The effect of CoBS immunosuppression on GVHD. (A) Lymphocyte proliferation was inhibited in animals treated with CoBS. Representative example of R.5 showing forward-scatter versus side-scatter characteristics before transplantation (top) and on day 43 (bottom), during CoBS treatment. (B) Longitudinal analysis of the white blood cell (WBC) count, absolute neutrophil count (ANC) and absolute lymphocyte count (ALC) in R.4-R.7. (C) Survival advantage observed with CoBS immunosuppression. At the 30-day primary endpoint, all untreated animals had died, and 100% of CoBS-treated animals survived (P = .01). (D) Extended survival analysis of CoBS-treated versus untreated cohorts. Log-rank analysis indicated that overall survival of the CoBS-treated cohort (MST = 62 days) was statistically significantly different from the untreated cohort (MST = 11.6 days; P = .01). MST = mean survival time. (E) Photomicrographs of hematoxylin and eosin staining of the lungs, liver, and colon of the autologous transplant control (R.10), a representative CoBS-treated recipient who developed significant breakthrough GVHD (R.4), and a representative CoBS-treated recipient who showed minimal GVHD histopathology (R.5). Bar = 100 μm. These studies used an Olympus BX51 microscope (Olympus America), using a 10×/0.40 UPlan Apo lens (Olympus). Slides were mounted with Permount solution (Sigma-Aldrich) and photographs were taken with the Spot RT Slider imaging system (Spot Imaging Solutions). Image analysis was performed using Spot Advanced Version 4.6 (Spot Imaging Solutions), and image processing was performed using Photoshop CS4 extended v11.01 (Adobe). (F) Correlation of clinical disability (as measured by the activity score) with GVHD histopathology. The data fit a linear equation with an R2 = 0.7221. (G) Longitudinal analysis of weight loss in animals treated with CoBS immunosuppression.
