[Enhancement of in-vitro and in-vivo human T lymphocyte activation via cell painting with recombinant chimeric co-stimulatory B7-1:Fc gamma 1 Protein]

ABSTRACT

T cells express co-stimulatory molecules on their surfaces in both normal and pathologic settings; however, the possibility that expression of these co-stimulators may enable individual T cells to co-stimulate themselves has not been investigated. This study aims at assessing the possible potentiation of T cells activation and proliferation both in vitro and in vivo via enforced B7-1 expression on T lymphocytes using a non-genetic approach, i.e. cell painting, that comprises binding of recombinant B7-1 to plasma membrane-anchored palmitoylated protein A. A recombinant chimeric human co-stimulatory B7-1 protein that encompasses the extracellular domain of CD80 [B7-1] fused to the crystal fragment Fc gamma of human IgG antibody was engineered and produced in human kidney cell line 293. Recombinant protein A was chemically palmitoylated and inserted into cell membranes of T lymphocytes highly purified using negative selection. Subsequently, B7-1 Fc gamma 1 was added to protein A-decorated cells allowing its binding via the Fc portion. Quantitative transfer of B7-1 was assessed using flow cytometry, and in vitro proliferation and IL-2 production were measured. Moreover, in vivo engraftment of painted cells was evaluated using the immune deficient mouse model NOD/SCID/B2M K/O. Results from this study demonstrate the feasibility of "cell painting" using a two-step approach to quantitatively transfer palmitoylated protein A and B7-1 Fc gamma to T cells. B7-1 painted T lymphocytes exhibited augmented in vitroproliferation, enhanced IL-2 production, and superior in vivo engraftment. Cell painting technique enables arming T lymphocytes with co-stimulatory proteins that provide second signal inevitable for activation, proliferation, and production of IL-2. These results may pave the way for future applications that include but is not limited to cancer targeted immunotherapy and congenital and/or acquired immunodeficiency. This study was conducted in the laboratories of The School of Medicine, University of Pennsylvania, USA, and The National Commission for Biotechnology, Damascus between 2010 and 2012