Analysis of the transcriptional program of developing induced regulatory T cells

Abstract

CD25+ regulatory T cells develop in the thymus (nTregs), but may also be generated in the periphery upon stimulation of naive CD4 T cells under appropriate conditions (iTregs). To gain insight into the mechanisms governing iTreg development, we performed longitudinal transcriptional profiling of CD25+ T cells during their differentiation from uncommitted naive CD4 T cells. Microarray analysis of mRNA from CD25+ iTregs early after stimulation revealed expression of genes involved in cell cycle progression and T cell activation, which largely overlapped with genes expressed in CD25+ effector T cells (Teffs) used as a control. Whereas expression of these genes remained elevated in Teffs, it declined gradually in developing iTregs, resulting in a more quiescent phenotype in mature iTregs. A similar pattern of kinetics was observed for biological processes and for intracellular pathways over-represented within the expressed genes. A maximum dichotomy of transcriptional activity between iTregs and Teffs was reached at late stages of their maturation. Of interest, members of the FoxO and FoxM1 transcription factor family pathways exhibited a reciprocal expression pattern in iTregs and Teffs, suggesting a role of these transcription factors in determining T cell fate.

Figure 1. CD25+ iTreg development.

Human naive CD4 T cells depleted of CD25+ cells were cultured with autologous irradiated feeder cells for 10 days in the presence or absence of IL-4. (A) At days 3, 5, 7 and 10 cells were analyzed for CD25 expression. (B) CD25+ and CD25- populations were magnetically sorted at the days indicated. Foxp3 mRNA expression was assessed in both CD25+ and CD25- populations by real-time PCR and its relative expression was calculated in relation to cyclophilin mRNA. (C) At day 10, the proliferative and suppressive capacities of CD25+ and CD25- cells in response to CD3 stimulation were assessed by thymidine incorporation. Data are shown as a mean ± SEM of three independent experiments (A, B) or as a mean ± SD of one representative experiment performed in triplicates (C).

Figure 2. Number of regulated transcripts in CD25+ T cells during 10 days of culture.

Bars indicate numbers of probes with at least two-fold differential expression between CD25+ iTregs or Teffs and their corresponding CD25- cell population at each analyzed time point.

Figure 3. iTreg and Teff specific transcripts during development.

(A) Number of probes specifically expressed in iTregs and Teffs at different times during differentiation. (B) Hierarchical clustering was performed on 93 iTreg-specific and 142 Teff-specific probes. Red and green colors indicate high and low levels of expression, respectively. Rows correspond to individual transcripts. Columns reflect results from three individual donors denoted as 1 to 3. Cytokine clusters are highlighted by blue boxes (IL8 and IL17A at day 3; and IL3, IL4, IL9, IFNG and IL13 at day 5). Black boxes denote a cluster of inflammation-related surface proteins.

Figure 4. GO term enrichment analysis of biological processes in developing iTregs and Teffs.

GO term analysis was performed to identify biological processes significantly over-represented in iTregs and Teffs. Enriched processes in iTregs (black bars) and Teffs (grey bars) at different times organized by their significance score (-log10 p value) are shown. Red and green boxes indicate unique GO terms for iTregs and Teffs, respectively. Yellow boxes represent GO terms enriched in the gene lists of both, iTregs and Teffs.

Figure 5. Pathway analysis in developing iTregs and Teffs.

Pathway analysis was performed to identify biological pathways significantly over-represented in iTregs and Teffs. Enriched processes in iTregs (black bars) and Teffs (grey bars) at different times organized by their significance score (-log10 p value) are shown. Red and green boxes indicate unique pathways for iTregs and Teffs, respectively. Yellow boxes represent pathways enriched in the gene lists of both, iTregs and Teffs.

Figure 6. Pathways characteristic for developing iTregs or Teffs.

(A, B) Significance score (-log10 p value) of three pathways characteristic for iTregs or Teffs development, “calcineurin-regulated NFAT-dependent transcription in lymphocytes” (A), “FoxO family signaling” and “FoxM1 transcription network” (B) during the 10 days of culture. Grey bars indicate significance scores in iTregs, white bars in Teffs. (C, D) Relative expression of the transcription factors FoxO3a and FoxM1 as a ratio of their mRNA expression levels in iTregs to those in Teffs at each respective time point determined by microarray (C) and by real-time PCR (D). Data are shown as mean ± SD from three (C) and five (D) experiments. * p<0.05.