CTLA4-Ig immunosuppressive activity at the level of dendritic cell/T cell crosstalk

Abstract

Immunosuppressive cytotoxic T lymphocyte associated antigen-4 immunoglobulin fusion proteins (CTLA4-Ig) block the CD28:CD80/86 costimulatory pathway. On a cellular level, CTLA4-Ig is understood to dampen T cell responses. As a mechanism, CTLA4-Ig has been reported to affect dendritic cell (DC) function via inducing the immunosuppressive indoleamine 2,3 dioxygenase (IDO) pathway and promoting a DC regulatory phenotype. We here probed cellular mechanisms of CTLA4-Ig immunoregulation in an allogeneic setting using C57BL/6 splenic or bone marrow derived DCs (BMDCs) as stimulators of allogeneic Balb/c derived T cells. To address whether CTLA4-Ig immunosuppression affected DCs, we pre-exposed C57BL/6 splenic or BMDCs to CTLA4-Ig and removed unbound CTLA4-Ig before co-culture with allogeneic T cells. CTLA4-Ig disappeared rapidly (within 4 h) from the cell membrane by combined internalization and dissociation. These CTLA4-Ig pre-exposed DCs were fully capable of stimulating allogeneic T cell proliferation, suggesting that CTLA4-Ig does not impair the DC stimulatory capacity. Only the presence of CTLA4-Ig during DC/T cell co-culture resulted in the expected inhibition of proliferation. C57BL/6 splenic or BMDCs exposed to CTLA4-Ig did not display IDO activity. We conclude that CTLA4-Ig immunosuppressive activity does not depend on a DC regulatory phenotype but on its presence during DC/T cell interaction.

Fig. 1

Effect of CTLA4-Ig on costimulatory molecule expression in C57BL/6 splenic and BMDCs. Splenic DCs (left column) were incubated for 24 h with CTLA4-Ig (40 μg/ml). BMDCs (middle, right column) were incubated for 24 h without or with LPS stimulation (100 ng/ml) and were subsequently exposed to CTLA4-Ig (100 μg/ml, 2 h). After washing, DCs were stained for MHC class I and II, CD80 and CD86 and analyzed by flow cytometry. Shaded histograms, isotype control; open histograms, DCs without (dotted line) or with (black line) CTLA4-Ig. MFI, mean fluorescence intensity. One representative experiment of 3 experiments is shown.

Fig. 2

CTLA4-Ig is rapidly removed from the DC surface by internalization and dissociation. (A) C57BL/6 BMDCs were incubated without (shaded histogram) or with 100 μg/ml CTLA4-Ig for 4 h, washed and stained with a phycoerythrin (PE) labeled anti-IgG antibody and analyzed by flow cytometry either immediately after CTLA4-Ig exposure (dotted line) or after a further 16-hour incubation period at 37 °C (left panel, black line, estimating internalization) or at 4 °C (right panel, black line, estimating dissociation). The inserts in the histograms depict the MFI. One representative experiment of 3 experiments is shown. (B) C57BL/6 BMDCs were incubated with 100 μg/ml CTLA4-Ig as in (A), washed and incubated at 37 °C (filled rectangles) or at 4 °C (open circles) for increasing time periods, indicated as hours. Then, cells were stained with anti-human IgG-PE, as above. The level of expression was examined as the MFI of histogram analyses. (C) LPS (100 ng/ml) stimulated C57BL/6 BMDCs (24 h), were incubated with CTLA4-Ig (100 μg/ml) for 2 h and washed. Subsequently, cells were cultured in complete medium without CTLA4-Ig for further 2 and 16 h and the expression levels of CD80 and CD86 were analysed by flow cytometry. Shaded histogram, isotype control; open histograms, DCs without (dotted line) or with (black line) CTLA4-Ig. One representative experiment of 2 experiments is shown. MFI, mean fluorescence intensity.

Fig. 3

Effect of CTLA4-Ig on IFN-γ production in C57BL/6 splenic and BMDCs. C57BL/6 splenic DCs (left panel) were incubated with CTLA4-Ig (40 μg/ml) for 24 h, C57BL/6 BMDCs (right panel) were incubated with or without LPS (100 ng/ml), CTLA4-Ig (50 μg/ml) and/or human IgG1 (50 μg/ml) for 24 h. Cell culture supernatants were examined for IFN-γ release by ELISA. *Below detection limit.

Fig. 4

CTLA4-Ig does not induce IDO activity in C57BL/6 splenic and BMDCs. (A) Upper panel: IDO mRNA expression in splenic DCs was analyzed by RT-PCR. Total RNA of murine Baf3 cells was used as negative control and GAPDH as internal standard; NTC, non template control. Lower panel: IDO protein expression was analyzed in the same DCs as described above. Human DCs stimulated by LPS/IFN-γ as described served as positive and murine CD3 + T cells as negative controls. Internal standard, GAPDH. All samples were run on one gel. One representative of three experiments is shown. (B) Mean concentrations of tryptophan (black bars, left scale) and kynurenine (gray bars, right scale) in cell culture supernatants of the same DCs were determined by HPLC (n = 3). (C) Upper panel: BMDCs were left unstimulated or were exposed to increasing concentrations of CTLA4-Ig (left panel) or to LPS (1000 ng/ml) and increasing concentrations of IFN-γ (right panel, given as U/ml) for 24 h; expression of IDO protein was examined by immunoblotting as in (A); lower panel: The relative increase of IDO protein expression in relation to the internal standard, GAPDH, was calculated as arbitrary units using the ImageJ freeware. One representative of two experiments is shown. (D) The concentrations of tryptophan in unstimulated (black bars) or LPS/IFN-γ stimulated (gray bars) cultures were determined in cell culture supernatants. (E) In parallel to (D), cell culture supernatants were examined for the concentrations of nitrite. *Below detection limit.

Fig. 5

CTLA4-Ig inhibits the allogeneic response stimulated by C57BL/6 splenic DCs or BMDCs. (A) C57BL/6 splenic DCs were co-cultured with allogeneic CFSE labeled Balb/c T cells at a 1:10 DC:T cell ratio in the absence or presence of CTLA4-Ig (100 μg/ml) for 6 days. T cells were identified as CD3⁺ cells and were analyzed for CD25 expression and CFSE dilution (i.e. proliferation). Inserted numbers indicate the fraction of CD25⁺CFSE⁻ T cells. (B) Summary (mean ± SEM) of three independently performed experiments (as in A) in which splenic DCs (left) or BMDCs (right) were used as stimulator cells. ***P < 0.001.

Fig. 6

CTLA4-Ig does not confer a DC regulatory phenotype but acts at the level of DC/T cell interaction. (A) C57BL/6 splenic DCs were exposed to CTLA4-Ig (100 μg/ml, 24 h) or maintained in medium only and washed prior to co-culture with allogeneic Balb/c T cells. T cells were analyzed after 6 days of DC/T cell co-culture in the absence of CTLA4-Ig. One representative of three experiments is shown. (B) BMDCs were exposed to increasing concentrations of CTLA4-Ig for 24 h. Thereafter, DCs were washed and co-cultured with allogeneic Balb/c T cells in the presence (black squares, solid line) or absence (open circles, dashed line) of CTLA4-Ig for 6 days. T cells were analyzed as in (A). One representative of three experiments is shown. (C) Purified Balb/c CD3⁺ T cells were stimulated with plate bound anti-CD3/anti-CD28 for 48 h in the absence or presence of increasing concentrations of CTLA4-Ig. T-cell proliferation was assessed by CFSE dilution. One representative of two experiments is shown.