Korean J Lab Med.  2011 Oct;31(4):231-237. 10.3343/kjlm.2011.31.4.231.

CD4+CD25highFoxP3+ Regulatory T-cells in Hematologic Diseases

• Moon HW
• Kim BH
• Park CM
• Hur M
• Yun YM
• Kim SY
• Lee MH

• KMID: 1781685
• DOI: http://doi.org/10.3343/kjlm.2011.31.4.231

Abstract

BACKGROUND
CD4+CD25+ regulatory T-cells (Tregs) play a critical role in immune responses. We explored the status of Tregs in neoplastic and autoimmune hematologic diseases. We also evaluated the technical aspects of Treg measurement in terms of sample type and detection markers.
METHODS
A total of 68 subjects were enrolled: 11 with AML, 8 with MDS, 10 with autoimmune diseases, and 39 controls. Tregs were analyzed in peripheral blood (PB) and bone marrow (BM) samples from each subject. Flow cytometry and the Human Regulatory T cell Staining Kit (eBioscience, USA) for CD4, CD25, and FoxP3 (forkhead box P3) were used.
RESULTS
The CD4+CD25high/CD4 and CD4+CD25highFoxP3+/CD4 populations were significantly correlated (P<0.0001). The AML and high-risk MDS groups had significantly larger CD4+CD25high/CD4 and CD4+CD25highFoxP3+/CD4 populations in PB than the autoimmune (P=0.007 and 0.012, respectively) and control groups (P=0.004 and 0.006, respectively). Comparable findings were observed in BM. The CD4+CD25highFoxP3+/CD4 population was significantly larger in PB than in BM (P=0.0003).
CONCLUSIONS
This study provides comparison data for Tregs in AML, MDS, and autoimmune hematologic diseases, and would be helpful for understanding the different immunologic bases of various hematologic diseases. Treg measurement using CD4, CD25, and/or FoxP3 in PB rather than in BM seems to be practical for routine hematologic purposes. Large-scale analysis of the diagnostic role of Treg measurement is needed.

Keyword

Regulatory T-cells; FoxP3; Hematologic disease; Peripheral blood; Bone marrow

MeSH Terms

Adolescent
Adult
Aged
Aged, 80 and over
Autoimmune Diseases/diagnosis/immunology
Bone Marrow Cells/cytology
Female
Flow Cytometry
Forkhead Transcription Factors/*metabolism
Hematologic Diseases/*diagnosis/immunology
Humans
Interleukin-2 Receptor alpha Subunit/*metabolism
Leukemia, Myeloid, Acute/diagnosis/immunology
Leukocytes, Mononuclear/cytology
Male
Middle Aged
Myelodysplastic Syndromes/diagnosis/immunology
T-Lymphocytes, Regulatory/immunology/*metabolism

Figure

• 

  Fig. 1 Analysis of Tregs using flow cytometry. Mononuclear cells were stained with CD4-FITC, CD25-PE, and FoxP3-PE-Cy5 antibodies. After gating the lymphocyte population, the CD4+CD25high population and the CD4+CD25highFoxP3+ population were sequentially gated and analyzed. The percentage of FoxP3+ cells (CD4+CD25highFoxP3+) within the CD4+ population was calculated. In the case shown above, the proportion of CD4+CD25high cells is 2.8% of CD4+ population and 92.8% of that population is Foxp3 positive (2.59% of CD4+ population), defined by isotype control.Abbreviations: SSC, side scatter; FSC, forward scatter; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PE-Cy5, phycoerythrin-cyanin 5; FoxP3, forkhead box P3.

• 

  Fig. 2 Correlation and difference between the CD4+CD25high/CD4 and CD4+CD25highFoxP3+/CD4 populations in peripheral blood. (A) The 2 populations were significantly correlated (Y=0.9326X - 0.2901, R=0.9674, P<0.0001). (B) The CD4+CD25high/CD4 population (5.56±2.82%) was slightly larger than the CD4+CD25highFoxP3+/CD4 population (4.90±2.67%) with mean difference of 0.66%.Abbreviation: FoxP3, forkhead box P3.

• 

  Fig. 3 Peripheral blood CD4+CD25high/CD4 (A) and CD4+CD25highFoxP3+/CD4 populations (B) in each group. Both PB CD4+CD25high/CD4 and PB CD4+CD25highFoxp3+/CD4 populations were significantly larger in the AML and high-grade MDS groups compared with the autoimmune and control groups, by Student's t-test. Shown are the mean (middle bar) and 2SD (error bar) for each group.Abbreviations: PB, peripheral blood; FoxP3, forkhead box P3; MDS-H, high-grade MDS; MDS-L, low-grade MDS; AI, autoimmune group.

• 

  Fig. 4 Bone marrow (BM) CD4+CD25high/CD4 and CD4+CD25highFoxP3+/CD4 populations in each group. (A) The BM CD4+CD25high/CD4 population was significantly larger in the AML and high-grade MDS groups than in the autoimmune and control groups. (B) The BM CD4+CD25highFoxP3+/CD4 population was significantly larger in the AML and high-grade MDS groups than in the autoimmune and control groups. The low-grade MDS group had significantly smaller populations of CD4+CD25high/CD4 and CD4+CD25highFoxP3+/CD4 cells compared with the high-grade MDS group. Shown are the mean (middle bar) and 2SD (error bar) for each group.Abbreviations: BM, bone marrow; FoxP3, forkhead box P3; MDS-H, high-grade MDS; MDS-L, low-grade MDS; AI, autoimmune group.

• 

  Fig. 5 Comparison of Tregs (CD4+CD25highFoxP3+/CD4) in peripheral blood (Y axis) and bone marrow (X axis). (A) The 2 populations were significantly correlated (Y=0.9560X+1.5534, R=0.8440, P<0.0001). (B) The percentage of PB Tregs (5.52±3.41%) was significantly higher than that of BM Tregs (4.16±3.00%) (mean difference 1.37±1.83%, P=0.0003 by paired t-test).Abbreviations: PB, peripheral blood; BM, bone marrow; Treg, regulatory T-cells.

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