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Purpose and Appropriate Sample Types

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published Omips
  5. Literature Cited
  6. Supporting Information

The present panel was optimized to investigate the frequency and phenotype of regulatory T-cells (Treg), as well as the activation status of CD4+ and CD8+ T-cells in peripheral blood mononuclear cells (PBMC) from healthy individuals, without the use of intracellular staining (i.e., excluding the use of the canonical Treg marker, FoxP3). The panel has been developed using cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested.

Background

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published Omips
  5. Literature Cited
  6. Supporting Information

Two OMIPs designed for the investigation of Treg have recently been published (1, 2). These use the expression of surface CD25 and intracellular FoxP3 to identify the Treg subset of CD4+ T-cells. This panel is designed to avoid experimental complexity induced by fixation, permeabilization, and intracellular staining. Notably this panel is potentially compatible with live cell sorting needed for functional and genomic interrogation of Treg. A combined measurement of the IL-2Rα chain (CD25) and the IL-7R α-chain (CD127) was used, with Treg being defined as CD25hi CD127 (3). Only about 13% of this population is FoxP3 (unpublished data) (3, 4). Treg were further characterized functionally and phenotypically by the expression of CD39, CD73, CD45RA, CD45RO, and PD-1. The nucleoside triphosphate dephosphorylase CD39 has been shown to identify cells with in vitro immunosuppressive capacity (5, 6). CD73 is an ecto-5′-nucleotidase that, together with CD39, contributes to the inhibitory function of Treg and other suppressive immune cells, as well as the tumor microenvironment, by generating adenosine (7–9). CD45RA (10) and CD45RO (11) were included in order to define naïve and activated Treg, respectively, while PD-1 was selected as it has been demonstrated to negatively regulate Treg function (12).

While the primary objective of the present panel was to identify and characterize Treg, some of the markers included also provide information on the differentiation and activation status of other CD4+ and CD8+ T-cells. For instance, though CD39 is mainly expressed on immunosuppressive Treg, it is also found on pathogenic CD4+ CD25 effector memory T-cells (13). CD45RA, CD45RO, and CD127 are differentially expressed during T-cell differentiation, while CD25 and PD-1 are expressed in an activation-dependent manner.

Finally, CD38 and HLA-DR were included; differential expression of these two T-cell activation markers identifies subsets disparately correlating with disease control, particularly in HIV-1 infection (14, 15).

To explore the expression of activation markers on Treg, gates were set on total CD4+ T-cells (Fig. 1B) and then applied to Treg (Fig. 1C). For dim markers such as HLA-DR and PD-1, careful choice of the second marker visualized in the dot plot allowed identifying the best threshold for separating positive and negative cells. Neither isotype nor fluorescence minus one (FMO) controls were used as many years of working with these markers have taught us what the staining patterns typically look like and where to set gate boundaries.

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Figure 1. Example staining and gating. A: Identification of CD4+ and CD8+ T-cells in healthy donor PBMC. After selecting live CD3+ single cells, any cells nonspecifically labeled with dye aggregates are excluded (gray box) and a lymphocyte gate set. CD4+ and CD8+ T-cells are then selected for further analysis. B: Evaluation of CD4+ and CD8+ T-cell activation, and identification of Treg. The expression level of CD25, CD38, CD39, CD45RA, CD45RO, CD73, CD127, HLA-DR, and PD-1 are investigated within CD4+ and CD8+ T-cell subset as gated in (A). Within CD4+ T-cells, Treg are CD127 while expressing high levels of CD25. C: Treg phenotype. The analysis of CD39 and CD73, CD45RA, and CD45RO, and CD38, HLA-DR, and PD-1 on their surface provides insight into maturation states, regulatory potential, and activation of the cells, respectively. Gates were set on total CD4+ T-cells (see B) and were then applied to Treg. D: Exploration of Treg activation. The pie chart illustrates the proportion of total Treg expressing any given combination of the activation markers measured.

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Similarity to Published Omips

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published Omips
  5. Literature Cited
  6. Supporting Information

OMIP-004 and -006 were also designed for the investigation of human Treg. 1, 2

Table 1. Summary Table for application of OMIP-015
PurposeT-reg characterization and T-cell activation
SpeciesHuman
Cell typesPBMC
Cross-referencesn.a.
Table 2. Reagents used for OMIP-015
SpecificityCloneFluorochromePurpose
  1. BV, brilliant violet; QD, quantum dot; PE, R-phycoerythrin; Cy, cyanine; APC, allophycocyanin; eF, eFluor; CF, cyanine-based fluorescent dye; FITC, fluorescein; Ax, Alexa; AqBlu, LIVE/DEAD Fixable Aqua Dead Cell Stain.

CD3OKT3BV785Lineage
CD4OKT4QD605
CD8RPA-T8QD585
CD25M-A251PE-Cy5T-reg
CD127eBioRDR5APC-eF780
CD39eBIOA1PE-Cy7T-reg functionality
CD73AD2PE
CD38HIT2PE-CF594Other activation/differentiation
CD45RA5H9QD705
CD45ROUCHL1FITC
HLA-DRG46-4Ax680
PD-1EH12.2H7BV421
Dead cells-AqBluDump

Literature Cited

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published Omips
  5. Literature Cited
  6. Supporting Information
  • 1
    Biancotto A,Dagur PK,Fuchs JC,Langweiler M,McCoy JPJr. OMIP-004: In-depth characterization of human T regulatory cells. Cytometry Part A 2012; 81A: 1516.
  • 2
    Murdoch DM,Staats JS,Weinhold KJ. OMIP-006: Phenotypic subset analysis of human T regulatory cells via polychromatic flow cytometry. CytometryPart A 2012; 81A: 281283.
  • 3
    Seddiki N,Santner-Nanan B,Martinson J,Zaunders J,Sasson S,Landay A,Solomon M,Selby W,Alexander SI,Nanan R, et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006; 203: 16931700.
  • 4
    Liu W,Putnam AL,Xu-Yu Z,Szot GL,Lee MR,Zhu S,Gottlieb PA,Kapranov P,Gingeras TR,Fazekas de St Groth B, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006; 203: 17011711.
  • 5
    Dwyer KM,Hanidziar D,Putheti P,Hill PA,Pommey S,McRae JL,Winterhalter A,Doherty G,Deaglio S,Koulmanda M, et al. Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a regulatory memory phenotype. Am J Transplant 2010; 10: 24102420.
  • 6
    Schulze Zur Wiesch J,Thomssen A,Hartjen P,Toth I,Lehmann C,Meyer-Olson D,Colberg K,Frerk S,Babikir D,Schmiedel S, et al. Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease. J Virol 2011; 85: 12871297.
  • 7
    Alam MS,Kurtz CC,Rowlett RM,Reuter BK,Wiznerowicz E,Das S,Linden J,Crowe SE,Ernst PB. CD73 is expressed by human regulatory T helper cells and suppresses proinflammatory cytokine production and Helicobacter felis-induced gastritis in mice. J Infect Dis 2009; 199: 494504.
  • 8
    Beavis PA,Stagg J,Darcy PK,Smyth MJ. CD73: A potent suppressor of antitumor immune responses. Trends Immunol 2012; 33: 231237.
  • 9
    Deaglio S,Dwyer KM,Gao W,Friedman D,Usheva A,Erat A,Chen JF,Enjyoji K,Linden J,Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007; 204: 12571265.
  • 10
    Hoffmann P,Eder R,Boeld TJ,Doser K,Piseshka B,Andreesen R,Edinger M. Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood 2006; 108: 42604267.
  • 11
    Haas J,Fritzsching B,Trubswetter P,Korporal M,Milkova L,Fritz B,Vobis D,Krammer PH,Suri-Payer E,Wildemann B. Prevalence of newly generated naive regulatory T cells (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis. J Immunol 2007; 179: 13221330.
  • 12
    Franceschini D,Paroli M,Francavilla V,Videtta M,Morrone S,Labbadia G,Cerino A,Mondelli MU,Barnaba V. PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV. J Clin Investig 2009; 119: 551564.
  • 13
    Moncrieffe H,Nistala K,Kamhieh Y,Evans J,Eddaoudi A,Eaton S,Wedderburn LR. High expression of the ectonucleotidase CD39 on T cells from the inflamed site identifies two distinct populations, one regulatory and one memory T cell population. J Immunol 2010; 185: 134143.
  • 14
    Giorgi JV,Ho HN,Hirji K,Chou CC,Hultin LE,O'Rourke S,Park L,Margolick JB,Ferbas J,Phair JP. CD8+ lymphocyte activation at human immunodeficiency virus type 1 seroconversion: development of HLA-DR+ CD38− CD8+ cells is associated with subsequent stable CD4+ cell levels.The Multicenter AIDS Cohort Study Group. J Infect Dis 1994; 170: 775781.
  • 15
    Meditz AL,Haas MK,Folkvord JM,Melander K,Young R,McCarter M,Mawhinney S,Campbell TB,Lie Y,Coakley E, et al. HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo. J Virol 2011; 85: 1018910200.

Supporting Information

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published Omips
  5. Literature Cited
  6. Supporting Information
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CYTOA22230_OMIP-015_online.pdf2046KSupporting Information

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