Global expression profiling of peripheral Qa-1-restricted CD8αα+TCRαβ+ regulatory T cells reveals innate-like features: implications for immune-regulatory repertoire

Abstract

Among peripheral regulatory T cells, CD8(+) T cells also play an important role in the maintenance of immune homeostasis. A subset of CD8(+) Treg that express αβ T cell receptor (TCR) and CD8αα homodimers can recognize TCR-derived peptides in the context of the class Ib MHC molecule Qa-1. To gain a better understanding of the nature and phenotype of CD8αα(+)TCRαβ+ Treg, a global gene expression profiling using microarray, real-time quantitative polymerase chain reaction, and flow-cytometric analysis was performed using functional Treg clones and lines. The study findings show that CD8(+) Treg shared gene profile expressed by innate-like lymphocytes, including murine intraepithelial lymphocytes and thymic CD8αα(+)TCRαβ+ T-cell populations. In addition, this subset displays differential expression of several key regulatory molecules, including CD200. CD8αα(+) Treg expressed higher levels of a number of natural killer cell-related receptors and molecules belonging to the TNF superfamily. Collectively, peripheral class Ib-reactive CD8αα(+)TCRαβ+ T cells represent a unique regulatory population different from class Ia major histocompatibility complex-restricted conventional T cells. These studies have important implications for the regulatory mechanisms mediated by the CD8(+) Treg population in general.

FIGURE 1

Scatter plot displays the log of raw intensity values for the genes of interest between CD8αα+TCRαβ+ Treg vs. conventional CD8αβ+TCRαβ+ T cell populations. Genes with a fold change expression greater then 2 are shown on the left and right hand side of the outer diagonals whereas the center diagonal indicates less than 2 fold change. Immunoregulatory genes are shown in red whereas the remaining ones are shown in blue. The complete microarray data has been submitted to the public database GEO: submission number GSE22985.

FIGURE 2

Relative expression of some characteristic genes enriched among clonal as well as polyclonal populations of CD8αα+TCRαβ+ Treg cells. mRNAs were isolated from respective CD8αα+ Treg clone (2D11) and lines (XT14 and PL1), and expression of > 50 genes was analyzed by real-time PCR and only expression of some of the most relevant genes is shown. Gene expression from the OVA-reactive CD8αβ+TCRαβ+ OT−1 T cells was used as control. The data were representative of one of two independent experiments and are presented as relative fold change after normalization against internal control gene L32.

FIGURE 3

CD8αα+TCRαβ+ Treg showed elevated cell surface expression of immunoregulatory molecules CD200, HVEM (A) and Ly49 (B) cell surface molecules. CD8αα+TCRαβ+ Treg (2D11) and OVA-reactive CD8αβ+TCRαβ+T cells are stained with the indicated antibodies or relevant isotype controls and analyzed by flow cytometry. The thick line represents staining of CD8αα+TCRαβ+ Treg and the thin line represents staining of CD8αβ+TCRαβ+ T cells. An isotype control is also included and is represented by the dotted line. While y-axis denotes the % max fluorescence, x-axis shows fluorescence intensity relative to staining with anti-HVEM or anti-CD200 antibodies (3A) and several anti-Ly-49 markers as indicated on the top (3B). Data are representative of three independent experiments.

FIGURE 3

CD8αα+TCRαβ+ Treg showed elevated cell surface expression of immunoregulatory molecules CD200, HVEM (A) and Ly49 (B) cell surface molecules. CD8αα+TCRαβ+ Treg (2D11) and OVA-reactive CD8αβ+TCRαβ+T cells are stained with the indicated antibodies or relevant isotype controls and analyzed by flow cytometry. The thick line represents staining of CD8αα+TCRαβ+ Treg and the thin line represents staining of CD8αβ+TCRαβ+ T cells. An isotype control is also included and is represented by the dotted line. While y-axis denotes the % max fluorescence, x-axis shows fluorescence intensity relative to staining with anti-HVEM or anti-CD200 antibodies (3A) and several anti-Ly-49 markers as indicated on the top (3B). Data are representative of three independent experiments.

FIGURE 4

Differential expression profile of some key immune-related transcripts in CD8αα+TCRαβ+ Tregs. mRNAs were isolated from CD8αα+ Treg clone 2D11 and conventional OVA-reactive CD8αβ+ T cell clone OT1. Expression of mRNA for the indicated molecules enriched in CD8αα+ Tregs (A) as well as selected NK-related genes (B) was examined by real-time PCR. The data are representative of one of two independent experiments and are presented as relative fold change after normalization against internal control gene L32.

FIGURE 4

Differential expression profile of some key immune-related transcripts in CD8αα+TCRαβ+ Tregs. mRNAs were isolated from CD8αα+ Treg clone 2D11 and conventional OVA-reactive CD8αβ+ T cell clone OT1. Expression of mRNA for the indicated molecules enriched in CD8αα+ Tregs (A) as well as selected NK-related genes (B) was examined by real-time PCR. The data are representative of one of two independent experiments and are presented as relative fold change after normalization against internal control gene L32.