Purpose and Appropriate Sample Types
WE designed and optimized an inter-convertible flow cytometry panel for the analysis and sorting of human CD4+ regulatory T cells (Treg) utilizing all of the major, currently accepted marker combinations for Treg identification. The panel is optimized for use with peripheral blood mononuclear cells (PBMC). In addition, the panel allows for the identification, classification, and isolation of activated, antigen-experienced Treg, and provides estimates of proliferation and suppressive capacity. To this end we created two, easily inter-convertible sub-panels: ATREG—which includes intra-nuclear markers, and BTREG—using surface markers only. Both sub-panels have been tested on cryopreserved PBMC from healthy donors and those with human autoimmune diseases, as well as on long-term cultured human Treg and non-Treg cell lines and clones.
Background
Identification, enumeration and isolation of human Treg remains challenging due to the lack of a single Treg-specific surface marker. Additional complexity is added by the intra-nuclear expression of the Treg lineage marker, FoxP3, its upregulation in activated human non-Treg effector cells, and its constitutive low-level expression in a distinct population of CD4+ cytokine-secreting non-suppressive cells (1,2). Currently, different marker combinations are in use in basic and clinical research, including in clinical trials (3). The populations defined by these different marker combinations are not completely inter-changeable, leaving room for diverging interpretations. They may also lack sensitivity, specificity, or both, depending on the population or marker combination chosen. This has hampered the meaningful interpretation of study results as it poses problems for the generalizability and validity of claims of reliable Treg identification. An additional problem may be disease-innate alterations in expression levels of molecules currently used to define Treg, such as CD127 and CD25 in chronic inflammatory/autoimmune diseases, following therapeutic interventions in cancer immunotherapy, or the absence of certain Treg subpopulations in tumor samples, for example, FoxP3int/CD45RApos (4–6). In human systemic lupus erythematous (SLE), for instance, CD25 expression may be high but not necessarily reflecting an increase in Treg numbers, necessitating the inclusion of non-CD25 containing marker combinations (e.g., FoxP3/HELIOS) for a more reliable and differentiated approach toward Treg phenotyping and enumeration (4).
Here, we incorporated cell surface and intra-nuclear markers commonly used for the delineation of all major, commonly accepted human Treg classification schemes into one single panel (ATREG). This enables the detection and characterization of Treg according to all currently accepted major Treg definitions (and beyond). In addition, several non-essential, generally somewhat less commonly used markers in Treg classification provide further information on homing, activation status, suppressive activity (CCR7/CD38/HLA-DR/CD39); and markers that enable sorting of presumably thymic human Treg (TIGIT/CD226) were added. ATREG is easily convertible into a derivate panel which uses surface markers exclusively in order to enable sorting of all major cell populations (BTREG) and a large variety of their subsets.
Priority was given to the reliable identification of the following Treg-Defining Populations (TDP) which has been suggested for use in clinical trials (3):
TDP-1 = CD127lo/CD25hi/FoxP3pos
TDP-2 = CD45RApos/FoxP3low” “resting Treg” (rTreg) and FoxP3high/CD45RAneg “activated” Treg (aTreg)
TDP-3 = FoxP3pos/HELIOSpos, presumably thymic Treg
These essential Treg populations identifiable in ATREG are recapitulated in the surface panel BTREG through the use of surface markers that correlate with their existence and represent analogs to the marker combinations used in the intracellular panel as shown in Table 3.
Table 3.
Treg-defining populations
| TREG- DEFINING POPULATION |
ATREG (INCLUDES INTRA-NUCLEAR STAINING) CD3+CD4+ |
CORRESPONDING BTREG ANALOG (SURFACE STAINING ONLY) CD3+CD4+ |
REFERENCES AND REMARKS |
|---|---|---|---|
| TDP-1 | CD127lo | CD127lo | (3,7) |
| CD25hi | CD25hi | ||
| FoxP3pos | |||
| TDP-2 | FoxP3lo/hi | CD25lo/hi | (1,3) |
| CD45RApos/neg | CD45RApos/neg | ||
| TDP-3 | FoxP3pos/ | CD127loCD25hi | (3,8,9) |
| Heliospos | CD226pos/neg | ||
| TIGITpos/neg | |||
The panel enables the further characterization of TDP in terms of the following properties: (i) Activation status (CD38, HLA-DR), (ii) Naïve/memory phenotype/antigen-experienced/inexperienced (CCR7, CD45RA), (iii) ontogeny (presumably thymic versus induced: HELIOS, TIGIT/CD226), (iv) suppressive capacity and stability (CD39) (10), and (v) proliferation (Ki67). Ki67 was considered essential given its usefulness in delineating activated Treg populations which may be of prognostic value in malignant disease (3). It was therefore included in the intra-nuclear subpanel ATREG.
Following the testing of a wide variety of potential candidate reagents in single titration experiments, the panel was optimized in a series of individual, separately performed “add-in experiments,” sequentially adding one new antibody/staining/concentration in each experiment. All optimizations were performed with FMO controls to enable meaningful assessment of cell populations through accurate gating. FMO controls also were highly useful in assessing performance of reagent combinations in terms of brightness/sensitivity, spillover, and background signals in each “add-in” experiment performed (Fig. 1; Tables 1 and 2).
Figure 1.
Identification, classification, analysis and isolation of human CD4+ regulatory T cells. (A) CD4+ T cells. Following exclusion of doublets, dead and CD8+ cells, a small lymphocyte gate is set and cells are gated either as direct descendants of CD3+/CD4+ cells (giving rise to TDP2 and 3, as well as SE2), or CD3+/CD4+ CD127lo/CD25hi/FoxP3+ (defining TDPl), or CD3+/CD4+CD127lo/CD25hi (defining SE1 and giving rise to SE3). (B) Regulatory T cells. Gates are shown for all Treg-defining populations, their predecessors (blue), and their surface equivalents (red). Predecessor populations for the surface-staining variant of the panel prior to the CD127lo/CD25hi gating level are omitted. Dotted lines signify their conceptual relationship with the CD4+ T cell analog from the intra-nuclear staining version of the panel. (C) Treg function and ontogeny. All Treg-defining populations can be assessed for activation (HLA- DR), proliferation (Ki67), immunologic memory (CCR7/CD45RA) and presumed thymic origin (i.e., HELIOS+ or TIGIT+). The surface staining version of the panel (BTREG) allows sorting of Treg “surface equivalents” SE1–3 as well as their respective activated (HLA-DR/CD38), naive/central-/peripheral –memory/TEMRA (CCR7/CD45RA), thymic (TIGIT+), and presumably functionally stable subsets under pro-inflammatory conditions (CD39). Exclusion of CD226 expressing cells (in particular CD226+TIGIT− cells) can be used to enrich Treg with superior purity and stability in in-vitro culture. Gates are set according to FMO controls. Where differential expression levels of antigens are critical, that is, CD25/FoxP3 “high versus low,” gates were set according to appearance of the population of relevance as well as according to experience acquired through the use of reference Treg and T effector cell clones.
Table 1.
Summary table for applications of OMIP-053
| Purpose | Human CD4+ Treg identification, |
| enumeration, sorting, and classification | |
| Species | Homo sapiens |
| Cell type | PBMC, long-term cultured Treg |
| Cross-references | OMIP-004, 006, 015 |
Table 2.
Reagents used for OMIP-053
| SPECIFICITY | CLONE | FLUOROCHROME | PURPOSE |
|---|---|---|---|
| Dead cells | N/A (dye) | Fixable blue | Viability |
| CD8 | SK1 | Brilliant violet 650™ | Exclusion |
| CD3 | SK7 | PerCP/Cy5.5 | Lineage |
| CD4 | SK3 | Brilliant violet 510™ | Lineage |
| CD127 | A019D5 | Brilliant violet 421™ | Treg identification (TDP I), sorting |
| CD25 | M-A251 | PE/dazzle™ 594 | Treg identification (TDP I and II), sorting |
| CD45RA | HI100 | Brilliant violet 711™ | Treg identification (TDP II), Treg activation status; |
| Naïve/memory classification | |||
| CCR7 | G043H7 | PE | Naïve/memory classification |
| HLA-DR | L243 | FITC | Activation |
| OPEN | OPEN | AF700 | Open |
| FoxP3 (ATREG PANEL) | 236A/E7 | APC | Treg identification (TDP I–III) |
| HELIOS (ATREG PANEL) | 22F6 | PE/Cy7 | Treg identification (TDP III) |
| Ki-67 (ATREG PANEL) | B56 | BUV395 | Proliferation |
| CD38 (BTREG PANEL) | HB7 | BUV395 | Activation |
| CD39 (BTREG PANEL) | A1 | APC/Fire™ 750 | Suppressive capacity and stability under inflammatory conditions |
| CD226 (DNAM-1) (BTREG PANEL) | HB7 | PE/Cy7 | Exclusion of functionally unstable Treg for in vitro expansion; tTreg identification |
| TIGIT (BTREG PANEL) | MBSA43 | APC | tTreg identification |
Similarity to Published OMIPs
OMIP-004
OMIP-006
OMIP-015
Our panel is related to the previously published OMIPs listed above. In contrast to those, and specific to this panel, are the following properties: It allows for (i) identification of the major Treg defining populations in one single panel (ATREG), (ii) sorting of their corresponding population equivalents identified by surface markers only (BTREG), including sorting for presumably thymic Treg (TIGIT/CD226), and for Treg with presumably superior functional stability in in vitro expansion (CD226−); and under pro-inflammatory conditions (CD39+), as well as (iii) assessment of Treg and CD4 T effector cell proliferation.
Acknowledgments
Grant sponsor: National Eye Institute of the National Institutes of Health, Grant number K08EY025324
Footnotes
Additional Supporting Information may be found in the online version of this article.
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