there are no combinations of markers unique to MDSCterm·def. Presently, the only method allowing for separation of neutrophilsterm from PMN-MDSCterm is gradient centrifugation using 1.077 g l −1 density (standard Ficoll gradient used for the isolation of mononuclear cells). PMN-MDSCterm are enriched in low density (mononuclear cell fraction), whereas neutrophilsterm are high-density cells 15 . The limitations of this approach are unavoidable. Low-density fraction contains not only PMN-MDSCterm, but some activated neutrophilsterm and probably some PMN-MDSCterm can pass through gradient and contaminate high-density fraction of neutrophilsterm. Therefore, CD11b + CD14 − CD15 + /CD66b + cells in low-density fraction contain both PMN-MDSCterm and neutrophilsterm. These cells are heterogeneous in their morphology (containing both mature and immature cells) 18 . Since functional, biochemical and genomic characterization is performed on entire population of cells, and single-cell functional analysis is not yet feasible, the precise nature of PMN-MDSCterm remains unclear. An important goal for future studies is to define cell-surface markers and gating strategies that uniquely identify the different populations of MDSCterm·def. One of the most pressing unmet issues is to determine markers of MDSCterm·def that would allow detection of cells in unseparated peripheral blood.
It is important to emphasize that the method for collecting and analysing MDSCterm·def can influence the results: freezing of samples lead to substantial loss of cells, especially PMN-MDSCterm 19 , and thus comparison with freshly isolated cells is necessary.
Furthermore, one of the main challenges in the field is the phenotypic characterization of MDSCterm·def in tissues by immunohistochemistry without isolation and functional characterization of the cells. In mice, Gr-1 or Ly6G staining is used to identify tumour-associated MDSCterm·def. However, the distinction between neutrophilsterm or monocytesterm and MDSCterm·def in frozen or paraffin-embedded tissues is impossible. In human samples similar confusion of MDSCterm·def with monocytesterm and neutrophilsterm exists. Similarly, CD33, a myeloid marker that sometimes is used for identification of MDSCterm·def, does not allow for distinction of MDSCterm·def from macrophages, dendritic cells or other myeloid cells in tissues. Recently, a combination of CD33 with S100A9 was suggested to identify MDSCsterm 20 . However, although this pair of markers excludes most dendritic cells and macrophages (due to their low expression of S100A9), it does not permit a clear distinction of MDSCterm·def from neutrophilsterm. Thus, at this moment, clear phenotypic characterization of human and mouse MDSCterm·def by immunohistochemistry is lacking.
It is important to emphasize that phenotypic evaluation is a starting point for the analysis of MDSCterm·def (see the proposed algorithm below) since it defines population of cells in both naive and pathologically changed hosts. However, expansion of myeloid compartment under pathologic conditions is a critical, first step that allow for further evaluation of MDSCterm·def. For instance, in healthy donor cells with typical PMN-MDSCterm phenotype in the peripheral blood mononuclear cell fraction are practically undetectable and cells with M-MDSC phenotype are present in much smaller numbers than in patients with either melanoma or chronic infections 11 . In mice, similar situation exists in spleen. The proportion of splenic MDSCsterm increases from 1–2% in naive mice to 10–15% or more in many cancers. In bone marrow and peripheral blood of mice the differences in the proportion of the cells are smaller (usually increase is <twofold) (refs 8 , 21 ).
Functional assays to define MDSCterm·def The ability to suppress immune cells is an important characteristic of MDSCterm·def. Although MDSCterm·def were implicated in suppression of different cells of the immune system such as NK and B cells 22 23 24 , inhibition of T cells is the ‘gold' standard for evaluation of MDSCterm·def function and inhibition of T-cell activity appears to be sufficient for designation of cells as MDSCterm·def, provided that they meet the phenotypic criteria described above. Different methods for the evaluation of T-cell suppression are used in the literature (summarized in Table 2 ) and can be divided into two groups. One method assesses antigen-specific suppression and utilizes antigen-specific T cells, activated with cognate peptides or in an allogeneic mixed leucocyte reaction (MLR), in the presence of titrated numbers of purified input MDSCterm·def. The second, non-specific suppression, uses T cells activated by CD3 antibody, alone or in combination with CD28 (immobilized or presented by APC), or with lectins (ConA in mice or PHA in humans, although an influence of lectins on other cell types contained in the in vitro assay is a concern for potential technical biases). However, antigen-specific suppression assays are preferable when possible, since these assays are likely more relevant to in vivo conditions. Various methods have been used to determine T-cell function activity modulated by MDSCterm·def. Measurement of T-cell proliferation (either with 3 H-thymidine incorporation or by CFSE dilution) or inhibition of interferon (IFN)-γ production (ELISPOT or intracellular staining) provides experimental evidence supporting functions associated with MDSCterm·def. It is important to note that, under some experimental conditions, MDSCterm·def could cause inhibition of T-cell proliferation without affecting IFN-γ production and vice versa. The titration of input MDSCterm·def numbers in these tests (and the cognate loss of suppression as the cells are titrated down) is a critical experimental tool to help eliminating the possibility of artefacts due to MDSCterm·def viability and to aid in comparison between experiments. One of the critical issues to consider is the use of appropriate controls—cells with the same phenotype and preferably from the same tissues from either healthy donors or naive mice. It is important to point out that there are several exceptions since neutrophilsterm (cells with the phenotype similar to PMN-MDSCterm) are rarely present in the PBMC fraction of healthy donors. In contrast to PMN-MDSCterm from tumour-bearing mice and cancer patients, neutrophilsterm are largely absent from lymph nodes of naive mice and healthy donors.
The evaluation of tolerogenic activity of MDSCterm·def in vivo by using adoptive transfer of MDSCterm·def and antigen-specific T cells to syngeneic recipients, with subsequent stimulation of antigen-specific T cells with cognate peptide, provides the most comprehensive way to study different pathways of MDSCterm·def activity. However, this method requires considerable effort and probably is not feasible for routine evaluation of MDSCterm·def activity. Examples of the in vivo tolerogenic use of MDSCsterm could be found in several publications 25 26 .
In humans, evaluation of MDSCterm·def effects on antigen-specific immune responses is difficult due to the limitation in generation of antigen-specific T cells. Recent advances allow for transduction of human T cells with lentiviruses expressing cloned T-cell receptors, which permits analysis of suppression in antigen-specific assays using cognate peptides in the presence of autologous antigen-presenting cells and MDSCterm·def. Currently, a three-way allogeneic MLR is a reliable method for functional assessment of human MDSCterm·def. This assay utilizes cells obtained from a pair of unrelated healthy donors: one is the source of T cells, the other one provides APCs. The pair is selected based on strong T-cell proliferative or IFN-γ responses of the responder, and aliquots of cells can be stored for use in subsequent iterative experiments. MDSCsterm from cancer patients are tested in MLR at different ratios compared with responder T cells. The assay is based on the premise that allogeneic MLR requires presentation of epitopes in the context of MHC class II and class I, so that suppression of responses reflects the ability of MDSCterm·def to prevent antigen-specific T-cell immune responses 27 .
Measurement of human antigen-non-specific suppression of T-cell responses by MDSCterm·def is based on the same principles as in mice, and utilizes either anti-CD3/CD28 antibody or activation with lectins. In several studies, evaluation of MDSCterm·def activity was substantiated by antibody-mediated depletion of candidate cells from PBMC before analysis of T-cell proliferation. Although this approach is less attractive, due to technical limitations in complete MDSCterm·def removal and the unintended depletion of non-MDSC that may share cross-reactive cell-surface molecules, it may provide valuable information about MDSCterm·def functional activity. To provide for better comparison between different studies, it is very important to include detailed information about the experimental protocol used for all steps involved in the purification of MDSCterm·def. Since some antibodies used for positive isolation of MDSCterm·def may modify function of these cells, it is preferable (when possible) to use negative-selection approaches. In any case, it is important to select control groups to account for possible impact of isolation techniques on MDSCterm·def function.
A number of molecules produced by MDSCsterm have been implicated in suppression (discussed below) including: arginases, NO, ROS, IDO, TGFβ and PGE 2 , among others. Although important for a thorough understanding of MDSCterm·def suppressive mechanism(s), evaluation of their expression cannot substitute for functional assays. In different settings MDSCsterm utilize different mechanisms of suppression and it is difficult to predict which will be more prevalent. It is also challenging to ascertain what level of production of any given effector molecule is sufficient for the MDSCterm·def suppressive activity.
Biochemical and molecular characteristics of MDSCsterm One of the most controversial aspects of MDSCterm·def biology is why cells with the morphology and phenotype similar to neutrophilsterm and monocytesterm should require a designation of PMN-MDSCterm and M-MDSC. The main consideration reflects the potent immune-suppressive activityterm·var of MDSCsterm, the basis to define them as functionally different from monocytesterm and neutrophilsterm. However, in recent years studies have identified unique biochemical and molecular characteristics of MDSCterm·def, which demonstrate that they are pathologically activated. Classical activationterm of neutrophilsterm and monocytesterm has evolved to protect the host from bacteria and viruses, as well as to provide support for the remodelling of tissues after injury or after resolved inflammation. This activation state is characterized by robust phagocytosis, respiratory burst activity and release of pro-inflammatory cytokines. Myeloid activation is relatively short-lived and terminated on cessation of the stimulus. In contrast, pathological activationterm is the result of persistent stimulation of the myeloid compartment with relatively low-strength signals coming from tumours or sites of chronic inflammation. Myeloid cells generated under these conditions are poorly phagocytic, produce high levels of reactive oxygen and nitrogen species, and predominantly anti-inflammatory cytokines 9 . As a result, these cells are not able to perform effectively the normal functions of myeloid cells and acquire potent immune-suppressive potential. Reflecting their potent immunosuppressive character it possible that the main role of MDSCterm·def is in the protection of the host from extensive tissue damage caused by uncontrolled immune response associated with unresolved inflammation or infection. Tumours can hijack and amplify this activity to protect themselves from elimination by the immune system.
Since MDSCterm·def are a continuum of cells in different stages of differentiation, assignment of biochemical and molecular MDSCterm·def markers will depend on presently incompletely defined processes that govern differentiation stages of altered myelopoiesis, and some markers might thus be shared by cells with different phenotypes or with mature activated monocytesterm or neutrophilsterm. For example, conversion of monocytesterm to granulocytes, a process known as transdifferentiation, occurs in tumour-bearing but not tumour-free mice 28 (or at least not at the same rate). Analogously, genes contributing to the immune-suppressive phenotype, normally confined to some cell types under steady state, might be activated in MDSCsterm 29 .
On the basis of mostly mouse studies, four types of molecular and biochemical parameters are often associated with immune-suppressive MDSCterm·def as opposed to monocytesterm and neutrophilsterm ( Table 3 ). (1) Transcription factors and apoptosis regulators; (2) a signature of pro- and anti-inflammatory cytokines and cytokine receptors; (3) chemoattractants and related receptors affecting MDSCterm·def trafficking; and (4) enzymes and metabolic by-products contributing to MDSCterm·def immune-regulatory functions. Table 3 also indicates which molecular and biochemical parameters are often linked with the specific MDSCterm·def populations (considering PMN-MDSCsterm, M-MDSCs and total MDSCsterm). This grouping is not exhaustive, but is intended to incorporate key and non-redundant hubs related to MDSCterm·def biology. Although numbers of different parameters are implicated in MDSCterm·def biology and may help to define a molecular signature of MDSCsterm, some parameters are important for the development of the MDSCterm·def field and, thus, may help to identify MDSCsterm (highlighted in Table 3 ). Several assays need to be integrated to evaluate these biomarkers, which exploit gene and protein expression, as well as multiple post-translational modifications. Transcription factors may require assessment of their activity (DNA binding, phosphorylation). Technologies based on the detection of mRNA translation might be insufficient to classify MDSCterm·def as several mediators of MDSCterm·def function are regulated epigenetically or post-translationally. Epigenetic changes, such as histone modification related to myeloid differentiation, are an intensively studied and promising area of investigation, and have the potential to facilitate tracking cell stages during fate mapping analyses 30 . However, currently there is no clear indication about epigenetic markers that can discriminate specific MDSCterm·def subsets. A similar consideration is valid for microRNAs, even though in our experience microRNA signatures can be a useful classifier of myeloid cell subsets under different pathological conditions 31 .
A panoply of cytokines can either influence the properties and/or are produced by one or more subsets of MDSCsterm. These cytokines may establish an autocrine feed-forward loop that sustains MDSCterm·def accumulation, since they are not only released during chronic inflammation inducing MDSCterm·def, but are also released by MDSCterm·def. Chemokines and their receptors might complement the assessment of the overall inflammatory state, however, no chemokine is truly specific for MDSCsterm.
An immune-regulatory activity of MDSCsterm depends on the metabolic consumption and conversion of the amino acids L -arginine and L -tryptophan, by the activity of inducible enzymes such as arginase 1 (ARG1), nitric oxide synthase 2 (NOS2/iNOS). Since most available antibodies to these enzymes are fraught with the lack of specificity and need to access the intracellular compartment where their targets are located, detection of these enzymes is often performed by RT–PCR. However, RNA and protein levels are not necessarily coincident, and the mere presence of either RNA or protein does not confirm enzyme activation. For this reason, biochemical analyses of downstream metabolites (such as NO, kinurenines, ornithine, urea and polyamines) might be an alternative assay. MDSCsterm are also high producers of soluble reactive species. Reactive oxygen species exemplified by superoxide anion (O 2 − ) and peroxide hydrogen (H 2 O 2 ), are generated by the activity of NADPH oxidase (NOX) family members, in which NOX2 is likely active in myeloid cells. Reactive nitrogen species, such as the free-radical peroxynitrite (ONOO – ), are by-products of the combined activity of iNOS, ARG1 and NOX, and can induce a number of detectable covalent alterations in select aromatic amino acids of proximal proteins by enzyme-independent nitration/nitrosylation ( Table 3 ). The complexity of the post-translational modifications induced by peroxynitrite, the ‘nitrome', is not fully appreciated yet, but detection of nitrotyrosine and nitrotryptophan may be convenient for evaluating overall reactive nitrogen species production in biological samples, provided that more effective/specific antibodies are developed in the future. Finally, the clinical use of new checkpoint-blockade inhibitors for the therapy of human cancers, supports evaluation of their ligands (for example, PD-L1) in MDSCsterm, whose expression is influenced by local environmental factors such as inflammatory cytokines or hypoxia 32 33 .
MDSCterm·def versus tumour-associated macrophages and neutrophilsterm Tumours contain a complex landscape of myeloid cells, which is associated with tumour progression and response to therapy 34 35 . However, the specific role of individual components of this landscape remains obscure. While the literature on tumour-associated myeloid cells assigned an unequivocal tumour-promoting activity to M-MDSCs and PMN-MDSCsterm 12 36 , both tumour-associated macrophages (TAM) and tumour-associated neutrophilsterm (TAN) can exert either tumour-promoting or anti-tumour activities in different cancers 37 38 . This dual capacity stems out from the apparent phenotypic plasticity of TAM and TAN, which is modulated through their transcriptional re-programming operated by distinct micro-environmental signals, at different stages of tumour progression 37 38 39 40 .
This raises an important question of how to distinguish these cells. In mice, PMN-MDSCterm and TAN can be separated from mononuclear cells within the CD11b + myeloid cell fraction by the expression of Ly6G granulocytic cell marker and eosinophils can be distinguished by the expression of sialic acid-binding immunoglobulin-like lectin F 41 42 . Mononuclear cells in tumours likely exist in various differentiation phases from monocytesterm/M-MDSCs towards TAM. Molecularly, this process is accompanied by the upregulation of anti-apoptotic molecules cFLIP and A1, as well as the enzyme ARG1 (refs 32 , 43; Fig. 3 ). Phenotypically, TAM can be distinguished from M-MDSCs by increased relative expression of F4/80, low-to-intermediate expression of Ly6C and low or undetectable expression of S100A9 protein. When compared with M-MDSCs, TAM express higher amounts of IRF8, a marker of terminal macrophage differentiation, and increased M-CSF receptor, CD115 (ref. 44 ). Most of the published data indicate that cells with the phenotype of inflammatory monocytesterm (CD11b + Ly6C hi Ly6G − ) in tumours have potent immune-suppressive activityterm·var and thus can be attributed to M-MDSCs 45 . However, whether this is indeed the case, can be clarified when specific markers of M-MDSCs are identified. There is evidence that PMN-MDSCsterm and M-MDSCs can be distinguished from neutrophilsterm and monocytesterm due to their elevated ER stress response 46 47 .
In the absence of functional tests, the distinction between neutrophilsterm and PMN-MDSCsterm is impossible, since these cells share the same phenotype. This limitation of phenotypical distinction between PMN-MDSCsterm and neutrophilsterm only partially alleviated with additional staining with some antibodies. Compared with mature neutrophilsterm, some PMN-MDSCsterm in the blood/spleen of mice have lower expression of the receptors for Fc (CD16/CD32) and complement (C5aR) and higher expression of the transcription factor retinoic-acid-related orphan receptor, an inhibitor of neutrophilterm maturation 37 , In some tumour models, some PMN-MDSCsterm, in contrast to neutrophilsterm from naive mice, may express CD115 and CD244 (ref. 9 ). However, these markers have limited direct value for delineation of these cells due to apparent heterogeneity of PMN-MDSCsterm. A whole-transcriptomic analysis provided a pattern of gene expression that allows for discrimination between spleen PMN-MDSCsterm from tumour-bearing mice, and spleen and bone marrow neutrophilsterm from tumour-free naive mice 9 48 .
The situation with TAN is different. TAN are a heterogeneous population of cells with some cells demonstrating pro-tumourigenic and some anti-tumour activity 38 49 . The distinction between TAN and PMN-MDSCsterm is currently impossible, since these cells share the same phenotype. A nomenclature on circulating neutrophilsterm and TANs was recently suggested based on different parameters, such as gradient density, morphology function and tissue localization 50 . While concepts about cell subsets are certainly helpful for future discussions, mechanistic evidence about distinct maturation and differentiation steps involving neutrophilsterm are still preliminary. For instance, the terms anti-tumourigenic N1 and pro-tumourigenic N2 mouse neutrophilsterm were introduced to describe different populations of TAN 18 38 . However, the phenotype and functional characteristics of N2 neutrophilsterm are very similar to that of PMN-MDSCsterm, which necessitate better characterization of these cells. This notion is supported by data demonstrating potent immune-suppressive activityterm·var by TAN, which defines these cells as PMN-MDSCsterm 51 52 53 . A whole-transcriptomic analysis demonstrated a substantial difference between TAN and spleen neutrophilsterm and PMN-MDSCsterm 37 . However, the transcriptomic analysis of true tumour PMN-MDSCsterm will not be possible until markers separating these cells from TAN are discovered.
In humans, macrophage-specific markers CD68 and CD163, as well as low or absent expression of S100A9 can be used to discriminate between TAM and tumour M-MDSCs. The challenges in distinguishing between tumour PMN-MDSCsterm and TAN are the same as in mice: no clear cell-surface markers exist so far to allow for direct separation between TAN and PMN-MDSCsterm.
Proposed experimental algorithm for MDSCterm·def reporting Immune-suppressive activityterm·var has been long considered as the major characteristic of MDSCsterm, and the lack of unique phenotypic markers made it previously impossible to characterize these cells as MDSCsterm if immune-suppressive activityterm·var was not detected. These cells have been referred to as TAN or monocytesterm 38 54 , but recently this designation has been refined. More sophisticated biochemical and gene-expression profiling has allowed for characterization of these cells as pathologically activated, immature myeloid cells distinct from myeloid cells found in the steady state. Immune-suppressive activityterm·var, although a critically important attribute of MDSCterm·def, is not always associated with the cells present in tumour-bearing hosts or in conditions of chronic inflammation. For example, early stages of cancer or initial stages of chronic inflammation may be associated with accumulation of cells with phenotypic ( Table 1 ) and biochemical characteristics ( Table 3 ) of MDSCterm·def but lacking potent suppressive activity. Moreover, it is possible that even in advanced-stage cancer, not all cells with a MDSCterm·def phenotype possess immune-suppressive activityterm·var. Although the nature of the factors responsible for acquisition of the immune-suppressive phenotype of MDSCterm·def is not entirely clear, non-suppressive MDSCsterm can nonetheless regulate various aspects of tumour onset and progression 5 12 36 . In fact, recent studies demonstrated that in the setting of chronic inflammation cells with an MDSCterm·def phenotype, but lacking suppressive activity contribute to the early stages of tumour inflammation 20 55 . Since the term ‘MDSCterm·def' presumes suppressive activity, this term should not be used to identify cells lacking this function. Therefore, when describing these cells we suggest the term MDSC-like cells (MDSC-LC; Fig. 4 ).
For defining MDSCterm·def populations in mice and humans, we propose an algorithmic approach that first focuses on the phenotypic characterization of the cells ( Table 1 ). If cells meet these criteria, then suppressive activity must be determined ( Table 2 ). The presence of suppressive activity would define these cells as MDSCterm·def. However, if cells lack suppressive activity, then major biochemical characteristics of these cells need to be evaluated ( Table 3 ). On the basis of the available data we suggest the following criteria that could help to define MDSC-LC in the absence of suppressive activity. The analysis should include appropriate controls: cells with the same phenotype from healthy individuals or naive mice. These criteria include changes in at least two critical transcription factors and regulators—such as, IRF8, phospho-STAT3, c/EBPb, S100A8/A9, Rb1—and upregulation of at least one critical cytokine/receptor—such as, IL-10, TGFβ, IL-4R—or immune-regulatory molecule—such as,ARG1, NOS2, NOX2, PNT, PGE 2 . The number of parameters can be further expanded as new information becomes available. The absence of relevant biochemical characteristics would then exclude these cells as MDSCterm·def, whereas the presence of these markers would allow characterization of these cells as MDSC-LC ( Fig. 4 ). Definition of molecular pathways and markers for MDSC-LC will clarify steps of the differentiation pathway induced in MDSCterm·def by growing tumours and will help to dissect tumour-promoting functions of MDSCterm·def. Furthermore, a better understanding of the developmental complexity of these cells may provide additional information on the tumour-promoting functions of MDSCterm·def that either act in a dependent or independent manner to regulate adaptive and/or innate immunity.
Additional information How to cite this article: BrontepersName, V. et al. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat. Commun. 7:12150 doi: 10.1038/ncomms12150 (2016).
This work was supported by NIH grants CA 141438 and CA 084488 to D.I.G., GM021248 to S.O.-R., CA 184185 to P.C.R., grants from the Italian Ministry of Education, Universities and Research (FIRB cup: B31J11000420001; FIRB cup: RBAP11H2R9_005; PRIN cup: 20103FMJEN_003) to V.B. and A.S.; the Italian Association for Cancer Research (AIRC 6599, 12182, 12886, 14103, 14194, 14103 and 15585) and the Italian Ministry of Health (FINALIZZATA cup: E35G1400019001) to V.B., M.P.C., A.S., S.M.; Action BM1404 Mye-EUNITER supported by COST (European Cooperation in Science and Technology) to S.B. and V.U.; the German Research Association (DFG, grant 2278/2-1) to S.B., The American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital and Cancer Center Core grant P30 CA21765 to P.M. and the DKFZ-MOST Cooperation in Cancer Research (CA157) to V.U.
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Figure 1 Gating strategy for the identification of mouse MDSCterm·def subsets.
Gating strategy used to define MDSCterm·def subpopulations in BM, blood and spleen of C57Bl/6 tumour-free or MCA203 tumour-bearing mice. After exclusion of doublets (not shown), live CD11b + cells were gated and the proportion of Ly6C and Ly6G cells was evaluated.
Figure 2 Gating strategy for the identification of MDSCterm·def subsets in the peripheral blood of healthy donors and melanoma patients.
Doublets were excluded and live PBMC were gated (not shown). ( a ) CD14 + HLA-DR −/lo M-MDSC. Monocytesterm were gated on the basis of FSC and SSC parameters and HLA-DR downregulation was defined by FMO control. ( b ) Lin − HLA-DR − CD33 + eMDSCterm·def. ( c ) CD14 − CD15 + CD11b + PMN-MDSCterm.
Figure 3 Overview of MDSCterm·def involvement in myeloid cell differentiation in cancer.
In cancer and chronic inflammation, the bone marrow and spleen increase the output of mature and immature myeloid cells that comprise a spectrum between monocytesterm and neutrophilsterm. In mice, MDSCterm·def toward the monocytic end of the spectrum (M-MDSC) are CD11b + Ly6C + Ly6G − , while towards the neutrophilterm end of the spectrum (PMN-MDSCterm) are CD11b + LyG + Ly6C − . Within solid tumours M-MDSC develop through intermediate steps towards macrophages where Ly6C is progressively downregulated and MHCII, F4/80 and CX3CR1 are upregulated. Under chronic inflammation, monocytic lineages show an increasing requirement for anti-apoptotic survival pathways (mediated primarily by GM-CSF signalling) to block the intrinsic mitochondrial death pathway. A similar scheme is likely to occur in humans; however, the cell markers are different.
Figure 4 Algorithm for identification of cells as MDSCterm·def.
Step-by-step approach to identify cells as MDSCterm·def for reporting. It is important, wherever possible, to use cells with the same phenotype from control mice or healthy donors as controls.
Table 1 Minimal phenotypic characteristics necessary to identify cells as MDSCterm·def.
Mouse Phenotype Human (in PBMC fraction) Phenotype Total MDSCterm·def (not sufficient for MDSCterm·def characterization) Gr-1 + CD11b + Total (mixed) MDSCterm·def Not clearly determined PMN-MDSCterm CD11b + Ly6C lo Ly6G + PMN-MDSCterm CD14 − CD11b + CD15 + (or CD66b + ) M-MDSC CD11b + Ly6C hi Ly6G − M-MDSC CD11b + CD14 + HLA-DR low/− CD15 − eMDSCterm·def Not clearly determined e-MDSC Lin − (CD3/14/15/19/56)/HLA-DR − /CD33 + eMDSCterm·def, early-stage MDSCterm·def; MDSCterm·def, myeloid-derived suppressor cell; M-MDSC, monocytic-MDSC; PBMC, peripheral blood mononuclear cell; PMN-MDSCterm; polymorphonuclear-MDSC.
Although phenotype is the first necessary step for defining MDSCterm·def, please note that, it cannot be used as the sole parameter for distinction between PMN-MDSCterm and neutrophilsterm and M-MDSC and monocytesterm.
It is important, wherever possible, to use cells from control mice or healthy donors as controls.
Table 2 Minimal functional characteristics necessary to identify cells as MDSCterm·def.
Mouse functional tests Human functional tests Type of immune response Assays Autologous system Allogeneic system · Inhibition of antigen-non-specific function (anti-CD3/CD28 or ConA induced)· Inhibition of antigen-specific function using antigen-specific T cells (induced after immunization with peptides or from transgenic mice) · Inhibition of 3 H-thymidine incorporation or CFSE dilution· Inhibition of CTL activity· Inhibition of IFN-γ production by T cells in ELISPOT or intracellular staining· Inhibition of expression of CD3ζ chain on T cells· Inhibition of IL-2 production · Inhibition of anti-CD3/CD28 (or PHA) induced T-cell proliferation or IFN-γ production (in ELISPOT or by intracellular staining) by the addition of candidate MDSCterm·def populations· Improved T-cell proliferation after removal of candidate MDSCterm·def populations · Inhibition of proliferation or IFN-γ production by T cells (in ELISA, ELISPOT or by intracellular staining) by the addition of selected MDSCterm·def populations CTL, cytotoxic T lymphocyte; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; MDSCterm·def, myeloid-derived suppressor cell.
It is important, wherever possible, to use cells from control mice or healthy donors as controls.
For either antigen-specific or antigen-non-specific response, one assay is usually sufficient.
Table 3 Biochemical and molecular parameters associated with MDSCterm·def characterization.
Class of biomarkers Biomarker Detection technology Mainly found in Reference Transcription factors and apoptotic regulators ↓ IRF8 * Phospho-STAT3 * cEBP/β * S100A8/9 * RBPhospho-STAT5ROR/RORC1sXBP, CHOP FC, PPTM (FC, ELI), FAELI, P, T, FAELI, FC, IHC, P, TIF, P, T, FCPTM (FC, IHC, P), FA FC, PP, T MDSCMDSCMDSCMDSCM-MDSC>PMN-MDSCMDSCPMN-MDSCMDSC 56 57 58 59 26 31 60 61 28 † 62 44 46 47 Genes and molecules contributing to the immune-regulatory activity ARG1 * NOS2/NO * NOX2/ROS * PNT/RNS * VEGFPGE 2 PD-L1 E, FC, IHC, P, TFC, IF, IHC, P, TE, FC, P, TPTM (IHC), E, FCFC, IHCELIFC, P, T M-MDSC M-MDSCPMN-MDSCMDSCMDSCM-MDSCMDSC 63 64 65 64 8 66 46 59 67 27 68 65 69 70 71 72 73 Cytokines and receptors IL-10 * TGFβ * IL-4R (CD124) * ELI, FC, TELI, FC, T, PFC, T MDSCM-MDSCM-MDSC 74 75 66 76 63 77 Cytokines involved in MDSCterm·def development These cytokines are not produced by MDSCterm·def. However, they are important for the evaluation of MDSCterm·def microenvironment. GM-CSFG-CSFIL-13IL-1 ELI, TELI, TFCELI MDSCPMN-MDSCM-MDSCMDSC 21 78 79 21 79 80 63 81 22 74 75 E, enzyme assay; ELI, enzyme-linked immunosorbent assay; FA, functional activity (for example, DNA binding); FC, flow cytometry (including ICS, intracellular staining); IF, immunofluorescence; IHC, immunohistochemistry; IL, interleukin; MDSCterm·def, myeloid-derived suppressor cell; M-MDSC, monocytic-MDSC; P, protein detection in cell extracts or supernatants (that is, by western blot, mass spectrometry); PMN-MDSCterm; polymorphonuclear-MDSC; PTM, post-translational modification; T, transcript analysis (that is, by RT–PCR, RNA-seq or in situ hydridization).
It is important, wherever possible, to use cells from control mice or healthy donors as controls.
PMN-MDSCsterm are compared with neutrophilsterm and M-MDSCs are compared with monocytesterm.
Total population of MDSCsterm in most of these studies was compared with Gr-1 + CD11b + cells from control mice. Characteristics described in the table are the same for MDSCterm·def and MDSC-LC.
* Parameters crucial for MDSCterm·def biology and, thus, for their identification.
† In this study Rb expression was largely compared between the groups of M-MDSC and PMN-MDSCterm.