How Pure is Pure Enough: Effects of Red Blood Cell and Platelet Contamination on T-cell Culture

Red blood cells (RBCs) and platelets (PLTs) have historically been considered immunologically inert. Recently, enough evidence has accumulated to suggest an important immunoregulatory role for these cells, both in vivo and in vitro. The latter is particularly important in the context of manufacturing novel cellular immunotherapies, including chimeric antigen receptor (CAR) T-cell therapies. T-cells are a sub-set of lymphocytes, a type of white blood cells that play a major role in the adaptive immune response. By reprogramming these cells to seek out and destroy target malignant cells, CAR T-cell therapies for cancer achieve remission rates as high as 90%. To manufacture such a highly effective treatment, T-cells must first be separated from whole blood and ultimately expanded in culture. RBCs serve as dynamic reservoirs of immunological signaling proteins, able to absorb and release at least 46 different cytokines. The presence of RBCs during culture has a significant impact on T-cell proliferation and survival. Contamination of T-cell culture with residual PLTs results in significant suppression of CD4+ cells and upregulation of regulatory T-cells, a type of T-cell that suppresses the function of other T-cells to prevent autoimmune responses. Additionally, residual PLTs modulate a variety of cytokines including IL-2, IL-5, IL-10, IL-17, IFN-γ and TNF-α. There is a wide range of cell separation methods currently employed in both research and clinical settings. And the number of residual RBCs and PLTs present during T-cell culture is almost entirely determined by the method used to isolate T-cells from whole blood. Given their significant immunoregulatory effects, the level of RBC and PLT contamination necessitates careful consideration when choosing a T-cell isolation method, in addition to the typical performance metrics such as T-cell recovery and purity, separation throughput, and operating costs. This thesis examines the known effects of RBCs and PLTs on expansion of T-cells in culture to help evaluate the allowable number of these contaminating cells, and facilitate a well-informed decision when selecting a separation method for manufacturing CAR T-cell therapies.