Fig 1.
LiClPAM3 dendritic cells (DCs) display characteristic features of a tolerogenic phenotype with high IL-10:IL-12 ratio.
Bone marrow-derived C57Bl/6N DCs were cultured for 9 days with GM-CSF. Cells were gated for CD11c+ (A) and subsequently analyzed by flow cytometry for percentage of CD40, CD80, CD86, and MHCII cells (black lines, grey area depicts unstained cells). The percentage of CD40+ or CD86+ cells was lower in LiClPAM3 DC compared to LPS DC (* p<0.05) (B). Supernatants obtained after 9 days of DC culture were analyzed for cytokines (** p<0.01) (C). LiClPAM3 DCs have a higher IL-10:IL-12 ratio compared to LPS and control DC (** p<0.01) (D). Results are of 6 independent experiments.
Fig 2.
LiClPAM3 dendritic cells (DCs) are potent T cell stimulators and induce Th1 and Th17 differentiation in vitro.
The T cell stimulatory capacity of (C57Bl/6N) LiClPAM3 DCs was tested in mixed lymphocyte reaction. (Balb/c) T cell proliferation was analyzed by dilution of CFSE signal (A). LiClPAM3 DC stimulation resulted in higher T cell proliferation at days 3, 4, 5, and 6 compared to control DC stimulation (*p<0.05) (B). T cell differentiation was analyzed by measuring cytokine in co-culture supernatants in ELISA. Interferon-γ (C), but not IL-17 (D) production, by LiClPAM3 DC stimulated T cells was higher compared to LPS DC stimulated T cells at day 4 (* p<0.05). IL-4, IL-6 and IL-23 levels did not differ between groups (E). Results are of 3 independent experiments.
Fig 3.
LiClPAM3 DC pre-treatment results in accelerated allograft rejection in a fully MHC mismatched heterotopic heart transplant model.
Balb/c recipient mice were intravenously infused with 106 C57Bl/6N donor-derived DC or syngeneic DC seven days prior to transplantation. Graft survival was daily assessed by transabdominal palpation of the donor heart. LiClPAM3 DC pre-treatment (n = 9) resulted in accelerated allograft rejection compared to control DC pretreated mice (n = 10) (**p<0.01).
Fig 4.
LiClPAM3 DC produce high levels of CXCL-1 in culture, which sustains in co-culture with MHCII-depleted splenocytes.
CXCL-1 was measured in (C57Bl/6N) DC culture supernatant after 9 days of culture (A). LiClPAM3 DC produce large amounts of CXCL-1 during culture, significantly higher as compared to LPS DC (**p<0.01). In supernatant of LiClPAM3 DC stimulated (Balb/c) T cells (B) higher levels of CXCL-1 were found as compared to LPS DC stimulated conditions (** p<0.01). Results are of 5 independent experiments.
Fig 5.
LiClPAM3 DC derived CXCL-1 is not involved in T cell activation or proliferation.
Balb/c MHCII-depleted splenocytes were co-cultured for 4–6 days with C57Bl/6N LiClPAM3 DC in the presence of a CXCL-1 neutralizing antibody, or an isotype control. Neutralization of DC derived CXCL-1 did not affect T cell proliferation (A) or activation (B) (n = 5). Selective chemical CXCR2 blockade by SB225002 did not result in reduced T cell proliferation (C) or activation (D) caused by CXCR2 blockade, compared to vehicle only (n = 3).