Fig. 6. Treatment with ALPN-101 at onset of xenogeneic aGVHD and huPBMC-NSG model with human leukemia cells.

(A to C) Effect of ALPN-101 on health [BW (A) and GVHD severity score (B)] and survival (C) of the human PBMC-NSG GVHD model. NSG mice were irradiated at 350 cGy at day −1 and then transplanted with 5 × 10⁶ human PBMCs at day +1. Mice were treated with either Fc control every other day from days +7 to +14 (n = 9), or 20 μg of ALPN-101 every other day from days +7 to +14 (n = 9), or 100 μg of ALPN-101 (n = 15) every other day from days +7 to +14. (D to F) Human hematopoietic cell engraftment (D), Lin⁻HLA-DR+ DC frequencies (E), and CD4⁺CD146⁺CCR5⁺ T cell frequencies (F) in the GI tract of NSG recipient mice. Three NSG mice from each group in (A) to (C) were analyzed at day 14 after HCT, comparing the Fc control and ALPN-101–treated groups. (G) Concentration of human FLT3L in plasma from three NSG mice from (A) to (C) at the indicated times after HCT. (H) Study design for evaluation of ALPN-101 GVHD prophylaxis in mice with leukemia. NSG mice were irradiated at 300 cGy and injected with 1 × 10⁶ human leukemic cells of the line MOLM-14-EGFP on day −3 and then transplanted with 4 × 10⁶ human PBMCs on day 0 (day of PBMC transplant). Two groups of mice did not receive PBMCs, while one did not receive any treatment, the other group was treated with ALPN-101 from days −1 to +14 (n = 15); the other mice all received PBMCs and were treated with either ALPN-101 only from days −1 to +14 (n = 20), Fc control every other day from days −1 to +14 (n = 20), 20 μg of ALPN-101 on day −1 and +1 (n = 20), or 20 μg of ALPN-101 every other day from days −1 to +14 (n = 20). (I) Survival of leukemia-recipient mice. Pie charts represent the percentage of surviving mice or cause of death for each group. (J) Clinical GVHD score in the three groups of leukemia-recipient mice receiving PBMCs. (K) Percentage of GFP⁺MOLM-14 leukemic cells in the BM of mice at death. In (A), (B), (D) to (G), and (I) to (K), data are shown as mean ± or + SEM. In (A), (B), (D) to (G), and (I) to (K), statistical significance was determined by ANOVA with Bonferroni’s correction. Kaplan-Meier method using the log-rank test for comparison was used for survival analysis in (C).