Differential role of all-trans retinoic acid in promoting the development of CD4+ and CD8+ regulatory T cells

Abstract

It is known that ATRA promotes the development of TGF-β-induced CD4(+)Foxp3(+) iTregs, which play a vital role in the prevention of autoimmune diseases; however, the role of ATRA in facilitating the differentiation and function of CD8(+)Foxp3(+) iTregs remains elusive. Using a head-to-head comparison, we found that ATRA promoted expression of Foxp3 and development of CD4(+) iTregs, but it did not promote Foxp3 expression on CD8(+) cells. Using a standard in vitro assay, we demonstrated that CD8(+) iTregs induced by TGF-β and ATRA were not superior to CD8(+) iTregs induced by TGF-β alone. In cGVHD, in a typical lupus syndrome model where DBA2 spleen cells were transferred to DBA2xC57BL/6 F1 mice, we observed that both CD8(+) iTregs induced by TGF-β and ATRA and those induced by TGF-β alone had similar therapeutic effects. ATRA did not boost but, conversely, impaired the differentiation and function of human CD8(+) iTregs. CD8(+) cells expressed the ATRA receptor RAR and responded to ATRA, similar to CD4(+) cells. We have identified the differential role of ATRA in promoting Foxp3(+) Tregs in CD4(+) and CD8(+) cell populations. These results will help to determine a protocol for developing different Treg cell populations and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

Keywords: Autoimmunity; Foxp3; GVHD; TGF-β; all-trans retinoic acid; regulatory T cells.

Figure 1.. ATRA increased the percentages of Foxp3 expression on TGF-β-primed CD4⁺, but not on CD8⁺ cells.

(A) CD8⁺CD62L⁺CD25⁻Foxp3⁻(GFP⁻) and CD4⁺CD62L⁺CD25⁻Foxp3⁻(GFP⁻) cells isolated from C57BL/6 Foxp3^gfp reporter mice were stimulated with immobilized anti-CD3 (1 μg/ml), soluble anti-CD28 (1 μg/ml), IL-2 (100 U/ml), or TGF-β (2 ng/ml), with (CD4_TGFβ+ATRA or CD8_TGFβ+ATRA) or without ATRA (50 nM) (CD4_TGFβ or CD8_TGFβ) for 3 days. Foxp3 (GFP) expression was examined by flow cytometry. Left: typical FACS histograms. Right: summary of data showing the frequency of Foxp3⁺ cells from TGF-β-primed CD4⁺ or CD8⁺ cells. *P < 0.05, NS. (B) The expression levels of regulatory T-cell associated markers including CD25, GITR, CTLA-4, and TNFR2 on CD4_TGFβ, CD8_TGFβ, CD4_TGFβ+ATRA, or CD8_TGFβ+ATRA cells were analyzed by flow cytometry. The graph data indicate the mean ± sem of 3 separate experiments showing the frequency of the indicated markers gated on the CD4 or CD8 cell populations.

Figure 2.. CD8⁺ cells had the same response to ATRA as did CD4⁺ cells.

(A) The expression of ATRA RARα mRNA was determined by quantitative RT-PCR on naive or activated CD4⁺ and CD8⁺ cells. These cells were activated with TCR and IL-2, with or without TGF-β and with or without ATRA. Data are the mean ± se of 3 separate experiments. (B) CD4_TGFβ, CD8_TGFβ, CD4_TGFβ+ATRA, or CD8_TGFβ+ATRA cells were induced as in Fig. 1A, and the expression of CD44 and CD62L gated on CD4 or CD8 was analyzed by flow cytometry. Dot plots are representative of 3 independent experiments. (C) The expression of α₄β₇ and CCR-9 on CD4_med, CD8_med, CD4_TGFβ, CD8_TGFβ, CD4_TGFβ+ATRA, or CD8_TGFβ+ATRA cells was analyzed by flow cytometry. Data are representative of 3 independent experiments showing staining (black) plus isotype control (gray).

Figure 3.. ATRA enhanced the suppressive activity of CD4⁺ iTregs, but not of CD8⁺ iTregs, in vitro.

(A) CD4_TGFβ, CD8_TGFβ, CD4_TGFβ+ATRA, or CD8_TGFβ+ATRA cells generated as in Fig. 1A were added to cultures of soluble anti-CD3 (0.025 μg/ml), irradiated APCs (1:1), and CFSE-labeled responder T cells for 3 days. Suppressive activity by each cell population was demonstrated and compared by percentages of CFSE-diluted cells. A histogram representative of 3 separate experiments in 1:2 ratios of Treg-to-responder T cells is shown. (B) The same culture as in (A), but the graded ratio of Tregs was added to the culture. The graph shows the mean ± sem of results in 3 independent experiments. *P < 0.05. **P < 0.01.

Figure 4.. ATRA promoted the development of function of TGF-β-induced CD4⁺ iTregs, but not of CD8[supb]+ iTregs, in vivo.

CD4_TGFβ, CD8_TGFβ, CD4_TGFβ+ATRA, or CD8_TGFβ+ATRA cells were induced as in Fig. 1A. Freshly isolated splenocytes (80 × 10⁶) from DBA/2 mice or together with 5 × 10⁶ of various CD4⁺ or CD8⁺ cell populations, as indicated, were intravenously transferred into C57BL/6xDBA/2 F1 mice. PBS was used for the negative control and marked as the model group. Each group had 6 mice, and the experiment was repeated twice. (A) Survival was monitored weekly. A Kaplan-Meier survival curve is shown. (B) The levels of anti-dsDNA IgG antibody in sera were measured by ELISA 4 weeks after cell transfer. (C) The proteinuria levels were examined 8 weeks after cell transfer. Data are combined from 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.. ATRA promoted the function of TGF-β-induced human CD4⁺ iTregs, but not CD8₊ iTregs, in a humanized xeno-GVHD model.

(A) Human CD4⁺CD45RA⁺ or CD8⁺CD45RA⁺ cells isolated from peripheral PBMCs were stimulated with anti-human CD3/CD28 beads 1:10 (1 bead:10 cells) with IL-2 (50–100 U/ml), with or without TGF-β1 (5 ng/ml) and with or without ATRA (100 nM) for 5 days. Foxp3 expression was determined by flow cytometry. Histogram data are representative of 3 independent experiments. (B) Kaplan-Meier survival estimates of NOD/SCID mice that received 20 × 10⁶ CD25⁻ hPBMCs or plus 5 × 10⁶ CD4_TGFβ, CD8_TGFβ, CD4_TGFβ+ATRA, or CD8_TGFβ+ATRA cells. (D) Body weight of xeno-GVHD model mice treated with or without conditioning cells, as described in (A). Data were collected from 3 independent experiments. *P < 0.05, **P < 0.01.