FITC-conjugated Immuno-brite fluorospheres (Beckman Coulter, Fullerton, CA) were used to calculate the number of molecules bound to the CD64-transduced aAPCs. To determine the efficiency of the CD32 and CD64 aAPCs in activating T cells, the aAPCs were loaded with various concentrations of FLT3-IN-4 anti-CD3 and anti-CD28 antibodies (murine IgG2a isotype). for adoptive immunotherapies and vaccinations. INTRODUCTION Professional antigen-presenting cells (APCs), such as dendritic cells (DCs) and lymphocytes, are composed of diverse subsets with specific effector functions. Two main lineages of DCs are acknowledged: the myeloid lineage, which includes Langerhans cells and interstitial DCs, and the plasmacytoid lineage.1 According to current concepts, DCs undergo stepwise activation/ maturation.2 Immature DCs are essential to the maintenance of peripheral tolerance to self antigens and to the development of active immunity.3 In tissue, DCs become activated in response to microbial signals (toll-like receptor ligands) and other micro-environmental signals, triggering migration and differentiation into a mature antigen-loaded DC. The ability to generate DCs from blood monocytes or other precursors has enabled studies screening adoptive transfer of FLT3-IN-4 antigen-bearing DCs in humans. Injection of mature DCs can boost T-cell immunity in healthy volunteers,4 and injection of immature DCs can lead to the inhibition of T-cell responses via the induction of regulatory T cells.5 Therefore, DC immunization is of considerable interest for immunotherapy of cancer and auto-immunity. However, generation of DCs is usually laborious and expensive. The culture process is resource rigorous and utilizes a cocktail of cytokines, and there is variability among donors.6 In addition, there have been reports of dysfunctional DCs in cancer patients.7 We as well as others have demonstrated the successful expansion of human lymphocytes using artificial APCs (aAPCs) in place of natural APCs.8C12 We have chosen the K562 cell collection as a scaffold because the cells do not express major histocompatibility complex molecules, which therefore prevents allogeneic responses. The cells also contain adhesion molecules that enhance FLT3-IN-4 T cellCaAPC interactions. In this study, we have created an improved aAPC system to express a diverse array of co-stimulatory and human lymphocyte antigen (HLA) molecules. Previous studies exhibited expression of up to three genes of interest in aAPCs using retroviral transduction methods.12,13 Here we use lentiviral vector technology to demonstrate stable surface expression of at least seven genes in the K562 cellCbased aAPCs. The aAPCs have an efficiency comparable to that of natural DCs for driving T-cell growth; they are especially efficient for activation of human CD8 T cells, for maintaining CD28 surface expression, and for growth of genetically FLT3-IN-4 altered T cells. Finally, the co-stimulatory ligands around the aAPCs enable efficient proliferation and growth of CD8 T cells without the use of exogenous cytokines or feeder cells as used in current cell culture processes. RESULTS K562 aAPCs with CD32 or CD64 The parental K562 collection was first stably transduced with the CD32 intermediate-affinity Fc receptor or the CD64 high-affinity Fc receptor. Single-cell clones were obtained by flow-sorting, and surface expression of the molecules was assessed by circulation cytometry (Physique 1a). After incubation with a mouse IgG1 or mouse IgG2a antibody and washing, only the CD64 aAPC retained expression of mouse IgG2a, and no detectable antibody remained on the CD32 aAPC. These results are consistent with reported properties of human CD32 and CD64.14 To test the relative binding of anti-CD3, a mouse IgG2a monoclonal Tmem26 antibody, CD64 and CD32 aAPCs were incubated with anti-CD3 (OKT3) and washed. We found that only the CD64 aAPCs experienced detectable surface expression of anti-CD3 (Physique 1b). In previous studies using plasmid-transfected K562 cells, we found that CD32-expressing aAPCs were able to stimulate T cell proliferation, but only when the anti-CD3 was left in the culture medium, which FLT3-IN-4 is usually consistent with the low-level binding of the anti-CD3 to CD32.9 When CD64 aAPCs were loaded with anti-CD3, washed, and then placed back into culture, quantitative flow cytometry demonstrated that this binding and surface expression of the antibody were stable and remained at high levels for at least 24 hours (Figure 1c). Open in a separate window Physique 1 Lentiviral-mediated expression of CD64 and CD32 in K562 artificial antigen-presenting cells (aAPCs)(a) Expression of CD64 and CD32 in K562.