CD127low/- and FoxP3+ Expression Levels Characterize Different Regulatory T-Cell Populations in Human Peripheral Blood Sandra Klein, Cosima C. Kretz,

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CD127low/- and FoxP3+ Expression Levels Characterize Different Regulatory T-Cell Populations in Human Peripheral Blood Sandra Klein, Cosima C. Kretz, Peter H. Krammer, Annegret Kuhn Journal of Investigative Dermatology Volume 130, Issue 2, Pages 492-499 (February 2010) DOI: 10.1038/jid.2009.313 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Gating strategy for CD4+CD25+FoxP3+ cells. To define the number of circulating CD4+CD25+FoxP3+ cells, human peripheral blood mononuclear cells (PBMCs) from normal healthy donors (NHDs) were purified from peripheral blood samples and stained for CD4, CD25, and FoxP3. CD4+ T cells were gated out of lymphocytes (a), and the percentage of CD4+CD25+FoxP3+ cells among the CD4+ T cells (b) was determined. Journal of Investigative Dermatology 2010 130, 492-499DOI: (10.1038/jid.2009.313) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Frequency of CD4+CD25+CD127low/- cells. To define the number of circulating CD4+CD25+CD127low/- cells, human peripheral blood mononuclear cells (PBMCs) from normal healthy donors (NHDs) were purified from peripheral blood samples and stained for cell surface expression of CD4, CD25, and CD127. The PBMCs were gated for CD4+ T cells (data not shown; see Figure 1a), and the percentage of CD4+CD25+CD127low/- cells among the CD4+ T cells was determined using a dot plot (a) or contour blot (b). It is not obvious, however, where to set the boundary between CD127low/- and CD127+ expression in either analysis. Therefore, a clear population of CD4+CD25+CD127low/- cells might not be gated consistently over time. To determine whether the permeabilization procedure had any influence on the quantification of CD4+CD25+CD127low/- cells, cells from the same NHDs were stained with and without permeabilization (c), but no significant difference was observed (P=0.4328). Journal of Investigative Dermatology 2010 130, 492-499DOI: (10.1038/jid.2009.313) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of FoxP3 and CD127 in different CD4+ subsets. To define the expression of Foxp3 and CD127 in different CD4+ subsets, human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood and stained for CD4, CD25, FoxP3, and CD127. Expression of FoxP3 in CD4+CD127low/- and CD4+CD127+ T-cell subsets was determined. Boxes represent arbitrary designations of CD127+ vs. CD127low/- cells. Each number in the histograms indicates the percentage of gated cells expressing FoxP3. As shown in P14, a high number of CD4+CD127+CD25++ cells obviously expressed FoxP3, and a low number of CD4+CD127+CD25+/- cells expressed FoxP3 (P13). Journal of Investigative Dermatology 2010 130, 492-499DOI: (10.1038/jid.2009.313) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Correlation between CD4+CD25+FoxP3+ cells and CD4+CD25+CD127low/- cells in normal healthy donors (NHDs). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of NHDs and stained for CD4, CD25, aCD127, and FoxP3. PBMCs were gated for CD4+ T cells (data not shown; see Figure 1a), and CD4+CD25+FoxP3+ cells and CD4+CD25+CD127low/- cells among the CD4+ T cells were determined. Subsequently, the correlation between these two populations was examined (a, b). Of the CD4+CD25+FoxP3+ cells (a, P3), 30.3% (a, P3, cells shown in yellow) did not fall in the CD4+CD25+CD127low/- population (b, P6) but instead fell outside of this population (b, cells shown in yellow). Conversely, 34.0% (b, P6, cells shown in green) of the CD4+CD25+CD127low/- cells (b, P6) could not be detected in the CD4+CD25+FoxP3+ population but instead fell outside of this population (a, P5, cells show in green). Journal of Investigative Dermatology 2010 130, 492-499DOI: (10.1038/jid.2009.313) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Correlation between CD4+CD25+FoxP3+ cells and CD4+CD25+CD127low/- cells in patients with systemic sclerosis (SSc). To define the frequency of circulating CD4+CD25+FoxP3+ cells and CD4+CD25+CD127low/- cells in SSc, peripheral blood mononuclear cells (PBMCs) from patients with the disease were purified from peripheral blood samples and stained for CD4, CD25, FoxP3, and CD127. PBMCs were gated for CD4+ T cells (data not shown; see Figure 1a), and the percentage of CD4+CD25+FoxP3+ cells among the CD4+ T cells (a, P5) and that of CD4+CD25+CD127low/- cells among the CD4+ T cells (b, P6) were determined. Moreover, the correlation between CD4+CD25+FoxP3+ cells and CD4+CD25+CD127low/- cells was evaluated in patients with SSc (c) using the same strategy as explained in Figure 6. As indicated in the circle diagrams, 35.5% of the CD4+CD25+FoxP3+ cells could not be detected by the CD4+CD25+CD127low/- strategy. Conversely, 40.7% of the CD4+CD25+CD127low/- cells could not be detected by the CD4+CD25+FoxP3+ strategy. Journal of Investigative Dermatology 2010 130, 492-499DOI: (10.1038/jid.2009.313) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Numbers of Tregs in normal healthy donors (NHDs) and systemic sclerosis (SSc) patients using the CD4+CD25+FoxP3+ or CD4+CD25+CD127low/- strategy. To investigate quantitative differences in Treg populations in NHDs versus patients with SSc, peripheral blood mononuclear cells (PBMCs) from both groups were stained for CD4, CD25, FoxP3, and CD127. Statistical analysis revealed no significant difference regarding the frequency of CD4+CD25+FoxP3+ Tregs among the CD4+ cells between NHDs and patients with SSc. There was also no significant difference in the percentage of CD4+CD25+CD127low/- cells between NHDs and SSc patients. In addition, no significant difference could be found between CD4+CD25+FoxP3+ cells and CD4+CD25+CD127low/- cells in NHDs and SSc patients. Journal of Investigative Dermatology 2010 130, 492-499DOI: (10.1038/jid.2009.313) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions