FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be cultured from colonic mucosa of patients with Crohn's disease

Abstract

Summary CD4(+)CD25(+) regulatory T cells (T(regs)) are involved in the maintenance of peripheral tolerance and ensure a balanced immune response competent of fighting pathogens and at the same time recognizing commensals as harmless. This feature is lost in Crohn's disease (CD). The forkhead/winged helix transcription factor FoxP3 is a master gene for T(reg) function and defects in the FoxP3 gene lead to a clinical picture similar to inflammatory bowel disease (IBD). Murine colitis can be cured by adoptive transfer of T(regs) and ex vivo-generated gut-specific T(regs) represent an attractive option for therapy in CD. Thus, defective T(regs) could contribute to the development of CD. We cultured biopsies of colonic mucosa in the presence of high concentrations of interleukin (IL)-2 and IL-4 to overcome the anergic nature of naturally occurring CD4(+)CD25(+) T(regs) in the mucosa. We investigated the expression of FoxP3 and regulatory potential of gut-derived CD4(+)CD25(+) T cells cultured from patients with CD and healthy individuals. The FoxP3 expression was analysed by reverse transcriptase polymerase chain reaction (RT-PCR), and the suppressive effect of FoxP3(+)CD4(+)CD25(+) T cells on proliferation and cytokine production of autologous CD4(+) T cells was assessed by flow cytometry. Cultured gut-derived T cells with CD4(+)CD25(+) phenotype expressed FoxP3 and were able as the freshly isolated T(regs) from peripheral blood to suppress proliferation and cytokine production of autologous CD4(+) T cells. Thus, we demonstrate that FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be propagated in vitro from inflamed mucosa of CD patients, which may be of interest in adoptive immunotherapy.

Figures

Fig. 1

Upper panel, the freshly isolated PKH67-labelled autologous CD4+CD25− peripheral blood lymphocytes (PBLs) proliferate upon stimulation with platebound OKT-3 and soluble anti-CD28, which is illustrated by the gradual movement of the PKH67bright mother population to a PKH67dim population of daughter cells (sharing the membrane PKH-labelling upon cell division). After 4 days 75% of the PBLs have undergone mitosis. The proliferation is inhibited by co-culture (ratio 1 : 1) with freshly isolated CD4+CD25+ PBLs (not labelled, appearing as the indicated PKH-negative population) and cultured gut-derived CD4+CD25+ T cells (PKH-negative), but not with the cultured CD4+CD25− (PKH-negative) counterpart, indicating that suppressive properties are confined to the CD4+CD25+ T cell population. As indicated, 2% of the PBLs have initially escaped PKH-labelling. The dot-plots are gated on lymphocytes on forward side-scatter as surrogate marker for living cells. This experiment is representative for four Crohn's disease (CD) patients.

Fig. 2

CD4+CD25− peripheral blood lymphocytes (PBLs) from a patient with Crohn's disease (CD) were labelled with PKH67 (a) and proliferated for 4 days upon anti-CD3/CD28 stimulation (b). Proliferation of the responder population was not inhibited by co-cultured autologous gut-derived CD4+CD25− T cells (ratio 1 : 1) (c). Proliferation was inhibited by co-cultured CD4+CD25+ T cells (d). The suppressive function of the CD4+CD25+ T cells was not abrogated by neutralizing antibody against Fas ligand (FasL) (e). However, proliferation of the responder population could be partly restored by exogenous interleukin (IL)-2, indicated by the increase in daughter cells (f). Dot-plots gated on living cells.

Fig. 3

When co-cultured (ratio 1 : 1), gut-derived CD4+CD25+ T cells suppress the interferon (IFN)-γ and tumour necrosis factor (TNF)-α production of CD3/CD28-stimulated CD4+CD25− peripheral blood lymphocytes (PBLs). Within the first 4 days of culture, the suppressive effect is comparable to that of freshly isolated CD4+CD25+ T cells from peripheral blood. The cytokine levels are means of duplicate wells determined by cytometric bead array (CBA, Human TH1/TH2 kit, Becton Dickinson). Error bars indicate standard error of mean.

Fig. 4

Flow cytometric evaluation of the immunomagnetic separation of cultured gut-derived T cells from a patient with Crohn's disease (CD). The CD4+CD25+ T cells expressed higher levels of glucocorticoid-induced tumour necrosis factor receptor (GITR) than the CD4+CD25− subset, but equivalent levels of Fas ligand (FasL). Dot plots of 20 000 events gated on living cells.

Fig. 5

Nuclear transcription factor FoxP3 is expressed preferentially in the CD4+CD25+ T cell subset. FoxP3 expression was evaluated by reverse transcription-polymerase chain receptor (RT-PCR) in T cell subsets separated by MACS beads in cultured gut-derived T cells and peripheral blood from a Crohn's disease (CD) patient. The human 293 embryonic kidney cells (T293) and K562 served as negative controls.