Subpopulations of bovine WC1(+) gammadelta T cells rather than CD4(+)CD25(high) Foxp3(+) T cells act as immune regulatory cells ex vivo

Abstract

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4(+)CD25(high) T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4(+)CD25(high) T cells and gammadelta T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4(+)CD25(high) T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4(+)CD25(low) T cells, WC1(+), WC1.1(+) and WC1.2(+) gammadelta T cells, NK cells, CD8(+) T cells and CD14(+) monocytes. Only the WC1.1(+) and WC1.2(+) gammadelta T cells and CD14(+) monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4(+)CD25(high)Foxp3(+) and CD4(+)CD25(low) T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the gammadelta T cell population, more precisely in the WC1.1(+) and the WC1.2(+) subpopulation, major populations present in blood of cattle in contrast to non-ruminant species.

Figure 1.

CD4CD25, WC1.2⁺ and WC1.1⁺expression on stained bovine PBMC. (a) Representative dotplot of CD4FITC/CD25PE double-color surface stained PBMC (cow A) and gate regions for sorting and/or frequency analysis of CD4⁺CD25^- (gate R1), total CD25⁺ (gate R2), CD4⁺CD25^low (gate R3), CD4⁺CD25^high (gate R4) and CD4^-CD25^- (gate R5) cells. (b) Representative dotplots of WC1.2⁺, (gate R6) respectively (c) WC1.1⁺ (gate R7) γδ T cell subsets stained PBMC and gate regions for sorting and frequency analysis.

Figure 2.

Transcription of IL-10, TGF-β and Foxp3 in 7 sorted bovine cell subpopulations as determined by qRT-PCR. (a) PBMC were isolated from cows A-C, respectively PE stained and sorted CD3⁺, CD4⁺, CD8⁺, N24⁺, CD21⁺, CD14⁺, NKp46⁺ cells were tested. Results show mean fold changes in gene expression (±1SEM) of triplicate samples from three representative experiments of specific cell populations compared to total PBMC, calculations based on REST^© (Relative Expression Software Tool) software [29] relative to the gene expression of β2-microglobulin. (b) MW sizes of products of qRT-PCR (6 independent PCR), for β2-microglobulin (171 bp), Foxp3 (88 bp), IL-10 (112 bp) and TGF-β (141 bp) (cow A) performed on bovine PBMC derived samples on a 2% agarose gel and compared to a MassRuler™ low range DNA ladder.

Figure 3.

Transcription of IL-10, TGF-β and Foxp3 in CD4CD25 and γδ T cell sorted bovine cell subpopulations as determined by qRT-PCR. (a) CD4⁺CD25^high, CD4⁺CD25^low, CD4⁺CD25^-, and CD4^-CD25^- cells were FACS sorted (CD4FITC/ CD25PE) from cow A–C and (b) WC1⁺, WC1.2⁺ and WC1.1⁺ PE stained γδ T cells were sorted from cow A and C. Results show mean fold changes in gene expression (±1SEM) of triplicate samples from three representative experiments of specific cell populations compared to total PBMC, calculations based on REST^© (Relative Expression Software Tool) software [29] relative to the gene expression of β2-microglobulin.

Figure 4.

Intracellular staining of bovine Foxp3, IL-10, and human Foxp3. (a) Histograms showing intracellular Foxp3 fluorescence intensity of gated human CD4⁺CD25^high, CD4⁺CD25^low and FACS sorted bovine CD4⁺CD25^high, CD4⁺CD25^low T cells after ICS with anti-human/mouse/rat Foxp3 mAb (150DAlexa 647, unfilled histogram) compared to a murine IgG1k isotype control mAb (MOPC-21Alexa 647, filled histogram). (b) Dotplots representing intracellular staining of IL-10 in bovine PBMC gated for live cells after Con A stimulation + Brefeldin A, stained with intracellular biotinylated anti-bovine IL-10 (IgG1) and StreptavidinPE as a second step and the relevant isotype control biotinylated anti-chicken CD107 (IgG1). (c) Dotplots representing intracellular staining of IL-10 in bovine PBMC gated for live cells after Con A stimulation + Brefeldin A, surface stained with anti-bovine CD21, NKp46, CD3, WC1, CD14 and goat anti-mouseFITC as a second step in combination with intracellular biotinylated anti-bovine IL-10 and StreptavidinPE as a second step.

Figure 5.

Ex vivo regulatory functions of 9 bovine cell populations in a co-culture assay. As a read out system (grey bars) proliferation of 35 000 CD4⁺CD25^- Tresp cells (population R1 in Fig. 1, indicated as R along the Y-axis), isolated from peripheral blood (cow A as a representative example) combined with 70 000 irradiated CD4^-CD25^- APC (population R5 in Fig. 1, indicated as A along the Y-axis) is shown. In white bars control results of co-culturing 30 000 (indicated as Reg 1 along the Y-axis), 60 000 (indicated as Reg 2 along the Y-axis) or 90 000 (indicated as Reg 3 along the Y-axis) potential regulatory cells (a) CD4⁺CD25^high, (b) CD4⁺CD25^low, (c) CD8⁺, (d) NKp46⁺, (e) WC1⁺, (f) W15B (WC1⁺ T cell line), (g) WC1.1⁺, (h) WC1.2⁺ and (i) CD14⁺ monocytes combined with 70 000 irradiated CD4^-CD25^- APC are shown. In black bars results are shown of co-cultures with the 9 potential regulatory cells (30 000–90 000 cells indicated as Reg 1-3 along the Y-axis) in combination with CD4⁺CD25^- Tresp (R) and irradiated CD4^-CD25^- APC (A). The data are representative experiments and are presented as dose dependent (30 000, 60 000 and 90 000) potential Treg proliferation or ratio dependent (potential Treg: Tresp = 0.9:1, 1.7:1 and 2.6:1, R1-R3:R) as the mean of proliferation on day 5 (+ 1 SD) in cpm. The stimulus used was plate-bound anti-bovine CD3 at a sub maximal concentration of 3 μg/mL. (p values reflect comparison of R1 + R (2.6:1) + A versus R + A, ** p ≤ 0.01).