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Keywords:

  • flow cytometry;
  • intracellular staining;
  • CD4+CD25+;
  • FOXP3;
  • regulatory T cells

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

Background:

By measuring multiple parameters on a single-cell basis, flow cytometry is a potent tool to dissect the phenotypes and functions of cell subsets. However, because this technique may be time-consuming, particularly for intracellular staining, it could be problematic for its use in daily routine or in large cohorts. Recently, a novel reagent has been developed to perform intracellular staining in one step. The objective of our study was thus to assess this new method in comparison with the reference technique by focusing on FOXP3 staining in clinical samples.

Methods:

Peripheral blood was collected from 15 HIV-1-infected patients, 5 critically ill patients, and 5 healthy volunteers and stained using the two different methods. Different subsets of FOXP3 positive cells were investigated by flow cytometry.

Results:

When comparing results obtained with the two techniques, no statistical differences between the percentages of CD4+FOXP3+, CD4+CD25+FOXP3+, and CD4+CD25+CD127−FOXP3+ cells were observed. Besides, a strong correlation between percentages of CD4+FOXP3+CD25+CD127− lymphocytes measured with both techniques was found in patients (r: 0.843, P < 0.001, intra-class correlation coefficient: 0.820, P < 0.001). Importantly, flow cytometry stainings obtained with the one-step method were very robust with an excellent intra-assay precision, a better discriminative power and correct stability and reproducibility of the staining even after blood storage.

Conclusions:

With a strong correlation between the percentages of FOXP3+ Tregs when compared with the reference method, a better staining quality, a shorter realization time and no need of isotype control, this one step procedure may represent an important improvement for a daily routine use of intracellular staining. © 2012 International Clinical Cytometry Society

Flow cytometry is considered as a powerful tool for cellular analysis, either for studying cell phenotypes or various immune cell functions (1). However, several issues exist when considering flow cytometric intracellular stainings, especially in the context of a daily routine use and in clinical research protocols including large cohorts of patients for whom samples need to be processed immediately (2). In particular, this technique is time-consuming due to permeabilization and multiple washing steps and most importantly, results are usually poorly reproducible and hardly standardizable. This renders intracellular flow cytometry use poorly suitable for multicentric clinical studies.

Recently, a novel reagent has been developed to perform flow cytometry intracellular staining in one step (including permeabilization and staining). The aim of this study was thus to test the robustness of this reagent in daily routine conditions in clinical samples.

Accumulated evidence suggests the essential functions of regulatory T cells (Treg) in the pathophysiology of many clinical conditions such as autoimmune diseases (3), transplantations (4), severe septic shock (5), cancer (6), HIV infection (7), and in the development and maintenance of immunologic tolerance (8). However, very few studies have been conducted in large prospective clinical cohorts. This could be partly explained as Treg can only be precisely identified by flow cytometry using intracellular FOXP3 staining, the key transcriptional factor for Treg (9, 10). Even if other phenotypes (such as CD4+CD25+ or CD4+CD25+CD127−) are known to be good surrogate identification strategy for Treg (11), FOXP3 remains to date, the only marker to appropriately identify those cells (9, 12).

In this study, we thus assessed the quality and reproducibility of FOXP3 intracellular staining by using this novel one-step protocol in comparison with the well-accepted reference method in clinical samples.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

Study Population

HIV-1-infected patients from the Hospices Civils de Lyon HIV cohort, located in Croix-Rousse Hospital and intensive care unit (ICU) patients from Lyon-Sud Hospital and Edouard Herriot Hospital (Hospices Civils de Lyon, Lyon, France) were included. Samples were collected in EDTA anticoagulant tubes. Analyses were performed on available residual blood after routine analyses were performed.

Fifteen HIV-1-infected patients [men = 10 (67%), median age = 50 years, inter-quartile range (IQR): 38–69] were included. They all received highly active anti-retroviral therapy (HAART) [median treatment duration = 7.65 years, IQR: 3.03–11.03] and were aviremic with a viral load (VL) at baseline below 50 copies/ml (Abbott RealTime HIV-1 assay and m2000 RT system platform). Their median CD4+ cell count at baseline was 573 cells/mm3 [IQR: 391–761].

Five ICU patients were included [men = 2 (40%), median age = 70 years, IQR: 29–79]. Three of them met criteria for septic shock diagnosis of the American College of Chest Physicians/Society of Critical Care Medicine (13) and two were trauma patients (Injury Severity Score: ISS > 25).

Five healthy volunteers [men = 2 (40%), median age = 34, IQR: 26–56], all members of the laboratory staff, were also included after informed consent was given.

No statistical differences were observed in clinical data for age (P = 0.317) and gender (P = 0.342) among HIV patients, ICU patients, and healthy donors.

Reference Method of Intracellular Staining

Samples were stained with Anti-Human Foxp3 Staining Set following manufacturer's recommendations (eBioscience, San Diego, CA). As already described (14), samples were first stained using surface markers: anti human phycoerythrin (PE)-labeled anti-CD127, PE-Texas Red (ECD)-labeled anti-CD4 and PE-Cyanine5 (PC5)-labeled anti-CD25 antibodies (Beckman Coulter, Hialeah, FL) and incubated for 15 min. Samples were then lysed using Versalyse lysing solution (Beckman Coulter) for 15 min. After a washing step, permeabilization was performed using Fix/Perm Buffer for 40 min. Following three more washing steps with permeabilization buffer and incubation with this buffer and normal rat serum for 15 min (blocking step), the FoxP3 intracellular staining using anti human FoxP3-FITC antibody (clone PCH101, eBioscience) was performed for 30 min. Samples were finally washed two times with permeabilization buffer. Rat IgG2a isotype control was used to evaluate non-specific staining.

One-Step Method of Intracellular Staining

The PerFix-no centrifuge assay Kit from Beckman Coulter was assessed. Staining of fresh whole blood was performed using PE-labeled anti-CD25, PE-Cyanine7 (PC7)-labeled anti-CD127, Alexa fluor 647 (AF647)-labeled anti-FoxP3, and Pacific Blue (PB)-labeled anti-CD4 (Beckman Coulter). FoxP3-antibodies (clone 259D) were purchased from BioLegend (San Diego, CA). According to the manufacturer's instructions, samples were first fixed with the fixative reagent and incubated for 15 min. Then, aliquots were simultaneously permeabilized thanks to the permeabilizing reagent and stained with fluorochrome-conjugated antibodies. After 60 min of incubation, samples were washed once with PBS and secondly with a solution containing formaldehyde.

One-Step Method Robustness

To study intra-assay precision, two sets of analyses were performed: (i) one sample stained 10 times and analyzed 10 times (ii) one sample stained once and analyzed 10 times. To assess the stability of the staining, expression of the markers was measured immediately after staining, then 2 h and 24 h later for five patients. To assess the effect of blood storage before staining, results from fresh whole blood or obtained after blood storage for 24 h at 4°C and at room temperature for three patients were compared.

Flow Cytometric Data Acquisition and Analysis

Cytometry analyses were performed on a NAVIOS flow cytometer using the NAVIOS software (Beckman Coulter). FOXP3 expression was examined in three different CD4+ subsets as followed: CD4+, CD4+CD25+, and CD4+CD25+CD127− lymphocytes.

Statistical Analysis

Statistical analyses were performed using SPSS (version 17.0; SPSS, Chicago, IL) and GraphPad Prism (version 5.03, GraphPad Software, La Jolla, CA) software. Normality of the parameters was assessed using the Kolmogorov–Smirnov test. T-test, Wilcoxon test for continuous variables and exact Fisher test for categorical variables were performed. Correlations were studied using the Pearson's correlation coefficient test and the intra-class correlation coefficient (ICC). The Bland–Altman approach was also used to assess the agreement between the methods. P-values were considered significant when lower than 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

As Treg, independently of FOXP3 expression, are usually described as either CD4+CD25+ or CD4+CD25+CD127− lymphocytes, FOXP3 expression was evaluated in these different subsets. Examples of plots representing co-expression of either FOXP3 and CD25 or FOXP3 and CD127 in CD4+ gated cells are shown in Figure 1.

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Figure 1. Examples of plots representing co-expression of FOXP3 and CD25 or FOXP3 and CD127 in CD4+ cells. Cells were first gated on SSC/CD4 (left histogram).

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No statistical differences between the percentages of CD4+FOXP3+, CD4+CD25+FOXP3+, and CD4+CD25+CD127−FOXP3+ lymphocytes measured with the two techniques were observed. This was true for the overall patient population as well as in each group individually (Table 1). In line, a strong correlation between percentages of CD4+FOXP3+ cells obtained with the two different techniques was identified in the global population (r: 0.763, P < 0.001, ICC: 0.759, P < 0.001). Likewise, percentages of CD4+CD25+FOXP3+ (r: 0.715, P < 0.001, ICC: 0.659, P < 0.001) and CD4+CD25+CD127−FOXP3+ lymphocytes (r: 0.843, P < 0.001, ICC: 0.820, P < 0.001) (Fig. 2) were nicely correlated between reference and one-step procedures. Moreover, Bland–Altman plots revealed good agreement between both methods regardless of the phenotype (Fig. 2).

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Figure 2. Correlation and Bland–Altman plots between Treg percentages measured by using reference and one-step techniques. For each Treg phenotype, correlation (left side—Pearson's correlation coefficient test) and Bland–Altman plot (right side—the black line indicates the average value of the differences between the two methods; the two dotted lines form the 95% reference range) are represented as (a) CD4+CD25+CD127−FOXP3+cells; (b) CD4+CD25+FOXP3+ cells, and (c) CD4+FOXP3+ cells.

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Table 1. Percentages of Positive Cells Among CD4+ Lymphocytes and Mean of Fluorescence Intensity (MFI) of FOXP3 for Different Treg Phenotypes
  Reference methodOne step methodP
  1. Results were obtained in HIV-infected patients (HIV, n = 15), critically ill patients (ICU, n = 5), healthy volunteers (HV, n = 5) and in the whole cohort (Total, n = 25). Results are presented as median and IQR (inter quartile range) between brackets. P values were calculated using student T-test.

Percentages among CD4+ cells
FOXP3+HIV9.26 [7.09–10.44]10.66 [9.62–12.27]0.072
ICU11.85 [9.11–17.32]12.65 [9.68–16.53]0.98
HV11.92 [9.45–12.53]11.65 [10–12.08]0.99
Total10.08 [7.89–11.89]11.29 [9.80–12.5]0.257
FOXP3+ CD25+HIV7.27 [5.28–9.09]6.91 [5.27–8.16]0.492
ICU10.51 [6.96–14.86]9.04 [5.85–10.32]0.314
HV5.96 [5.69–7.83]6.73 [6.41–8.11]0.481
Total7.34 [5.63–9.44]6.97 [5.97–8.59]0.33
FOXP3+ CD25+ CD127−HIV5.62 [4.63–6.46]6.32 [5.83–7.13]0.192
ICU9 [6.07–12.7]8.9 [6.35–10.68]0.753
HV5.72 [5.03–6.83]6.18 [5.22–7.28]0.666
Total5.74 [4.83–7.92]6.32 [5.84–8.46]0.529
Mean of Fluorescence Intensity
MFI Difference: FOXP3+ minus FOXP3−HIV1.81 [1.60–2.01]7.40 [6.16–9.52]<0.001
ICU2.6 [1.57–3.16]10.59 [6.51–17.43]0.019
HV1.27 [1.16–1.36]9.94 [5.67–10]0.003
Total1.63 [1.36–2.03]7.52 [6.18–10.13]<0.001
MFI Ratio: FOXP3+ versus FOXP3−HIV3.17 [2.71–3.46]10.84 [9.82–12.19]<0.001
ICU4.92 [3.3–5.35]12.64 [11.24–17.29]0.001
HV2.78 [2.64–2.94]12.5 [10–12.76]<0.001
Total3.17 [2.74–3.46]11.81 [10.35–12.58]<0.001

We then compared the cell separation/discriminative power of these two techniques for FOXP3+ lymphocytes gating. The signal to noise ratio was calculated for each technique by comparing the mean of fluorescence intensity (MFI) of FOXP3 positive versus negative cells among CD4+ lymphocytes (MFI ratios and differences). Interestingly, significantly different discriminative power was identified between these two techniques in each study group and in the whole study population (Table 1). We indeed observed that FOXP3+ cell identification after one-step intracellular staining was largely easier than with the reference method (Fig. 3).

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Figure 3. Representative biparametric histograms of CD4+FOXP3+ cells for one-step and reference procedures. Both procedures were simultaneously used in one healthy volunteer, one HIV-infected patient, and one ICU patient.

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When testing intra-assay precision, we observed that coefficients of variation (CV) of the percentage of CD4+FOPX3+ cells measured with the one-step technique ranged from 4 to 5.8%. Similar results were obtained when percentages of CD4+CD25+FOXP3+ lymphocytes were measured (CV between 3.3 and 6.4%).

When testing stability of the staining, we did not observe any statistical difference. We observed in five patients that mean percentage of CD4+FOXP3+ cells was 7.6% (standard deviation, SD: 1.7) at T0, 7.1% (SD: 1.1) at T+2 h, and 7.4% (SD: 1.4) at T+24 h (Fig. 4a). In the same patients, the mean MFI ratio between FOXP3− and FOXP3+ cells among CD4+ lymphocytes was 10.5 (SD: 2.2) at T0, 10.3 (SD: 1.5) at T+2 h, and 9.6 (SD: 2) at T+24 h (Fig. 4b). Similar results were obtained with other phenotypes of Treg (CD4+CD25+FOXP3+ and CD4+CD25+CD127−FOXP3+). These results indicated an excellent staining stability.

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Figure 4. One-step method robustness (example of CD4+FOXP3+ cells). (a,b) Staining stability was evaluated for the one-step technique at three time points (T0, T+2 h after staining, T+24 h after staining) in five HIV-infected patients for (a) % CD4+FOXP3+ cells and (b) MFI ratio between FOXP3+ and FOXP3- cells. (c,d). Effect of blood storage before staining was evaluated for (c) CD4+FOXP3+ cells and (d) MFI ratio between FOXP3+ and FOXP3− cells. The one-step technique was performed in three HIV-infected patients. Cells were stained at T0 and after 24 h storage at 4°C and 24 h storage at room temperature (RT). For each graph, individual values are presented.

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Finally, when blood storage before staining was tested, we did not observe any statistical difference. The mean percentage of CD4+FOXP3+ cells for three patients were respectively, 7.0% (SD: 0.8) at T0, 6.9% (SD: 1.6) after storage for 24 h at 4°C, and 7.1% (SD: 0.9) after 24 h storage at room temperature (Fig. 4c). The mean MFI ratio between FOXP3− and FOXP3+ cells among CD4+ population for these patients was found to be very stable at 10.7 (SD: 3.1) at T0, 11.7 (SD: 3.2) after 24 h storage at 4°C, and 10.6 (SD: 2.1) after 24 h storage at room temperature (Fig. 4d). We obtained the same results considering other phenotypes of Treg (CD4+CD25+FOXP3+ and CD4+CD25+CD127−FOXP3+).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

The main result of our study is to show that Treg percentages obtained using reference and one-step techniques are well correlated. This was observed whatever the phenotype used for Treg identification (i.e., FOXP3+ cells among CD4+, CD4+CD25+, or CD4+CD25+CD127− lymphocytes). Another important result was that flow cytometry staining obtained by using one-step intracellular staining appeared as a robust technique with good intra-assay precision and adequate staining stability even after blood storage.

Interestingly, this novel technique presents with better advantages than reference technique.

Indeed, this one-step intracellular staining procedure offers several improvements. Intracellular FOXP3 staining with this technique takes about 90 min to complete (CD4, CD25, CD127, and FOXP3 stainings) whereas 200 min are needed for the usual reference method. This is due to the steps of permeabilization and staining (extra and intracellular) that are done at the same time in the one-step technique therefore shortening additional incubation times and wash/centrifugations cycles. Importantly, this diminished number of washing cycles did not impact the non-specific background fluorescence that could have been expected to be amplified. In contrast, the signal to noise ratio between FOXP3+ and FOXP3− cells was even improved with the one-step approach (Table 1). This enables for a better discrimination of the positive FOXP3 cells amongst CD4+ population with the one-step method. This is without the requirement of an isotype control use. In contrary, such specific cell separation appears as virtually impossible in some patients with the reference technique (Fig. 2).

Therefore, specifically regarding FOXP3 staining in clinical samples, our results open new perspectives of Treg investigation either in large prospective clinical studies or in daily routine. This could also be relevant for the study of different Treg subsets such as cells co-expressing CD45RA, CD38, or CTLA4 (15, 16). Moreover, this new technique obviously allows for the staining of other intracellular markers whatever the cells/phenotypes considered and could be of major interest for various users of flow cytometry.

Our study presents some limitations. In particular, two different fluorochromes and two different clones were used for intracellular FOXP3 staining in the compared techniques. This could partly explain the difference in discriminative power between the methods. For example, some studies have pointed out that the clone used in the reference method was associated with a higher level of non-specific staining compared with other clones (17, 18). This obviously needs to be confirmed in a subsequent study. Moreover, we only studied the correlation between the methods in three different pathologic situations. This novel method may therefore not be suitable in other clinical conditions. Thus, these preliminary results should be validated in a larger cohort of patients and in other clinical contexts. Besides, standardization of this novel technique should be elaborated on.

CONCLUSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

By comparing a one-step intracellular staining procedure with a reference protocol, we obtained a strong correlation between percentages of FOXP3+ Treg. Moreover, with a better staining quality, a shorter realization time and no isotype control requirement, this one-step procedure appears adequate for a daily routine use and in prospective clinical studies. This one-step procedure of intracellular staining therefore may represent an important improvement in the study of intracellular molecules by flow cytometry in clinical studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

The authors would like to thank Anne Portier and Caroline Guignant for technical assistance.

DISCLOSURE

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED

This study was supported by Beckman Coulter through donations of laboratory equipments and supplies. This private company had no role in the study design, the collection or interpretation of the data. Similarly, Beckman Coulter had no role in the preparation of the manuscript or the decision to submit it for publication. Conversely, eBioscience did not supply any of the reagents for the reference method.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE
  9. LITERATURE CITED
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