Fig. 4. ICOS expression on activated T cells in the human PBMCs-NSG GVHD model correlates with disease severity, and the suppressive effects of ALPN-101 are not altered by CsA.

(A) Survival of mice receiving repeated doses (RD) or a single dose (SD) of the indicated test articles. NSG mice x-ray irradiated (100 cGy) and administered 10 mg of human γ globulin subcutaneously on day −1 and then transplanted intravenously with 10 × 10⁶ human PBMCs on day 0 were treated intraperitoneally with saline; 100 μg of ALPN-101 TIW × 4 (days 0 to 28) or once on day 0; or 100 μg of belatacept TIW × 4 (days 0 to 28) or once on day 0. (B) Blood from each mouse treated with saline or a single dose (SD) or repeat doses (RDs) of belatacept or ALPN-101 [as described in (A)] was evaluated by flow cytometry for percent cells positive for ICOS or CD28 in the population of human CD4⁺ cells. Because ALPN-101 blocks the binding of anti-ICOS and anti-CD28 antibodies used for flow cytometry, data from the ALPN-101 groups are omitted. (C) Percent ICOS⁺ or CD28⁺ human CD4⁺ cells are plotted versus the terminal DAI score for each mouse as described in (A). Linear regression curves were calculated and correlation coefficients (R²) are indicated for each dataset. Because ALPN-101 blocks the binding of the antibodies used for flow cytometry, data from the ALPN-101 groups are shown on the graphs but were omitted from the correlation analyses. See table S7 for additional correlation analyses. (D) Effect of CsA on ALPN-101 therapy. Percent survival and clinical scores for NSG mice x-ray irradiated (100 cGy) and administered 10 mg of human γ globulin SC on day −1, then transplanted intravenously with 10 × 10⁶ human PBMCs on day 0 (D0) and treated intraperitoneally with saline daily from day −1 to 13, then TIW through day 28; CsA 20 mg/kg daily from day −1 to 13, then TIW through day 28; 500 μg of ALPN-101 once on day 0; combination of 500 μg of ALPN-101 once on day 0 plus CsA 20 mg/kg daily from day −1 to 13, then TIW through day 28; or 75 μg of belatacept TIW × 4. In (A) (left), statistical significance in survival proportions between groups was determined by Mantel-Cox log-rank test. In (B), data are shown as mean ± SEM. In (D) (left), statistical significance in survival proportions between groups was determined by Mantel-Cox and Gehan-Breslow-Wilcoxon log-rank tests. In (D) (right), statistical significance was determined by two-way repeated measures ANOVA for treatment effect.