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. 2007 May;121(1):129-39.
doi: 10.1111/j.1365-2567.2006.02550.x.

CD27 expression discriminates between regulatory and non-regulatory cells after expansion of human peripheral blood CD4+ CD25+ cells

Richard C Duggleby  1 , Tovah N F Shaw, Lorna B Jarvis, Gurman Kaur, J S Hill Gaston
Affiliations

CD27 expression discriminates between regulatory and non-regulatory cells after expansion of human peripheral blood CD4+ CD25+ cells

Richard C Duggleby et al. Immunology. 2007 May.

Abstract

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25- cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25(high) and CD25(intermediate) CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T-cell populations such as those found in joints.

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Figures

Figure 1
Figure 1
Isolation and expansion of regulatory T cells. (a) A representative example of CD25 expression on CD4+ cells in (i) healthy control peripheral blood mononuclear cells (PBMC), (ii) isolated CD4+ CD25– cells, and (iii) isolated CD4+ CD25+ cells. CD25 expression is subdivided as described by Cao et al.; CD25 high (Hi) cells are defined as CD4+ cells with brighter CD25 staining than the brightest CD8+ CD25+ cells. CD25 negative (Neg) and CD25 intermediate (Int) cells are gated using isotype controls. The percentages of live cells, by forward and side scatter, in each sector are shown, with the percentages of CD4+ gated cells in each sector in brackets. Quadrants were set using isotype controls. (b) Regulatory activity of lines generated from CD4+ CD25+ cells following 2–8 weeks of expansion using: (1) PPD 100 or 300 U/ml interleukin (IL)-2 and irradiated, autologous PBMC, (2) anti-CD3/CD28 dynabeads, with or without irradiated CD4+ cells, or (3) plate-bound anti-CD3, soluble anti-CD28 and IL-2 (pαCD3/CD28), with one or two rounds of stimulation. Regulatory activity is measured as ‘percentage response’, i.e. effector cell responses to soluble anti-CD3 in the presence of expanded CD4+ CD25+ cells compared with their response when cultured alone. Median regulatory activities of lines obtained using the three different protocols are shown.
Figure 2
Figure 2
Regulatory activity is associated with high numbers of CD27+ cells. CD4+ CD25+ cells were isolated from seven healthy donors and CD4+ CD25– cells from six healthy donors and expanded to generate 20 and 23 lines, respectively, using the methods described in Fig. 1(b) In each case regulatory activity is shown as the logarithm of the ‘percentage response’, i.e. effector cell responses to soluble anti-CD3 in the presence of expanded cells compared with their response when cultured alone. (a) Scatter plot showing the correlation between regulatory activity and the proportion of CD27+ cells in lines derived from CD4+ CD25+ cells. An exponential curve fit is shown with a logarithmic transformation (inset) to achieve a linear relationship; Spearman rank correlation for log-transformed data: r = −0·819 (95% confidence interval, −0·93 to −0·58); r2 = 0·67. (b) Scatter plot showing the lack of correlation between regulatory activity and the proportion of CD27+ cells in lines derived from CD4+ CD25– cells. A logarithmic transformation of the data as used in (a) is also shown (inset).
Figure 3
Figure 3
The CD27+ cells in the lines derived from CD25+ cells are hyporesponsive and possess the highest regulatory activity. (a) Response to soluble anti-CD3 antibody by lines derived from CD4+ CD25+ and CD4+ CD25– cells, and from CD27+ and CD27– cells purified from these lines. Responses are expressed as a percentage of the response by autologous peripheral blood mononuclear cells (PBMC) or freshly isolated CD4+ CD25– cells tested in the same experiment. Boxes contain values falling between the 25th and 75th percentiles, with lines extending from the box indicating ranges in each subgroup. The lines within the boxes represent median values. P-values were determined by Mann–Whitney U-tests. (b, c) Regulatory activity of CD27+ cells and CD27– cells isolated from cell lines derived from CD4+ CD25+ (b) or from CD4+ CD25– cells (c), compared with the unseparated T-cell lines. Regulatory activity is expressed as described in the legend to Fig. 1(b). P-values were determined using Wilcoxon paired tests.
Figure 4
Figure 4
Cell lines derived from CD4+ CD25+ cells express high levels of forkhead box P3 (Foxp3) and high numbers of Foxp3+ cells are present in the CD27+ subpopulation. (a) Flow cytometric analysis of T-cell lines derived from CD4+ CD25+ (upper) and CD4+ CD25– (lower) cells after 2 weeks of expansion with plate-bound anti-CD3, soluble anti-CD28 and interleukin (IL)-2. Cells were stained for CD25, intracellular Foxp3 and CD4. The dot plots shown are gated on CD4+ cells with quadrants set using isotype controls. The percentages of cells in each quadrant are indicated, with the mean fluorescence intensity (MFI) of Foxp3 staining given in brackets. (b) Variability of Foxp3 labelling of CD27+ cells. The figure shows labelling from (a) and three additional lines from healthy controls derived from CD4+ CD25+ cells or CD4+ CD25– cells, after expansion for 14–20 days. Upper panel: MFI of Foxp3 labelling of CD27+ cells. Lower panel: the percentages of CD27+ cells that were Foxp3+. Analyses were gated on CD4+ cells if either line was >5% CD4–. (c)Foxp3 mRNA expression by freshly isolated CD4+ CD25– and CD4+ CD25+ cells (black bars), and by CD27+ and CD27– cells isolated from expanded CD25+ (grey bars) and CD25– (open bars) cell-derived cell lines after 4 weeks in culture. Foxp3 mRNA was measured relative to hypoxanthine phosphoribosyl-transferase (HPRT) mRNA by quantitative polymerase chain reaction (PCR), standardized using a Foxp3+ CD8+ T-cell clone (see Materials and methods).
Figure 5
Figure 5
Phenotype of T-cell lines after 5 weeks in culture. Flow cytometric analysis of CD4+ CD25+ cell-derived (upper panels) and CD4+ CD25– cell-derived (lower panels) cell lines after 5 weeks in culture and two rounds of stimulation with plate-bound anti-CD3, soluble CD28 and interleukin (IL)-2. All markers were analysed in conjunction with staining for CD27; the percentages of cells in each quadrant (set on isotype controls) are shown together with the mean fluorescence intensity (MFI) on the y-axis (brackets).
Figure 6
Figure 6
A high proportion of synovial CD4+ cells are CD27+. Percentages of CD4+ CD25–, CD4+ CD25int (‘int’ denoting ‘intermediate’) and CD4+ CD25high cells (as defined by Cao et al.37) that were CD27+ in peripheral blood mononuclear cells (PBMC) from six healthy controls (HC, open boxes), PBMC from eight reactive arthritis (ReA) patients (light grey boxes) and synovial fluid mononuclear cells (SFMC) from six ReA patients (dark grey boxes). Boxes contain values falling between the 25th and 75th percentiles, with lines extending from the boxes indicating the range. The lines within the boxes represent median values. P-values were determined by Mann-Whitney U-test.
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