Impact of HpSC-produced C3 on induction of MDSCs in vivo. Three hundred BALB/c islets were mixed with 2.5 × 10⁵ HpSCs isolated from C3^−/− or WT (B6) mice and transplanted into diabetic B6 recipients. Islet allografts alone served as controls (None). Islet graft survival was monitored by blood glucose levels as described in “Materials and methods.” For cellular analyses, the animals were sacrificed on POD 7. (A) HpSCs deficient in C3 lost their ability to protect islet allografts. (B) C3 deficiency in cotransplanted HpSC reversed attenuation of CD8⁺ T-cell infiltration in the islet allografts. The infiltrating cells were isolated from the islet allografts, and analyzed for CD4⁺ and CD8⁺ T cells by flow cytometry (left panels, the number is the percentage of positive cells in the total cell population) and immunohistochemistry. The positive cells were counted under a microscope. A total of 10 hpf’s were randomly selected in each graft and expressed as mean cells per hpf ± 1 SD (middle and right panels). (C) Fewer Treg cells are induced by cotransplanted C3^−/− HpSCs. T cells isolated from islet grafts, D-LNs, and spleen were stained for CD4, CD25, and Foxp3, and analyzed by flow cytometry by gating on CD4⁺ T cells. The number is the percentage of positive cells in the CD4⁺ cell population. (D) Impact of HpSCs produced C3 on key molecule expression on myeloid cells. CD11b⁺ cells purified from islet allografts were analyzed for expression of the indicated surface markers by flow cytometry and expressed as histograms. The filled areas are isotype controls. The number is the percentage of positive cells in the CD11b⁺ cell population. (E) Myeloid cells isolated from islet/C3^−/− HpSC grafts demonstrate high allostimulatory activity. CFSE-labeled B6 spleen T cells were cultured for 3 days with the irradiated CD11b⁺ cells isolated from the islet grafts in each group (at a ratio of 10:1). T-cell responses were determined by CFSE dilution and intracellular expression of IFN-γ in CD4 and CD8 T cells. The number is the percentage of positive cells in the T-cell subset. (Right panel) Generation of CTLs. B6 spleen T cells cultured, at a ratio of 10:1, for 5 days, with CD11b⁺ cells isolated from islet grafts that had been pulsed with BALB/c spleen cell lysates were used as effectors. P815 (H2^d), EL4 (H2^b), or R1.1 (H2^k) lymphoma cells labeled with the fluorescent dye BCECF were used as donor-specific, syngeneic, and third-party targets, respectively, and expressed as a percentage of H2^d-specific cytotoxicity ± 1 SD (n = 3). No cytotoxicity was generated against syngeneic and third-party targets (data not shown). (F) Impact of HpSC-produced C3 on differentiation of suppressive myeloid cells. CD11b⁺ cells isolated from islet grafts from each group were added into CFSE-labeled BALB/c spleen T-cell (2 × 10⁵) culture (at a ratio of 1:10) in which T-cell responses were elicited by addition of anti-CD3 mAb (2 μg/mL) for 3 days. No addition of CD11b⁺ cells served as control. CD4⁺ or CD8⁺ T-cell responses were determined by CFSE dilution (proliferation) and intracellular staining for IFN-γ. Expression of annexin V was analyzed on T-cell (CD3⁺) populations, and T-cell absolute numbers were calculated based on flow analysis data (right panel). The number is the percentage of positive cells in the T-cell subset or whole T cells. The data are representative of three separate experiments.