Figure 3.

Peripheral CD4⁺ CD25^high FOXP3^high T cells in the dog show a regulatory phenotype and suppressive function in vitro. Following activation of mononuclear cells with concanavalin A (Con A) and interleukin-2 (IL-2; 10 U/ml), FACS™ was used to sort the 5% of CD4⁺ T cells with the highest and the 20% of CD4⁺ T cells with the lowest CD25 expression. (A 5% rather than a 2% gate was chosen to optimize both FOXP3 enrichment and numbers of cells sorted for functional assays.) The CD25^high cells were consistently enriched in cells expressing FOXP3 relative to the CD25^intermediate or CD25⁻ (CD25^neg) cells [a(i)]. The histograms show respective expression of FOXP3 of each population, with median fluorescence intensity (MFI) and absolute cell counts; these results are representative of seven independent experiments. The 96-hr (t = 96) activation protocol increased the proportion and absolute number of FOXP3⁺ Helios⁺ cells, as revealed by co-staining with the anti-murine/human Helios monoclonal antibody 22F6 [a(ii)]. (b) The phenotype of CD4⁺ T cells from a dog both immediately following FACS™ (‘post-sort’) and on the day of CD25^high and CD25^neg cell admixture following pre-activation with Con A, for the purposes of the regulatory T-cell assay (‘pre-assay’). The CD4⁺ CD25^high fraction, in this case approximately 55% FOXP3⁺, retains a population of FOXP3^high cells at the point of admixture, whereas the CD25^neg fraction contains only a small population of FOXP3^intermediate cells, likely to represent activated conventional T cells. The tables to the right of the dot plots show respective summary data (mean ± SEM [%]) for each of the quadrants from five independent experiments. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays (c) reveal that the ‘post-sort’ CD25^high T cells show greater expression of transcripts encoding FOXP3 and IL-10; greater expression of FOXP3 is also evident at the point of admixture of the cells (‘pre-assay’). All RT-qPCR assays were performed on lymph-node-derived cells and qPCR data are expressed as the natural logarithm (ln) of the ratio of gene expression Ct difference of CD25^high : CD25^neg cells, normalized to β₂-microglobulin; mean and standard deviation ln expression ratios are depicted, with geometric mean ratios shown on the right of the figure (n= 4). (An ln expression ratio of 0 represents equally abundant transcript in both populations). When the cells depicted in (b) were co-cultured with activated third party responder CD4⁺ T cells at a 1 : 1 ratio, the CD4⁺ CD25^high T cells were able to suppress the proliferation of the responder cells, whereas the CD4⁺ CD25^neg cells showed no suppressive properties (d). When cultured alone, the CD4⁺ CD25^high T cells showed anergy that could be broken by the addition of IL-2 (20 U/ml), while the CD4⁺ CD25^neg cells proliferated robustly with or without exogenous IL-2. Control co-cultures of CD25^high or CD25^neg CD4⁺ T cells maintained in complete medium without Con A admixed with similarly unstimulated third-party CD4⁺ T cells showed only background counts, ruling out any influence of a mixed leukocyte reaction. Error bars indicate standard errors of the mean of triplicate cultures (CD4⁺ T cells and co-cultures) or (difference in values/2) for duplicate monocultures of CD25^high or CD25^neg T cells. The figure to the right of the panel summarizes proportional suppression of proliferation for nine independent experiments; square symbols represent assays performed on peripheral blood mononuclear cells, circles represent assays performed on lymph node-derived cells, and the horizontal line indicates the mean value. (All co-culture assays were performed on cells derived from beagles.)