Fig. 5. Impact of ALPN-101 on T_eff and T_reg proliferation and on huDCs and huCD4⁺CD146⁺CCR5⁺ T cell populations in target organs.

(A to C) Effect of ALPN-101 on T cell proliferation. Human T_effs and T_regs were stained with antibodies recognizing FoxP3, CD4, PD-1, CD25, CD28, CD127, ICOS, PD-L1, and Helios to confirm their surface phenotype before culture. In (A), enriched T_regs were labeled with CellTrace Violet (CTV) and cultured with soluble anti-CD3 antibody, recombinant IL-2 (rIL-2), and K562 APCs (transfected with CD80 and treated with mitomycin C) in medium with saline (black) or with various concentrations of the test molecules: Fc control (brown), belatacept (blue), or ALPN-101 (green). (A) After 3 days, T_reg proliferation was assessed by CTV dilution by flow cytometry. Statistical differences between Fc control and the other test molecules were determined by an unpaired t test. The ALPN-101 treatment effect is significantly different from the Fc control group (P = 0.0037 at 1 nM, P = 0.0034 at 3 nM, and P = 0.0003 at 30 nM by unpaired t test) and also significantly different from the belatacept group (P = 0.004 at 1 nM and P = 0.007 at 3 nM). (B) T_regs labeled with CTV were mixed at the indicated ratios with T_eff labeled with CFSE and cultured with mitomycin C–treated, CD80^low K562 APCs, and soluble anti-CD3 antibody in medium containing added saline or 30 nM of each test molecule. After 4 days, T_eff proliferation was assessed by CFSE dilution by flow cytometry. The ALPN-101 treatment effect is significantly different from the Fc control group at all T_reg:T_eff ratios except 2:1 (P = 0.0001 at 0.67:1; P = 0.0345 at 0.22:1; P = 0.0158 at 0.074:1; P = 0.0313 at 0.024:1; P = 0.0196 at 0.0082:1, P = 0.0187 at 0.0027:1, and P = 0.0342 at 0:1, by unpaired t test). (C) Specific T_reg suppression activity for each culture condition was determined (see Materials and Methods). Concentrations are the same as in (B). Data are presented normalized to T_eff activity in the absence of T_regs. (D to F) Effect of ALPN-101 on health [BW (D) and GVHD severity score (E)] and survival (F) of the human PBMC-NSG GVHD model. NSG mice were irradiated at 300 cGy at day −1 and then transplanted with 3.5 × 10⁶ human PBMCs at day +1. Mice were treated every other day with Fc control (brown) or 100 μg of ALPN-101 from days −1 to +21 (12 doses total; blue) or 100 μg of ALPN-101 on days −1 and +1 (2 doses total; green). n = 10 mice in all groups. (G to I) Human hematopoietic cell engraftment (G), Lin⁻HLA-DR⁺ total DCs (H), and CD4⁺CD146⁺CCR5⁺ T cells (I) in the GI tract of NSG recipient mice. Five mice from (D) to (F) were analyzed at day 14 after HCT comparing Fc control and ALPN-101–treated groups. (J) Concentration of human FLT3L in plasma from NSG mice from (D) to (F) at days 7 and 14 after HCT (n = 3 mice per time point). Data are shown as mean ± SEM, except for survival curves and representative flow cytometry. In (A) to (C), statistical significance was determined by unpaired t test, and in (D), (E), and (G) to (J), it was determined by ANOVA with Bonferroni’s correction. Log-rank test (Mantel-Cox) was used for survival analysis in (F).