Engineering Artificial Antigen-presenting Cells to Express a Diverse Array of Co-stimulatory Molecules

Abstract

To facilitate the therapeutic application of antigen-presenting cells (APCs), we have developed a cell-based artificial APC (aAPC) system by engineering K562 cells with lentiviruses to direct the stable expression and secretion of a variety of co-stimulatory molecules and cytokines. Here we report the use of a combinatorial lentiviral gene transfer approach to achieve long-term stable expression of at least seven genes in the K562 parental cell line. Expression of various combinations of genes on the aAPC enables the precise determination of human T-cell activation requirements, such that aAPCs can be tailored for the optimal propagation of T-cell subsets with specific growth requirements and distinct functions. The aAPCs support ex vivo growth and long-term expansion of functional human CD8 T cells without requiring the addition of exogenous cytokines, in contrast to the use of natural APCs. Distinct populations of T cells can be expanded with aAPCs expressing CD137L (4-1BBL) and/or CD80. Finally, the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells. Therefore, K562-based aAPCs have therapeutic potential for adoptive immunotherapies and vaccinations.

Cutting Edge: Foxp3-Mediated Induction of Pim 2 Allows Human T Regulatory Cells to Preferentially Expand in Rapamycin

“…anti-CD28 Ab-coated beads (15) or by coculture with irradiated, anti-CD3 Ab-loaded, lentiviral vector-transduced K562 artificial APCs (aAPCs) expressing CD64 and CD86 as described previously (16). T cells were cultured in the presence of rhIL-2 (300 U/ml; Chiron) and, where indicated, rapamycin (100 ng/ml, Calbiochem).…”

“…To test this, we expanded enriched Treg cells (40 -80% Foxp3 positive) isolated from healthy donors using a previously described K562 cell-based aAPC. These KT64 86 aAPCs express CD64 to load an anti-CD3 agonist Ab and CD86 to engage CD28 (16). Enriched Tregs from nine donors were expanded by anti-CD3-loaded KT64 86 cells at a 2:1 ratio (Treg:aAPC) in the absence of rapamycin.…”

Clinical Application of <em>Sleeping Beauty</em> and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

“…Our approach to integrate a CAR transgene by electro-transfer of non-viral DNA plasmids from the SB system can be undertaken in quiescent primary T cells derived from PB and UCB. We and others have genetically modified K562 cells to serve as aAPC to efficiently propagate clinically-sufficient numbers of T cells for infusion 24,25 . The aAPC and tissue culture environment (e.g.…”

Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8+ T Cells Ex Vivo

“…Artificial antigen-presenting systems encompass both cell-based and acellular technologies. Several diverse scaffoldding systems such as genetically engineered insect cells, mouse fibroblasts, human tumor cell lines (3,4,(11)(12)(13), microbeads, artificial liposomes, and biomembrane derivatives (exosomes, immunosomes) (14, 15) have been developed. However, the self-limitation of insect cells at 37 o C (the viable temperature for D. melanogaster cells is 27 o C) could lead to massive release of D. melanogaster antigens and limit the duration of the contact between the T cell and the aAPC (16,17).…”

“…The K562 cell-based aAPCs have several advantages over other aAPCs based on beads, insect cells, or biomembrane derivatives, such as negative MHC expression, mycoplasma free, better formation of the immunological synapse as a result of the fluidity of its membrane, well growth in serum-free medium (3,4,11,13). Due to the preferential binding of B7-2 to CD28 and B7-1 to CTLA-4, and both CD28 and B7-2 constitutively expressed on T cells and APCs, respectively, relatively week interaction between CD28 and B7-2 may suffice as an early co-stimulatory interaction to initiate T cell activation (6,7).…”