Differential expression of regulator of G-protein signalling transcripts and in vivo migration of CD4+ naïve and regulatory T cells

Abstract

The immune response of T lymphocytes to pathogens is initiated in draining secondary lymphoid organs, and activated cells then migrate to the site of infection. Thus, control of naive and regulatory CD4+ T-cell migration is crucial; however, it is poorly understood in physiological and pathological conditions. We found that CD4+ subpopulations displayed characteristic regulator of G-protein signalling (RGS) gene expression profiles. Regulatory T cells express higher levels of RGS1, RGS9 and RGS16 than naive cells. These genes are up-regulated upon cell activation and their level of expression correlates with in vivo cell migration. Using parabiosis, we showed that regulatory T lymphocytes migrate less than naive T cells and that migrant naive T cells express even lower RGS levels than their static counterparts. Our results show an inverse correlation between the capacity to migrate and the levels of RGS1, RGS9 and RGS16 for both naive and regulatory T cells. Taken together, these results suggest a role for RGS molecules in chemokine-induced lymphocyte migration and demonstrate the peculiarity of regulatory T cells in terms of phenotype and migration ability, providing new insights into their function.

Figure 1

Differential chemokine responses of CD25⁺ and CD25⁻CD4⁺ T cells: no chemokine. Splenocytes were stained with anti-CD4 and anti-CD25 monoclonal antibodies and chemokine-induced migration towards chemokine (C-X-C motif) ligand 12/stromal cell derived factor 1α (CXCL12/SDF1α) (0·6 ng/ml, ligand of chemokine (CXC motif) receptor 4 (CXCR4)), chemokine (C-C motif) ligand 20 (CCL20)/macrophage inflammatory protein 3α (MIP3α) (6 ng/ml, ligand of CCR6) and chemokine (C-C motif) ligand (CCL19)/MIP3β (100 ng/ml, ligand of chemokine (C-C motif) receptor 6 (CCR6)) was evaluated for naïve T cells (CD4⁺ CD25⁻; open bars) and regulatory T cells (CD4⁺ CD25⁺; solid bars) in a transwell assay. Two independent experiments were performed with similar outcomes; results presented are the mean ± standard deviation of triplicate measurements.

Figure 2

Differential regulator of G-protein signalling (RGS) expression in CD25⁺ and CD25⁻ CD4⁺ T cells. Regulatory T cells (CD4⁺ CD25⁺ CD45RB^low), naïve T cells (CD4⁺ CD25⁻ CD45RB^high) and the remaining intermediate population of CD4⁺ CD25⁻CD45RB^low T cells were purified from the spleens of 8-week-old mice. Duplex quantitative PCR was performed for the 21 RGS genes and three domestic genes. Results of each gene expression are normalized for β-actin. The experiment was repeated twice and the PCR performed in duplicate. Elongation factor 1α (EF1α); regulator of G-protein signalling 10 long isoform (RGS10L); regulator of G-protein signalling 10 very-short isoform (RGS10VS); lbc (GEF protein) second cousin (lsc); RGS domain and PHOX domain containing protein (RGS-PX1).

Figure 3

Forkhead box protein P3 (Foxp3) is specifically expressed in regulatory T cells. Total RNA was extracted from sorted splenic naïve or regulatory T cells with RNAzol and reverse-transcribed. PCR reactions were performed as described in the ‘Materials and methods’.

Figure 4

The regulator of G-protein signalling (RGS) expression profile is modified after T-cell activation. (a) CD69 and CD25 are expressed on T cells after concavalin A (ConA) activation. Spleen cells were cultured in complete RPMI with or without ConA (2 µg/ml). The percentages of CD4⁺ CD69⁺ and CD4⁺CD25⁺ cells are shown in the upper right corner of each dot plot for the indicated times (3, 6 and 12 hr). Forward scatter (FSC). (b) RGS expression is modified after in vitro ConA stimulation. Splenic T cells from C57Bl/6 mice were activated in vitro with ConA (2 µg/ml). At the indicated times (3, 6 and 12 hr), cells were collected, mRNA was prepared and quantitative PCR was performed. The figure shows the fold induction of RGS1, RGS9, RGS11 and RGS16 expression during the time course of the experiment. No significant changes were observed for β-actin, elongation factor 1α (EF1α) or other RGS genes (data not shown).

Figure 5

Differential migration of regulatory and naïve CD4⁺ T cells. (a) Parabiosis between C57Bl/6 (Ly5²) and C57Bl/6.Ly5¹ congenic mice. Upper panels: 8-week-old female mice were joined laterally and their organs analysed after 6 days. The chimerism (% of cells coming from the other parabiont) was assessed by fluorescence activated cell sorter (FACS) in the spleen and the lymph nodes of each parabiont for the three CD4⁺ T-cell subpopulations: naïve (CD4⁺ CD25⁻ CD45RB^high), intermediate (Int.) (CD4⁺ CD25⁻ CD45RB^low) and regulatory (Reg.) (CD4⁺ CD25⁺ CD45RB^low) T cells. Results for two couples are presented (four animals): solid symbols represent C57Bl/6 animals, open symbols C57Bl/6.Ly5¹ animals, round symbols couple 1 and square symbols couple 2. Lower panels: same as upper panels but mice were thymectomized prior to parabiosis. (b) FACS profiles of cells obtained from a thymectomized C57Bl/6 (Ly5²) mouse which had been parabiosed for 6 days with a thymectomized C57Bl/6.Ly5¹ congenic mouse. The upper contour plot shows the CD45RB versus CD25 distribution of gated viable propidium iodide (PI^–) CD4⁺ spleen T cells and the histograms show the Ly5¹ profile of the gated R1 (CD45Rb^high CD25⁻) naïve and R2 (CD45Rb^low CD25⁺) regulatory T cells. The percentages of chimerism presented in Fig. 5(a) were obtained in this way. The lower dot plot shows the CD4 versus Ly5¹ distribution of lymph node T cells in Ly5² parabionts; Ly5¹⁺ cells represent exogenous lymphocytes. The contour plots (CD4 versus CD25) are gated on endogenous Ly5^1– CD4⁺ (R1) or exogenous Ly5¹⁺ CD4⁺ T cells (R2). Among the migrating CD4⁺ cells, regulatory T cells represent a small proportion (7·4%).

Figure 6

Differential expression of regulator of G-protein signalling (RGS) genes in mobile versus sessile CD4⁺ T cells. Spleen cells from C57Bl/6 (Ly5²) and C57Bl/6.Ly5¹ parabionts were stained for CD4, CD25, CD45RB, Ly5 and propidium iodide (PI) and five-colour sorted on a Moflo into ‘exogenous regulatory T cells’ (regulatory T cells expressing the Ly5 marker of the joined parabiont), ‘endogenous regulatory T cells’, ‘exogenous naïve T cells’, and ‘endogenous naïve T cells’. mRNA prepared from pools of sorted cells was used for quantitative PCR for the RGS1, RGS9 and RGS16 genes. Shown are the fold decrease of RGS gene expression in ‘exogenous cells’ compared to ‘endogenous cells’ in the naïve (upper part) and regulatory T cell (lower part) populations.