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. 2009 Oct;128(2):218-26.
doi: 10.1111/j.1365-2567.2009.03103.x.

Binding immunoglobulin protein-treated peripheral blood monocyte-derived dendritic cells are refractory to maturation and induce regulatory T-cell development

Valerie M Corrigall  1 , Olivier Vittecoq, Gabriel S Panayi
Affiliations

Binding immunoglobulin protein-treated peripheral blood monocyte-derived dendritic cells are refractory to maturation and induce regulatory T-cell development

Valerie M Corrigall et al. Immunology. 2009 Oct.

Abstract

Binding immunoglobulin protein (BiP) has been shown previously to have immunomodulatory functions. Herein we investigated whether BiP could affect the differentiation of monocytes into dendritic cells (DCs) and thence the development of regulatory T cells. Peripheral blood monocyte-derived DCs were matured with lipopolysaccharide in the presence or absence of BiP. DC development and T-cell changes were monitored by flow cytometry and regulatory T-cell function was measured by uptake of tritiated thymidine. More BiP-treated DCs (DC((BiP))s) expressed amounts of intracellular indoleamine 2,3-dioxygenase (IDO) and cell surface leucocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), retained CD14 expression but down-regulated expression of human leucocyte antigen (HLA)-DR and CD86, and produced copious amounts of interleukin (IL)-10, when compared with control DCs. T cells co-cultured with DC((BiP))s developed regulatory function with increased surface expression of CD4(+) CD25(hi) CD27(hi) but with no concomitant increase in forkhead box P3 (Foxp3). These T cells also showed significantly higher levels of intracellular cytotoxic T-lymphocyte antigen (CTLA)-4. The latter could be inhibited by the presence of the IDO inhibitor 1 methyl tryptophan. The addition of neutralizing anti-IL-10 antibody or the specific mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 reversed the inhibition of DC differentiation by BiP. In conclusion, BiP is an immunomodulator able to arrest inflammation through induction of tolerogenic DCs and subsequent generation of T regulatory cells.

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Figures

Figure 1
Figure 1
Binding immunoglobulin protein (BiP) alters expression of indolamine-2,3 dioxygenase (IDO), costimulatory molecules and regulatory molecules by dendritic cells (DCs). Peripheral blood monocytes were cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 in the presence or absence of BiP (20 μg/ml) for 7 days. Lipopolysaccharide (LPS; 500 ng/ml) was added for the final 48 hr to mature dendritic cells (mDCs). (a) Flow cytometry was used to detect intracellular expression of IDO after DC maturation in nine different experiments. (b) The expression of CD14 and CD86 on mDCs or mDC(BiP)s in a representative experiment and (c) the expression of leucocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) by mDCs and mDC(BiP)s in eight different experiments.
Figure 2
Figure 2
Co-culture of T cells with binding immunoglobulin protein (BiP)-treated dendritic cells (DCs) up-regulates regulatory markers. T cells were co-cultured with autologous mature DCs (mDCs) or BiP-treated mDCs (mDC(BiP)s) for 4 days. Data from a representative experiment are shown in (a) a dot plot of CD27+ CD25hi cells, gated for CD4+ cells only, and (b) a histogram representing intracellular CD4+ CTLA-4+ cells (dashed line) compared with isotype control cells (solid line) after co-culture with mDCs (ba) or mDC(BiP)s (bb). (c) T cells were cultured with autologous mDCs or mDC(BiP)s in the absence or presence of the indolamine-2,3 dioxygenase (IDO) inhibitor 1 methyl tryptophan (1MT) (200 μm). After 4 days the number of intracellular cytotoxic (ic) T-lymphocyte antigen (CTLA)-4+ CD4+ cells in each co-culture was measured by intracellular immunofluorescence and flow cytometry. Data from three different experiments are shown as mean ± standard deviation.
Figure 3
Figure 3
Co-culture with binding immunoglobulin protein (BiP)-treated dendritic cells (DCs) produces T cells with regulatory function. (a) A diagram of the protocol for the experiment is shown: after 4 days of pre-incubation with autologous mature DCs (mDCs) or BiP-treated mDCs (mDC(BiP)s), T cells were separated, washed and set up in autologous cultures with irradiated peripheral blood mononuclear cells (PBMC) (3000 rads) and responder T cells (1 : 1 : 1 ratio) (105 cells/well) in the presence or absence of anti-CD3 antibody (1/2000 dilution). (b) Data showing the proliferative response in a representative experiment carried out as described in (a). The data were recorded as counts per minute of tritiated thymidine added to the cultures for the final 18 hr. Cultures were set up in triplicate and data are shown as mean ± standard deviation. Four experiments were carried out.
Figure 4
Figure 4
The effect of interleukin (IL)-10 and mitogen-activated protein kinase (MAPK) inhibitors on dendritic cell (DC) maturation. Allogeneic proliferative responses by peripheral blood mononuclear cells (PBMC) in culture with irradiated early DCs (eDCs) (3000 rads), after only 3 days in culture with granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4 in the absence or presence of binding immunoglobulin protein (BiP) (20 μg/ml) at a ratio of 10 : 1 PBMC : early DCs (105 : 104 cells/well), were determined. The results are shown as allogeneic T-cell proliferation in response to eDCs or BiP-treated eDCs (eDC(BiP)s) (a) with the addition of neutralizing anti-IL-10 (1/100 dilution) antibody or (b) following pretreatment (2 hr) of monocytes with the p38 inhibitor SB203580 (SB) (10 μm) or the extracellular signal regulated kinase 1/2 (ERK1/2) inhibitor PD98059 (PD) (10 μm), prior to the addition of GM-CSF, IL-4 and BiP to the cultures. Proliferation was measured by uptake of tritiated thymidine and recorded as counts per minute (c.p.m.). Enzyme-linked immunosorbent assays (ELISAs) were used to detect IL-10 in (c) the culture supernatants of the experiments shown in (a) and (b), and (d) the culture medium from mature DCs (mDCs) or BiP-treated mDCs (mDC(BiP)s) differentiated for 7 days with GM-CSF and IL-4 with lipopolysaccharide (LPS; 500 ng/ml) added for the final 2 days.
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