Tr1-Like T Cells - An Enigmatic Regulatory T Cell Lineage

Abstract

The immune system evolved to respond to foreign invaders and prevent autoimmunity to self-antigens. Several types of regulatory T cells facilitate the latter process. These include a subset of Foxp3(-) CD4(+) T cells able to secrete IL-10 in an antigen-specific manner, type 1 regulatory (Tr1) T cells. Although their suppressive function has been confirmed both in vitro and in vivo, their phenotype remains poorly defined. It has been suggested that the surface markers LAG-3 and CD49b are biomarkers for murine and human Tr1 cells. Here, we discuss these findings in the context of our data regarding the expression pattern of inhibitory receptors (IRs) CD49b, TIM-3, PD-1, TIGIT, LAG-3, and ICOS on Tr1-like human T cells generated in vitro from CD4(+) memory T cells stimulated with αCD3 and αCD28 antibodies. We found that there were no differences in IR expression between IL-10(+) and IL-10(-) T cells. However, CD4(+)IL-10(+) T cells isolated ex vivo, following a short stimulation and cytokine secretion assay, contained significantly higher proportions of TIM-3(+) and PD-1(+) cells. They also expressed significantly higher TIGIT mRNA and showed a trend toward increased TIM-3 mRNA levels. These data led us to conclude that large pools of IRs may be stored intracellularly; hence, they may not represent ideal candidates as cell surface biomarkers for Tr1-like T cells.

Keywords: CD4+ T cell; IL-10; Tr-1 T cells; inhibitory receptors; peripheral tolerance.

Figure 1

(A) Naive and memory CD4⁺ T cells were isolated from PBMC from healthy donors by magnetic selection and stimulated with plate-bound 1 μg/ml αCD3 and 2 μg/ml αCD28 ± 100 ng/ml of IL-27. Intracellular staining for IL-10 was performed on days 3 and 7 after an additional 4-h stimulation with PMA/ionomycin in the presence of Golgi stop. Graphs show the percentages (mean value ± SEM, n = 3 donors) of viable CD4⁺IL-10⁺ T cells derived from the naive or memory cell subsets (left panel). A representative dot plot of CD4 and IL-10 staining on memory-derived CD4⁺ T cells on day 7 is shown in the right panel. (B) Expression of inhibitory receptors (IRs) on CD4⁺IL-10^+/− T cells derived from memory pool after 7 days of cell culture in the presence of 1 μg/ml αCD3 and 2 μg/ml αCD28 examined by flow cytometry. The black bars represent the average percentage of IL-10⁺ and white bars the IL-10⁻ cell fractions, respectively (mean + SEM, n = 3 donors). Right panel shows a representative dot plot of CD49b and LAG-3 expression on day 7 by memory CD4⁺IL-10^+/− stimulated with αCD3 and αCD28 ± IL-27.

Figure 2

(A) The expression of IRs on purified CD4⁺IL-10⁺/IL-10⁻ T cells. Magnetically sorted CD4⁺ T cells were cultured for 16 h in the presence of 1 μg/ml αCD3 and 2 μg/ml of αCD28 antibodies, then harvested, subjected to IL-10 cytokine secretion assay, and sorted by flow cytometry according to their IL-10 expression. Graphs represent the percentages of IL-10⁺ or IL-10⁻ T cells, expressing each IR determined by flow cytometry (n = 4). Purified CD4⁺IL-10^+/− T cells were rested for 48 h in the presence of 60 U/ml IL-2 and then were restimulated for 4 h with αCD3 and αCD28 antibodies. (B) mRNA levels of IRs on sorted CD4⁺IL-10^+/− T cells. Purified CD4⁺IL-10^+/− T cells were rested for 48 h in the presence of 60 U/ml IL-2 and then were restimulated for 4 h with αCD3 and αCD28 antibodies. Graphs show mean gene expression levels as relative values compared to HPRT-1 (n = 4). (C) Expression of IRs on CD4⁺IL-10^+/− T cell fraction at the point of RNA isolation as evaluated by flow cytometry. Figure shows percentages of viable CD4⁺IL-10^+/− T cells expressing the indicated marker (mean + SEM, n = 4). The significance has been analyzed using t test.