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Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.

Santegoets SJ, Dijkgraaf EM, Battaglia A, Beckhove P, Britten CM, Gallimore A, Godkin A, Gouttefangeas C, de Gruijl TD, Koenen HJ, Scheffold A, Shevach EM, Staats J, Taskén K, Whiteside TL, Kroep JR, Welters MJ, van der Burg SH - Cancer Immunol. Immunother. (2015)

Bottom Line: This resulted in a rationally composed ranking list of "Treg markers".Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells.In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Oncology, Leiden University Medical Center (LUMC), Leiden, The Netherlands, s.j.a.m.santegoets@lumc.nl.

ABSTRACT
Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of "Treg markers". Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

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Treg enumeration based solely on Foxp3 and Helios (def.2) or Foxp3 and CD45RA (def.3) led to an underestimation of CD25posCD127lowFoxp3pos def.1 Tregs through exclusion of def.1 Treg cells in the Foxp3posHeliosneg (def.2) or Foxp3intCD45RAneg non-Treg (def.3) populations. Overlap between the three most commonly used Treg definitions (def.1, def.2, and def.3) is given for a a representative HD and b,c six HDs. a Distribution of def.1 Tregs is shown in def.2 and def.3 populations (left), of def.2 Tregs is shown in def.1 and def.3 populations (middle), and of def.3 Tregs is shown in def.1 and def.2 populations (right). Percentage of Tregs analyzed via def.1, def.2, or a combination thereof b and via def.1, def.3, or a combination thereof c is depicted as percentage of CD4pos T cells. Overlap between the designated populations is calculated in relation to def.1 Tregs (set at 100) and given in the bar graph for each population
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Fig2: Treg enumeration based solely on Foxp3 and Helios (def.2) or Foxp3 and CD45RA (def.3) led to an underestimation of CD25posCD127lowFoxp3pos def.1 Tregs through exclusion of def.1 Treg cells in the Foxp3posHeliosneg (def.2) or Foxp3intCD45RAneg non-Treg (def.3) populations. Overlap between the three most commonly used Treg definitions (def.1, def.2, and def.3) is given for a a representative HD and b,c six HDs. a Distribution of def.1 Tregs is shown in def.2 and def.3 populations (left), of def.2 Tregs is shown in def.1 and def.3 populations (middle), and of def.3 Tregs is shown in def.1 and def.2 populations (right). Percentage of Tregs analyzed via def.1, def.2, or a combination thereof b and via def.1, def.3, or a combination thereof c is depicted as percentage of CD4pos T cells. Overlap between the designated populations is calculated in relation to def.1 Tregs (set at 100) and given in the bar graph for each population

Mentions: Figure 1a shows the expression of the different markers in def.1 Tregs. The gating strategy for the CD25posCD127lowFoxp3pos def.1 Treg subset is given for a representative HD in supplementary Fig. 2a. Cells expressing Foxp3 comprised 78.7 % (range 70.5–85.1 %) of the CD25posCD127low subpopulation. Due to variability in CD127 expression (Supplementary figure 2b, c), enumerating def.1 Tregs solely based on CD25 and CD127 is highly variable between HD and most likely leads to an overestimation of the number of Tregs (mean 17.6 %, range 7.2–30.4 %). Inclusion of Foxp3 resulted in less variation in the percentage of def.1 Tregs (mean 6.9 %, range 4.6–8.8 %) as would be expected among a group of HD, suggesting that simultaneous staining with CD25, CD127, and Foxp3 is needed for reliable measurement of def.1 Tregs. Further characterization of the CD25posCD127lowFoxp3pos subset revealed that 75 % of these cells were Helios positive (Fig. 1a). Moreover, the majority of CTLA-4 and Ki67 expressing CD4pos T cells were found in the CD25posCD127lowFoxp3pos population (data not shown). These observations add to the notion that bona fide Tregs are detected when the CD25posCD127lowFoxp3pos def.1 subset definition for Treg enumeration is used.Fig. 1


Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.

Santegoets SJ, Dijkgraaf EM, Battaglia A, Beckhove P, Britten CM, Gallimore A, Godkin A, Gouttefangeas C, de Gruijl TD, Koenen HJ, Scheffold A, Shevach EM, Staats J, Taskén K, Whiteside TL, Kroep JR, Welters MJ, van der Burg SH - Cancer Immunol. Immunother. (2015)

Treg enumeration based solely on Foxp3 and Helios (def.2) or Foxp3 and CD45RA (def.3) led to an underestimation of CD25posCD127lowFoxp3pos def.1 Tregs through exclusion of def.1 Treg cells in the Foxp3posHeliosneg (def.2) or Foxp3intCD45RAneg non-Treg (def.3) populations. Overlap between the three most commonly used Treg definitions (def.1, def.2, and def.3) is given for a a representative HD and b,c six HDs. a Distribution of def.1 Tregs is shown in def.2 and def.3 populations (left), of def.2 Tregs is shown in def.1 and def.3 populations (middle), and of def.3 Tregs is shown in def.1 and def.2 populations (right). Percentage of Tregs analyzed via def.1, def.2, or a combination thereof b and via def.1, def.3, or a combination thereof c is depicted as percentage of CD4pos T cells. Overlap between the designated populations is calculated in relation to def.1 Tregs (set at 100) and given in the bar graph for each population
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Related In: Results  -  Collection

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Fig2: Treg enumeration based solely on Foxp3 and Helios (def.2) or Foxp3 and CD45RA (def.3) led to an underestimation of CD25posCD127lowFoxp3pos def.1 Tregs through exclusion of def.1 Treg cells in the Foxp3posHeliosneg (def.2) or Foxp3intCD45RAneg non-Treg (def.3) populations. Overlap between the three most commonly used Treg definitions (def.1, def.2, and def.3) is given for a a representative HD and b,c six HDs. a Distribution of def.1 Tregs is shown in def.2 and def.3 populations (left), of def.2 Tregs is shown in def.1 and def.3 populations (middle), and of def.3 Tregs is shown in def.1 and def.2 populations (right). Percentage of Tregs analyzed via def.1, def.2, or a combination thereof b and via def.1, def.3, or a combination thereof c is depicted as percentage of CD4pos T cells. Overlap between the designated populations is calculated in relation to def.1 Tregs (set at 100) and given in the bar graph for each population
Mentions: Figure 1a shows the expression of the different markers in def.1 Tregs. The gating strategy for the CD25posCD127lowFoxp3pos def.1 Treg subset is given for a representative HD in supplementary Fig. 2a. Cells expressing Foxp3 comprised 78.7 % (range 70.5–85.1 %) of the CD25posCD127low subpopulation. Due to variability in CD127 expression (Supplementary figure 2b, c), enumerating def.1 Tregs solely based on CD25 and CD127 is highly variable between HD and most likely leads to an overestimation of the number of Tregs (mean 17.6 %, range 7.2–30.4 %). Inclusion of Foxp3 resulted in less variation in the percentage of def.1 Tregs (mean 6.9 %, range 4.6–8.8 %) as would be expected among a group of HD, suggesting that simultaneous staining with CD25, CD127, and Foxp3 is needed for reliable measurement of def.1 Tregs. Further characterization of the CD25posCD127lowFoxp3pos subset revealed that 75 % of these cells were Helios positive (Fig. 1a). Moreover, the majority of CTLA-4 and Ki67 expressing CD4pos T cells were found in the CD25posCD127lowFoxp3pos population (data not shown). These observations add to the notion that bona fide Tregs are detected when the CD25posCD127lowFoxp3pos def.1 subset definition for Treg enumeration is used.Fig. 1

Bottom Line: This resulted in a rationally composed ranking list of "Treg markers".Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells.In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Oncology, Leiden University Medical Center (LUMC), Leiden, The Netherlands, s.j.a.m.santegoets@lumc.nl.

ABSTRACT
Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of "Treg markers". Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

Show MeSH
Related in: MedlinePlus