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Keywords:

  • Chemokine receptors;
  • T cell memory subsets;
  • T helper cells;
  • T cell phenotyping;
  • Multicolor Flow Cytometry

Purpose and Appropriate Sample Types

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

This panel was developed for the enumeration of chemokine receptor expression on CD4 T cells from the naïve (TN), stem cell memory (TSCM), central memory (TCM), and effector memory (TEM) cell subsets (Table 1). Eight chemokine receptors were chosen based upon previously published observations implicating their preferential expression on T helper type 1 (Th1), Th2, Th17, or Th22 cells, and their role in controlling lymphocyte migration to secondary lymphoid tissues, B cell follicles or non-lymphoid tissues, both in the steady state and during immune responses to pathogens, autoantigens, or allergens (1). This panel has been optimized for use with fresh peripheral blood mononuclear cells (PBMCs) with the observation that 24 h after blood draw, the expression of some chemokine receptors is reduced. Optimal staining requires PBMCs to be processed and acquired within 5 h of blood draw and may be performed in a 96-well plate format for high throughput experiments.

Background

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

In early stages of the immune response, antigen is presented on dendritic cells in the context of costimulatory molecules and cytokines that polarize antigen-specific T cells to become specialized effector cells. T cell polarization is regulated by lineage-specifying transcription factors and leads to the up-regulation of a plethora of cytokines which characterize different types of effector T cells (2). Thus, Th1 cells under the influence of T-bet upregulate interferon-γ (IFN-γ), Th2 cells under the influence of GATA-3 upregulate interleukin-4 (IL-4), IL-5, and IL-13, while Th17 cells under the influence of retinoic acid-related orphan receptor γ t (RORγt) upregulate IL-17. Work from several laboratories has demonstrated that effector function and migratory capacities are coordinately regulated in differentiating T cells. Chemokine receptors, which are important determinant for lymphocyte (and in general leukocyte) migration and positioning at both steady state and during inflammation, are known to be differentially expressed on Th1, Th2, and Th17 cells and to mark T cells with skin- or gut-homing properties (3) (Fig. 1). Thus, CXCR3 is preferentially expressed on Th1 cells, CCR3, CCR4, and CRTh2 are expressed on subsets of Th2 cells, while CCR6 is expressed on Th17 cells. Recently, CCR10 has been shown to identify a subset ofskin-homing T cells producing IL-22 and therefore operationally defined as Th22 (4).

Chemokine receptors have been also used to identify different subsets within the population of CD45RA memory T cells. CCR7 discriminates between human TCM and TEM subset (5). Human and mouse studies demonstrated that TCM cells are involved in secondary responses, having high proliferative and reconstituting capacity, while TEM cells are present primarily in peripheral tissues and are endowed with immediate effector function, but show poor reconstituting capacity. CXCR5, a chemokine receptor that drives cell migration to B cell follicles, was found to be expressed on a subset of TCM cells in blood and on T cells that help B cells to produce antibodies in tonsils (6). A flow cytometry panel that identifies many combinations of chemokine receptors expressed on T cells is required in order to improve our understanding of the heterogeneity of T cell subsets and the role these subsets play in different types of protective or pathological immune responses.

The strategy used to develop a chemokine receptor panel was to make a “wish list” as described by Mahnke and Roederer (7) by first choosing the primary antigens that are best characterized and that identify broad subsets (CD3, CD4, CD14, CD16, CD19). The secondary antigens chosen were also high density antigens, but expressed more as a continuum, such as CD45RA, CCR7, and CD95, while the tertiary antigens were the least expressed (chemokine receptors) and therefore most difficult to detect. With this organization, fluorochromes were assigned based on the brightness and availability for the tertiary antigens, leaving the rest of thedetectors available for the well characterized antigens (Table 2)2.

Table 1. Summary table for application of OMIP-018
PurposePhenotype of CD4 T cell subsets via chemokine receptor expression
SpeciesHuman
Cell typesFresh PBMC
Bone marrow mononuclear cells
Cross referencesNone
Table 2. Reagents used in OMIP-018
SpecificityCloneFluorochromePurpose
  1. APC, allophycocyanin; Cy, cyanin; QD, quantum dot; PE, R-phycoerythrin; Bi, biotin; SAV, streptavidin; FITC, fluorescein isothiocyanate; BV, brilliant violet; TR, texas red; BH, BD Horizon; Percp, peridinin chlorophyll protein.

CD3S4.1PeCy5.5T-cell subset
CD4S3.5PE TR 
CD45RAMEM-56QD655Maturity
CCR7G043H7BV421 
CD95DX2Percp-eFluor 710 
CCR41G1PE Cy7Polarization
CXCR31C6/CXCR3PECy5 
CCR611A9Bi 
BiSAVQD800 
CXCR551505QD605 
CCR52D7/CCR5APC Cy7 
CCR361828 ratAPC 
CCR10314305 ratPE 
CRTh2BM16 ratFITC 
CD14M5E2BH V500Exclusion
CD163G8BH V500 
CD19HIB19BH V500 
Dead cells Aqua 

Since chemokine receptors are internalized and recycled at the cell membrane, it was important to define staining conditions for optimal detection. To ascertain the best staining conditions, different types of samples were used and all anti-chemokine receptor antibodies were titrated at a range of temperatures, 4°C, 18°C, and 37°C, and stained for 10–20 min prior to acquisition. It was noted that the quality of chemokine receptor staining is affected by a variety of factors, most notably the time post-blood draw, cryopreservation, and temperature during staining.

Human Subjects

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Buffy coats from healthy blood donors were obtained from the Swiss Red Cross Centers. Permission for experiments with human primary cells was obtained from the Federal office of Public Health (N. A000197/2 to F.S.).

Similarity to Published OMIPs

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

None to date.

thumbnail image

Figure 1. Staining patterns of human PBMCs. A: Overview of gating strategy. After exclusion of doublets, lymphocytes were identified based upon forward and side scatter properties. Live, CD3+ CD14–/CD16–/CD19− cells were then identified and Boolean gating performed for the exclusion of cell aggregates from each detector (e.g., as shown for CD45RA and CCR6). The panel was then analyzed for memory subsets and chemokine receptor expression. B: Identification of T naïve (CD45RA+CCR7+CD95–), T memory stem cells (TSCM) (CD45RA+CCR7+CD95+), T central memory (TCM) (CD45RA–CCR7+), and T effector memory (TEM) (CD45RA+/–CCR7–) subsets. C: Clear discrimination of T cell subsets as distinguished by chemokine receptor expression in naïve and memory compartments. CCR3 is not shown as with the antibody used was only detectable on basophils. All samples analyzed were obtained from healthy donors.

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Acknowledgements

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

The authors thank Laurent Perez for help with the CXCR5 QD605 in-house antibody conjugation, David Jarrossay for help with panel development and Suzanne Campion for critical reading of the manuscript. Authors would also like to thank the blood donors who made this work possible.

Literature Cited

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Acknowledgements
  7. Literature Cited
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22278_sm_SuppFig1.tif206KSupporting Information Figure 1.
CYTO_22278_sm_SuppFig2.tif140KSupporting Information Figure 2.
CYTO_22278_sm_SuppFig3.tif254KSupporting Information Figure 3.
CYTO_22278_sm_SuppInfo.doc36KSupporting Information
S_Table-1.doc48KSupporting Information Table 1.
S_Table-2.doc57KSupporting Information Table 2.
S_Table-3.doc24KSupporting Information Table 3.
S_Table-4.doc37KSupporting Information Table 4.
MIFlowCyt_Item-Checklist.doc49KSupporting Information: MIFlowCyt

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