Equine CD4+ CD25high T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Abstract

Summary Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4+ Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4+ CD25+ T cells, mainly in the CD4+ CD25high subpopulation. Proliferation of CD4+ CD25− sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co‐culture with CD4+ CD25high sorted cells in a dose‐dependent manner. The mechanism of suppression by the CD4+ CD25high cell population is mediated by close contact as well as interleukin (IL)‐10 and transforming growth factor‐β1 (TGF‐β1) and probably other factors. In addition, we studied the in vitro induction of CD4+ Treg and their characteristics compared to those of freshly isolated CD4+ Treg cells. Upon stimulation with a combination of concanavalin A, TGF‐β1 and IL‐2, CD4+ CD25+ T cells which express FoxP3 and have suppressive capability were induced from CD4+ CD25− cells. The induced CD4+ CD25high express higher levels of IL‐10 and TGF‐β1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4+ CD25high subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4+ CD25+ cells in horses and offers insights into ex vivo manipulation of Treg cells.

In vitro induction of functional allergen‐specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity

“…Given the impaired suppressive function of CD4 + CD25 high cells in IBH‐affected horses upon interaction with Culicoides ‐pulsed feeder cells, we examined the possibility to expand autologous functional Culicoides ‐specific CD4 + CD25 high cells through a combination of factors consisting of either rIL‐2 and rTGF‐β1, or retinoic acid and rapamycin. The single use of either has been shown to impair the expansion or have a detrimental effect on the function of Treg . Our experiments showed that treatment with either rIL‐2/rTGF‐β1, or RetA/Rapa was able to expand Tregs as shown before and largely restored the suppressive function in IBH‐N‐sup horses (Fig.…”

“…Given the impaired suppression of Culicoides ‐induced proliferation by CD4 + CD25 high cells in IBH‐N‐sup horses, we have investigated the possibility of restoring this suppression by expanding autologous functional Treg cells. We have shown previously that a combination of ConA, rIL‐2 and rTGFβ‐1 was able to expand CD4 + CD25 high cells with regulatory function from healthy horses in vitro . A possible role of retinoic acid and rapamycin on the expansion of regulatory CD4 + CD25 high cells has been recently shown in other species .…”

“…S1b, freshly isolated CD4 + T cells from IBH‐affected Icelandic horses ( n = 5) were sorted into CD25 − and CD25 high as mentioned above (magnetic cell separation and sorting). These two populations were cultured separately with Culicoides ‐ pulsed irradiated autologous PBMC in the absence or the presence of different factors as follow: (1) recombinant human IL‐2 (rIL‐2, 100 U/mL; Peprotech, London, UK) combined with recombinant human TGF‐β1 (rTGF‐β1, 2 ng/mL; Peprotech) ; (2) retinoic acid (RetA, 10 μ m , Sigma‐Aldrich) combined with rapamycin (Rapa, 10 n m , Sigma‐Aldrich). Four days later, the cultured CD4 + CD25 − or CD4 + CD25 high cells were stained for CD25 and re‐sorted.…”

“…They contribute to the control of allergen‐specific immune responses through suppression of effector T cells or dendritic cells that support the generation of effector T cells. In horses, we have recently reported that FoxP3 is constitutively expressed by a proportion of CD4 + CD25 high T cells . There were no differences between IBH‐affected and healthy horses in the proportion of circulating regulatory T cells.…”

Activation-Induced FoxP3 Expression Regulates Cytokine Production in Conventional T Cells Stimulated with Autologous Dendritic Cells

“…However, the possibility that FOXP3 ϩ effector T cells help to fine-tune the developing immune response is an attractive hypothesis that cannot be discounted, particularly because transduction of FOXP3 in naive human T cells imparts a regulatory phenotype (31,54). This is supported by the recent finding that stimulation of conventional equine T cells induced a population of CD4 ϩ CD25 ϩ T cells that were functionally suppressive and did not proliferate (20). However, these cells were activated in the presence of TGF-␤, so they may represent true Tregs rather than activated conventional T cells with transient Treg-like characteristics (40).…”