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. 2006 Apr 25;103(17):6659-64.
doi: 10.1073/pnas.0509484103. Epub 2006 Apr 14.

Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development

Marc A Gavin  1 , Troy R Torgerson, Evan Houston, Paul DeRoos, William Y Ho, Asbjørg Stray-Pedersen, Elizabeth L Ocheltree, Philip D Greenberg, Hans D Ochs, Alexander Y Rudensky
Affiliations

Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development

Marc A Gavin et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9373

Abstract

Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25+CD4+ regulatory T cell (T(R)) development and function in mice. In humans, its role in mediating T(R) development has been controversial. Furthermore, the fate of T(R) precursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human T(R) development. Unlike murine Foxp3- T cells, a small subset of human CD4+ and CD8+ T cells transiently up-regulated FOXP3 upon in vitro stimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, only ex vivo FOXP3+ T(R) cells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a T(r) developmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or without FOXP3 mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing a FOXP3 mutation enabling expression of stable FOXP3(mut) protein exhibited FOXP3(mut)-expressing cells among a subset of highly activated CD4+ T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from T(R) precursors.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Flow cytometric detection of Foxp3 in murine and human cells. (A and B) Normal or Foxp3-deficient mouse lymph node cells were stained for Foxp3 and cell-surface markers by using digoxigenin-conjugated mAb 3G3 (A) or Foxp3-specific rabbit antibody (B). CD4+ gated lymphocytes are shown. (C–E) Normal (1792 and 1745) or FOXP3-deficient (IPEX) PBMC were stained for FOXP3 and lymphocyte markers by using digoxigenin-conjugated mAb 3G3 (C) or digoxigenin-conjugated Foxp3-specific rabbit antibody (D and E). Both CD4+ and CD8+ gated lymphocytes are shown. Additional IPEX-1 PBMC were not available for subsequent analysis with rabbit antibody. High background staining of Foxp3− cells is a consequence of the three-step staining procedure.
Fig. 2.
Fig. 2.
Analysis of FOXP3 expression in activated human CD4+ and CD8+ T cells. (A–C) Total or CD25-depleted PBMC from donor 1745 were stimulated with 5, 100, or 1000 ng/ml anti-CD3. FOXP3 and CD25 expression on CD4+ and CD8+ cells were assessed at days 3, 7, and 10 of culture. Shown are expression profiles for 100 ng/ml anti-CD3 (A), 5 or 1,000 ng/ml anti-CD3 for CD4+ gated cells (B), and the plotted percentage of gated cells expressing FOXP3 (C). FOXP3 was detected with digoxigenin-conjugated FOXP3-specific rabbit antibody. (D) Total PBMC from donor 1745 were labeled with CFSE and stimulated with 100 ng/ml anti-CD3. FOXP3 expression was assessed at days 3 and 7 with digoxigenin-conjugated rabbit antibody. Data are representative of four separate experiments and three normal adult donors.
Fig. 3.
Fig. 3.
Induced FOXP3 does not suppress IL-2 or IFN-γ synthesis. (A) Freshly isolated or stimulated (100 ng/ml anti-CD3) total PBMC from donor 1745 were incubated with PMA, ionomycin, and monensin and stained for FOXP3 (rabbit IgG-digoxigenin), IL-2, IFN-γ, and surface markers as described in Materials and Methods. (B) The percentage of cytokine-expressing cells among FOXP3high, FOXP3low, or FOXP3− cells is plotted. The distinction between high and low FOXP3 expression was not made for CD4+ cells and CD8+ cells on day 0. Data are representative of three separate experiments.
Fig. 4.
Fig. 4.
FOXP3 expression in IPEX. (A and B) Normal (1745), IPEX-like (A), and IPEX (B) PBMC were stained for cell-surface markers and FOXP3 with mAb 3G3. Gated CD4+ cells are shown. Histograms show FOXP3 expression (A) and side scatter (B) on CD4+ cells expressing varying degrees of CD25 as delineated in the adjacent 2D plots. Because the staining procedure results in a decrease in forward scatter, side scatter is a better indicator of cell size. Staining was performed before the development of protocols by using digoxigenin-conjugated rabbit antibody, but additional PBMC from these patients were not available for further study. PBMC shown in A and B were stained in separate experiments. (C) Freshly isolated or simulated (100 ng/ml anti-CD3; 3 days) normal (2020) or IPEX-2-P2 PBMC were stained for FOXP3 with mAb 259D (14).
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