Effects of TNF and IL6 blockade on acute GVHD after alloHSCT in mice. (A-E) Lethally irradiated C57BL/6 mice received 5 × 10⁶ bone marrow (BM) cells with and without 40 × 10⁶ spleen cells (SCs) from donor BALB/c mice and were treated with either single or dual cytokine blockade. (A) Survival rate (BM: n = 4, other groups: n = 16 per group). (B) aGVHD clinical scores (n = 8 per group). (C) Representative images of H&E staining from colon samples at day 9 post-HSCT. Red arrows indicate crypt apoptosis, black arrows indicate lamina propial lymphocytes, orange arrows indicate intracryptal lymphocytes. The scale bar, 100 μm. (D) Pathology scores of GI tract samples (n = 5-7 per group). (E) Serum IL-6 and serum TNF concentrations at day 6 post-HSCT (n = 5-7 per group). (F-J) Lethally irradiated control and DIO BALB/c mice received 8 × 10⁶ BM cells and 25 × 10⁶ SCs from donor B10.D2 mice and were treated with either single or dual cytokine blockade. (F) Survival rate (n = 7-8 per group) (G) aGVHD clinical scores at day 6 post-HSCT (n = 7-8 per group). (H) Serum IL-6 and TNF concentrations at day 7 post-HSCT (n = 5-7 per group). (I) Representative images of H&E staining from colon samples at day 7 post-HSCT. The scale bar is 100 μm. Arrows indicate crypt apoptosis. Much milder lamina proprial lymphocytic infiltration is noted with dual cytokine blockade compared with single blockade. (J) Pathology scores of samples from panel H (n = 5-6 per group). Bar graphs depict mean ± SEM. Survival curves (A,F) were plotted on a Kaplan-Meier curve and analyzed by a log-rank test. Clinical scores were analyzed by 2-way analysis of variance (ANOVA) with Tukey's post hoc test for comparison among groups. A 1-way ANOVA test was used in panels E, D, H, and J. *P < .05; **P < .01; ***P < .001; ****P < .0001.