Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response

Abstract

The alloreactive immune response is a series of events initiated by the interaction of T cells with allogeneic dendritic cells (DCs), involving alloantigen recognition and costimulatory signals. In this study, we investigated the role of OX40 in alloreactivity in vitro. We first demonstrated that anti-OX40 ligand (anti-OX40L) monoclonal antibody (mAb) could markedly suppress the mixed leucocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC). To further define the contribution of the OX40/OX40L system to the MLR, we set up a co-culture system of CD4+ T cells and allogeneic monocyte-derived dendritic cells (DCs). After 2 days, OX40 expression was induced on CD4+ T cells and this induction was strongly inhibited by the addition of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein, suggesting that the expression of OX40 during alloreaction is dependent on CD28 signalling. Next we examined the effects of anti-OX40L mAb, CTLA-4-Fc fusion protein and anti-human leucocyte antigen (HLA)-DR mAb on the proliferative response of CD4+ T cells to allogeneic DCs. The proliferation of T cells was almost completely suppressed by anti-OX40L mAb, which was comparable with that of CTLA-4-Fc. Measurement of interleukin-2 (IL-2) production in the culture supernatants showed that suppression of a proliferative response was at least in part ascribed to reduced IL-2 production. Furthermore, purified OX40L- allogeneic DCs could induce considerable proliferation of CD4+ T cells, which was suppressed by anti-OX40L mAb. These results suggest that the OX40/OX40L system is crucial for induction of the allogeneic T-cell response and the OX40/OX40L system is subsequent to and dependent on CD28 signalling, but is crucial for the end outcome of the human alloreactive T-cell response.

Figure 1

Effects of anti-OX40 ligand (OX40L), anti-human leucocyte antigen (HLA)-DR, anti-CD18 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein on the two-way mixed leucocyte reaction (MLR). Peripheral blood mononuclear cells (PBMC) from two different donors were mixed and co-cultured in a 96-well culture plate for 7 days in the presence of the indicated reagents. Cells were pulsed with [³H]thymidine ([³H]TdR) (0·5 µCi/well) for the last 7 hr of culture and the incorporated radioactivity was measured in a liquid scintillation counter. Results are expressed as mean counts ± standard deviation (SD) of triplicate samples. The asterisk indicates a P-value of < 0·05 compared with the suppressed reactivity.

Figure 2

OX40 ligand (OX40L) expression on monocyte-derived dendritic cells (DC), in the presence or absence of stimulation with monocyte-conditioned medium (MCM). Purified monocytes were cultured in RPMI-1640 supplemented with granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and autologous plasma for 7 days to generate immature DCs. Then, cells were cultured for a further 2 days, with or without MCM. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human leucocyte antigen (HLA)-DR, biotinylated anti-OX40L (ik-1), and phycoerythrin (PE)-labelled streptavidin, and subsequent flow cytometric analysis, with a FACScan, was performed. The percentage of OX40L⁺ HLA-DR⁺ cells is indicated in the upper right corner of each dot-plot.

Figure 3

OX40 expression on CD4⁺ T cells stimulated by allogeneic dendritic cells (DCs) and the inhibitory effect of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein against the expression of OX40. CD4⁺ T cells and allogeneic irradiated mature DCs (mDCs) were co-cultured in a 96-well culture plate at a ratio of 200 : 1. (a) Control monoclonal antibody (mAb) (MOPC21) or (b) CTLA-4–Fc fusion protein was added to the culture at a final concentration of 100 or 10 µg/ml, respectively. On days 0, 2, 4, 6 and 8, cells were collected, stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, biotinylated anti-OX40L (ik-1) and phycoerythrin (PE)-labelled streptavidin, and subjected to flow cytometric analysis in a FACScan. The percentage of OX40⁺ CD4⁺ T cells is indicated in the upper right corner of each dot-plot.

Figure 4

Effects of anti-OX40 ligand (OX40L) monoclonal antibody (mAb) on CD4⁺ T-cell proliferation stimulated by allogeneic dendritic cells (DCs). (a) CD4⁺ T cells and allogeneic irradiated mature DCs (mDCs) were mixed and co-cultured in a 96-well culture plate for 5 days in the presence of the indicated reagents. Cells were pulsed with [³H]thymidine ([³H]TdR) (0·5 µCi/well) for the last 7 hr of culture and the incorporated radioactivity was measured in a liquid scintillation counter. Results are expressed as mean counts ± standard deviation (SD) of triplicate samples. The asterisk indicates a P-value of < 0·05 compared with the suppressed reactivity. (b) Effects of anti-OX40L mAb on the viability of DCs. DCs were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS) in the presence of 100 µg/ml control mouse IgG2a (UPC-10; open circle) or anti-OX40L mAb (ik-5; closed square) and the viable cell number was counted by using Trypan-Blue dye exclusion.

Figure 5

Effects of anti-OX40 ligand (OX40L) monoclonal antibody (mAb) on the proliferation of CD4⁺ T cells stimulated by purified OX40L⁻ mature DCs (mDCs). (a) OX40L⁻ mDCs were purified in a cell sorter and irradiated. CD4⁺ T cells and OX40L⁻ mDCs were mixed at a ratio of 1000 : 1 and co-cultured in a 96-well culture plate for 7 days in the presence of the indicated reagents. Cells were pulsed with [³H]thymidine ([³H]TdR) (0·5 µCi/well) for the last 7 hr of culture and the incorporated radioactivity was measured in a liquid scintillation counter. Results are expressed as mean counts ± standard deviation (SD) of triplicate samples. The asterisk indicates a P-value of < 0·05 for comparison with the suppressed reactivity. (b) CD4⁺ T cells and allogeneic OX40L⁻ mDCs were mixed at a ratio of 10 : 1 and co-cultured in a 96-well culture plate for 7 days. Cells were collected on days 3, 5 and 7, and the expression of OX40L was examined on mDCs.