Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Clin Exp Allergy. 2013 Aug;43(8):889-901. doi: 10.1111/cea.12131.
Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses.
We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it.
CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or tetanus toxoid as control antigen, in the presence of CD4(+) CD25(high) cells. Furthermore, Culicoides-specific CD4(+) CD25(high) regulatory cells were expanded or induced from CD4(+) CD25(-) cells in vitro in the presence of a combination of rIL-2 and rTGF-β1 (rIL-2/rTGF-β1) or of retinoic acid and rapamycin (RetA/Rapa). Proliferation was determined by [(3) H] thymidine incorporation and cytokine production measured by flow cytometry.
The ability of Culicoides- but not tetanus-stimulated CD4(+) CD25(high) cells to suppress proliferation of CD4(+) CD25(-) cells was significantly lower in IBH-affected horses (28%) than in healthy controls (86%). The decreased suppression in IBH-affected horses was associated with a significantly higher proportion of IL-4(+) cells and a lower percentage of FoxP3(+) IL-10(+) compared to controls. Addition of rIL-2/rTGF-β1 or of RetA/Rapa to Culicoides-stimulated CD4(+) CD25(high) cells from IBH-affected horses significantly increased the proportion of FoxP3(+) IL-10(+) cells. We also found that RetA/Rapa induced a more significant decrease in the frequency of IL-4(+) cells than rIL-2/rTGF-β1. Moreover, the suppressive activity of Culicoides-stimulated CD4(+) CD25(high) cells was significantly restored by both rIL-2/rTGF-β1and RetA/Rapa, albeit in an antigen-unspecific manner. In contrast, in vitro induced Culicoides-specific CD4(+) CD25(high) cells suppressed proliferation of CD4(+) CD25(-) cells in an antigen-specific manner.
The in vitro induction of functional allergen-specific Treg cells in IBH-affected horses suggests a potential therapeutic use of these cells in allergy.
昆虫叮咬过敏（IBH）是一种反复发作的马过敏性皮炎，类似于人类特应性皮炎，由库蠓属昆虫叮咬引起。先前的研究表明，IBH 受影响的马对库蠓过敏原的 T 细胞耐受性失调。
我们研究了 IBH 受影响的马中 CD4+CD25（高）细胞的抑制功能是否受损，以及可能恢复它的方法。
从外周血单核细胞（PBMC）中分离出 CD4+CD25（-）细胞，在 CD4+CD25（高）细胞存在的情况下，用照射的自体 PBMC 脉冲 Culicoides 或破伤风类毒素作为对照抗原进行刺激。此外，在 rIL-2 和 rTGF-β1（rIL-2/rTGF-β1）或维甲酸和雷帕霉素（RetA/Rapa）的组合存在下，从 CD4+CD25（-）细胞体外扩增或诱导 Culicoides 特异性 CD4+CD25（高）调节细胞。通过[3H]胸苷掺入测定增殖，通过流式细胞术测量细胞因子产生。
与健康对照组（86%）相比，IBH 受影响的马中，Culicoides 而非破伤风刺激的 CD4+CD25（高）细胞抑制 CD4+CD25（-）细胞增殖的能力显著降低（28%）。在 IBH 受影响的马中，与对照相比，IL-4+细胞的比例显著升高，FoxP3+IL-10+细胞的比例降低。向 Culicoides 刺激的来自 IBH 受影响的马的 CD4+CD25（高）细胞中添加 rIL-2/rTGF-β1 或 RetA/Rapa 显著增加了 FoxP3+IL-10+细胞的比例。我们还发现，RetA/Rapa 诱导的 IL-4+细胞频率下降比 rIL-2/rTGF-β1 更显著。此外，尽管以非抗原特异性方式，rIL-2/rTGF-β1 和 RetA/Rapa 均显著恢复了 Culicoides 刺激的 CD4+CD25（高）细胞的抑制活性。相比之下，体外诱导的 Culicoides 特异性 CD4+CD25（高）细胞以抗原特异性方式抑制 CD4+CD25（-）细胞的增殖。
在 IBH 受影响的马中诱导功能性过敏原特异性 Treg 细胞的体外诱导提示这些细胞在过敏中的潜在治疗用途。
