ProGP-1 enhances CD8⁺ T cell cytotoxicity following allogeneic SCT. (A) Irradiated B6D2F1 mice received whole spleen from donors mobilized with G-CSF or ProGP-1. In vivo cytotoxicity was determined as described in Methods (syngeneic, n = 3 per group; allogeneic, n = 5 per group). *P = 0.008 G-CSF allogeneic vs. ProGP-1 allogeneic. (B) At day 13 after SCT, whole spleen was utilized as the effector population in ⁵¹Cr-release CTL assays; host-type target, P815; donor-type target, EL4. **P < 0.05 vs. other groups. One of 2 identical experiments shown. (C) Irradiated B6D2F1 mice received whole spleen, CD4⁺- or CD8β⁺-depleted spleen, or CD4⁺- and CD8β⁺-depleted spleen from WT donors (n = 9 per group). Additional cohorts received whole spleen from Jα18^–/– donors or CD4⁺-depleted spleen from WT donors supplemented with purified CD4⁺ T cells from WT or Jα18^–/– donors (n = 5 per group). All donors were mobilized with ProGP-1. In vivo cytotoxicity was determined at day 12. **P < 0.05 vs. WT whole spleen. Combined data from 2 experiments shown. (D) CD8⁺ T cells were sort-purified at day 13 from recipients of whole WT spleen, CD4⁺-depleted spleen, or whole Jα18^–/– spleen and utilized as effectors in ⁵¹Cr-release CTL assays. **P < 0.05 WT nondepleted anti-host vs. WT CD4⁺-depleted anti-host or Jα18^–/– anti-host. (E) Irradiated B6D2F1 mice received grafts from WT or Jα18^–/– donors mobilized with G-CSF or ProGP-1; in vivo cytotoxicity index was determined at day 12 (n = 6 per group). ^#P = 0.002 WT ProGP-1 vs. Jα18^–/– ProGP-1. (F) CD8⁺ T cells were sort-purified at day 13 and utilized as effectors in ⁵¹Cr-release CTL assays; P815 target population. **P < 0.05 WT ProGP-1 vs. all other groups.

ProGP-1–activated NKT cells enhance host DC activation and subsequent CD8⁺-dependent GVL effects. (A) Irradiated B6D2F1 mice received whole spleen grafts from WT or Jα18^–/– donors mobilized with control diluent or ProGP-1. Total splenic DC numbers and costimulatory molecule expression were determined 24 hours after SCT. Purified DCs were stimulated as described in Methods and supernatant cytokine production determined by ELISA. Data presented as mean ± SEM. Change in MFI calculated as follows for individual samples: MFI-conjugated antibody of interest — MFI-conjugated isotype control. Data for Class II expression presented as MFI only. *P ≤ 0.05 vs. other groups. Combined data from 2 experiments shown. (B) Irradiated B6D2F1 mice received whole spleen (n = 20 per group) or T cell–depleted (TCD) spleen (n = 10 per group) from WT or Jα18^–/– donors mobilized with ProGP-1. At the time of transplant, mice also received a leukemic challenge of 5 × 10⁴ P815 cells. Leukemia death analyzed by Kaplan-Meier analysis. ^#P < 0.0001 vs. WT nondepleted grafts. Combined data from 2 experiments shown. (C) Irradiated B6D2F1 mice received whole spleen from WT or Jα18^–/– donors or CD8β-depleted spleen from WT donors. All donors were mobilized with G-CSF. At days 1 and 4 following transplantation, recipients also received either α-GalCer (C20:2) or vehicle as indicated. In vivo cytotoxicity index determined at day 12 (n = 10 per group except Jα18^–/– + C20:2, n = 5). Combined data from 2 identical experiments shown. **P < 0.01 vs. other groups.

Donor mobilization with Peg–G-CSF expands type 1 NKT cells, enhances in vitro responses to α-GalCer, and increases GVL effects. (A) C57BL/6 mice received control, G-CSF 2 μg/animal/day from days –6 to –1, or Peg–G-CSF 12 μg/animal on day –6 only (n = 8 per group). Whole spleen was then cultured with α-GalCer (C20:2) and supernatant IFN-γ determined by cytokine bead array after 24 hours. *P < 0.05 vs. control. Combined data from 2 experiments shown. (B) Irradiated B6D2F1 mice received whole spleen from WT or Jα18^–/– donors mobilized with G-CSF 2 μg/animal/day from days –6 to –1 or Peg–G-CSF 12 μg/animal on day –6 only. In vivo cytotoxicity index determined at day 12 (WT G-CSF, n = 14; WT Peg–G-CSF, n = 10; other groups, n = 5). **P < 0.01 vs. other groups. (C) Irradiated B6D2F1 mice received whole spleen from C57BL/6 donors mobilized with G-CSF 2 μg/animal/day from days –6 to –1, G-CSF 10 μg/animal/day from days –6 to –1, or Peg–G-CSF 12 μg/animal on day –6 only. In vivo cytotoxicity index determined at day 12 (n = 6 per group). **P < 0.01 vs. other groups. (D) Irradiated B6D2F1 mice received whole spleen from B6D2F1 or C57BL/6 donors mobilized with G-CSF 2 μg/animal/day from days –6 to –1 or Peg–G-CSF 12 μg/animal on day –6 only. At the time of transplant, mice also received a leukemic challenge of 5 × 10⁴ luciferase-transfected P815 cells. Leukemia-free survival by Kaplan-Meier analysis (syngeneic control, n = 8; other groups, n = 18). **P < 0.01 vs. Peg–G-CSF. Representative Xenogen images at day 20 shown.

ProGP-1 expands type 1 NKT cells and enhances in vitro responses to α-GalCer. (A) C57BL/6 mice received control diluent, G-CSF, or ProGP-1. The relative proportion, absolute number, and mean fluorescence intensity (MFI) of CD69 staining of type 1 NKT cells (defined as α-GalCer–loaded CD1d tetramer⁺/αβTCR⁺) were determined by FACS analysis (control, n = 3; G-CSF, n = 6; ProGP-1, n = 6). *P ≤ 0.05 vs. other groups. Combined data from 2 experiments shown. DN, double negative. (B) Whole spleen (5 × 10⁶ cells) was cultured with α-GalCer (C20:2) and supernatant IFN-γ determined by ELISA after 24 hours. **P < 0.05 vs. other groups. Combined data from 2 experiments shown. (C) NKT cells were sorted from control or ProGP-1–mobilized splenocytes as the CD11b^–, 7-aminoactinomycin D negative, TCR⁺, NK1.1⁺ population (circled). Sorted NKT cells (2.5 × 10⁴ cells) were cultured with purified DCs (2.5 × 10⁴ cells) and α-GalCer (C20:2); supernatant IFN-γ was determined by ELISA after 24 hours. **P < 0.05 vs. control. Combined data from 2 experiments shown.

Donor mobilization with ProGP-1 effectively separates GVHD and GVL effects following allogeneic SCT. Irradiated B6D2F1 mice received spleen grafts from B6D2F1 (syngeneic) or C57BL/6 (allogeneic) donors mobilized with control diluent, G-CSF, or ProGP-1 as indicated. At the time of transplant, mice also received a leukemic challenge of 1 × 10⁴ P815. Overall survival and death due to leukemic progression or GVHD by Kaplan-Meier analysis are indicated (ProGP-1 allogeneic, n = 30; G-CSF allogeneic, n = 51; control allogeneic, n = 4; control syngeneic, n = 16). *P < 0.05 vs. ProGP-1.

Enhanced cellular cytotoxicity following donor pretreatment with ProGP-1 is not attributable to Flt-3L signaling or the modulation of donor DCs. (A) GM cells and DCs from ProGP-1–mobilized WT C57BL/6 donors were cultured with nonmobilized C57BL/6 hepatic lymphocytes plus control diluent or α-GalCer (C20:2). *P < 0.05 vs. control. One of 2 identical experiments shown. (B) Irradiated B6D2F1 mice received whole spleen from WT donors mobilized with G-CSF or ProGP-1. Additional cohorts received whole G-CSF–mobilized spleen plus purified GMs or DCs from a ProGP-1–mobilized donor or ProGP-1–mobilized spleen depleted of CD11c⁺ DCs. In vivo cytotoxicity index determined at day 12 (G-CSF–mobilized donors, n = 9; G-CSF–mobilized donors + ProGP-1 DCs, n = 9; G-CSF–mobilized donors + ProGP-1 GM, n = 5; ProGP-1–mobilized donors, n = 4; ProGP-1–mobilized donors CD11c depleted, n = 4). *P < 0.01 vs. G-CSF–mobilized groups. (C) Irradiated B6D2F1 mice received whole spleen from WT donors mobilized with G-CSF, G-CSF + Flt-3L, or ProGP-1 as described in Methods. In vivo cytotoxicity index determined at day 12 (n = 4 per group). (D) Irradiated B6D2F1 mice received whole spleen from B6D2F1 or C57BL/6 donors mobilized with G-CSF or G-CSF + Flt-3L. At the time of transplant, mice also received a leukemic challenge of 1 × 10⁴ luciferase-transfected P815 cells. Overall survival by Kaplan-Meier analysis (control syngeneic, n = 11; other groups, n = 40). Representative Xenogen images at days 8 and 17 as described in Methods.

The enhanced cytotoxicity observed following donor pretreatment with ProGP-1 is mediated solely via the perforin pathway. (A) Irradiated B6D2F1 mice received whole spleen from WT, Perforin^–/–, TNFα^–/–, TRAIL^–/–, or FasL^–/– donors mobilized with G-CSF or ProGP-1. In vivo cytotoxicity index was determined at day 12 (n = 4 per group). *P < 0.05 ProGP-1 vs. G-CSF within matched donor groups. (B) CD8⁺ T cells were sort-purified at day 13 from recipients of WT or Perforin^–/– spleen and utilized as effectors in ⁵¹Cr-release CTL assays; P815 target population. *P < 0.05 WT ProGP-1 vs. all other groups. (C) Total CD8⁺ T cells per spleen at day 13 after transplant (n = 4 per group). (D) CD8⁺ T cells were sort-purified at day 13 from recipients of Perforin^–/– spleen and utilized as effectors in fluorolysis CTL assays as described in Methods. Host-type target, P815; donor-type target, EL4.