B7-1/2, but not PD-L1/2 molecules, are required on IL-10-treated tolerogenic DC and DC-derived exosomes for in vivo function

Abstract

Costimulatory molecules, such as B7-1/2 and PD-L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7-1/2 and PD-L1/2 to the function of IL-10-treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL-10 treatment of DC significantly downregulated surface expression of MHC II, B7-1, B7-2, and decreased levels of MHC I and PD-L2. IL-10 treatment of DC resulted in a modified costimulatory profile of DC-secreted exosomes with a reduction in B7-1, PD-L1 and PD-L2. We further demonstrate that absence of B7-1 or B7-2 on donor DC results in a loss of ability of IL-10-treated DC and their exosomes to suppress the delayed-type hypersensitivity response, whereas IL-10-treated DC deficient in PD-L1/2 as well as their secreted exosomes retained the ability to suppress delayed-type hypersensitivity responses. We conclude that B7-1 and B7-2, but not PD-L1 and PD-L2, on IL-10-treated DC and DC-derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.

Figure 1

Characterization of rIL10-treated tolerogenic DC. (A) DC were stained with PE-conjugated mAb specific for IA/IE, H2k^b, PD-L1, PD-L2, B7-1, B7-2, and CD11c and analyzed by FACS. Percent of positively stained cells was compared to isotype controls. Results are shown as mean±SD of four independent experiments. * p≤0.05, IL-10-treated DC compared to non-treated controls. (B) 10 μg of DC lysate from rIL-10-treated and control groups were run on SDS-PAGE gels under non-reducing conditions and immunoblotted for B7-1, B7-2 or beta-actin as control. (C) IL-10 treated and non-treated DC were stimulated with 100 ng/mL LPS overnight on day 7 of culture. IL-6, IL-23, IL-12 p70 and IL-12/IL23 total p40 levels in DC culture supernatant were determined on day 8 using ELISA. p≤0.05, DC.Non+LPS group compared to the other three groups. **p≤0.05, DC.IL-10+LPS group compared to the other three groups.

Figure 2

Characterization of exosomes from tolerogenic DC. Exosomes were purified from supernatant of control or rIL-10-treated DC. (A) Anti-CD81 mAb was preabsorbed onto latex beads which were subsequently incubated with 10 μg of DC-derived exosomes. Samples were stained with PE-conjugated mAb specific for IA/IE, H2k^b, PD-L1, PD-L2, B7-1, and B7-2, or isotype control (open black line). Results shown are representative of three independent experiments. Percentage of beads containing positive signal, as well as MFI (in parentheses) are displayed. (B) 5 μg of exosomes were run on SDS-PAGE gels under non-reducing conditions and immunoblotted for B7-1, B7-2 or CD9 as a loading control.

Figure 3

B7-1 and B7-2 are required on DC and DC-derived exosomes for activity whereas PD-L1 and PD-L2 are dispensable. Wild-type C57BL/6 or knockout strain-derived DC were treated with rIL-10 or left untreated, and exosomes were isolated from the enriched media. DC or exosomes were injected into the right hind footpad of KLH-immunized wild-type mice simultaneously with KLH challenge to both hind footpads. Dark bars = swelling post challenge in treated (right) paws, white bars = swelling in contralateral (left) untreated paws. (A) Loss of B7-1 or B7-2 on DC or DC-derived exosomes abrogates ability to suppress inflammation in the DTH model. **p≤0.001 for wild-type IL-10-treated DC and their secreted exosomes compared to all other groups. *p≤0.05, exosomes from IL-10-treated B7-1−/−, B7-2−/−, and double knockout DC conditions as compared to the wild type and their respective knockout untreated controls. B7-2−/− IL-10 treated DC are also significant at p≤0.05 compared to the B7-2−/− non-treated DC as well as the non-treated wild type DC. (B) PD-L1 and PD-L2 are not required on DC and DC-derived exosomes for therapeutic effect in the DTH model. *p≤0.05 for the treated paw of the IL-10-treated wild-type and PD-L DKO samples compared to controls. There are no statistical differences between the wild-type and PD-L DKO IL-10 treated DC and exosome groups.