Expansion and Transformation of Primary Acute Lymphoblastic Leukemia Cells into Antigen-Presenting Cells using a Novel Culturing System Enables the Generation of Leukemia-Reactive T Cell Responses that Are Effective in Vivo.
Authors: Nijmeijer Bart A. , Van Schie Marianke L.J. , Willemze Roel , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
Allogeneic cellular immunotherapy is generally ineffective in acute lymphoblastic leukemia (ALL). In vitro studies have suggested that this inefficacy may be the result of a lack of costimulatory molecule expression by ALL cells, resulting in the induction of T cell anergy. Activation of T cells by ALL cells that are transformed into adequate antigen-presenting cells (ALL-APC) may prevent the induction of T cell anergy and result in the generation of competent leukemia-reactive T cell responses for adoptive immunotherapy. However, in vitro modification of ALL cells was hampered by the fact that ALL cells from adult patients could not be cultured in vitro for prolonged periods of time. We have developed a novel serum-free culturing system for B-lineage ALL in which proliferation is initiated and sustained by ALL-cell derived growth factors. Long-term (>2 yrs) proliferation was induced in 12 out of 26 randomly selected primary samples from patients with ALL. The cell cultures ( Leiden cell lines) proliferated with a mean doubling time of 3.0 days (range 2.7–3.6 days). All Leiden cell lines presented the chromosomal abberations observed in the primary cells. The Leiden cell lines displayed an immune phenotype similar to the primary cells, exept for loss of CD34 expression. In vivo characteristics of Leiden cells were evaluated in NOD/scid mice. After intravenous inoculation, Leiden cell lines and primary cells showed identical homing patterns initially involving spleen and bone marrow, followed by the development of overt and progressive leukemia. A comparison of in vivo progression kinetics was performed for one of the Leiden cell lines and the corresponding primary cells. Weekly determination of leukemic cell counts in the blood of engrafted animals revealed that the cell line and the primary cells displayed similar doubling times in vivo of 6.3 and 7.7 days, respectively. To generate cells with improved antigen presentation function, Leiden cell lines were exposed to various activating agents. Stimulation with CpG containing oligonucleotides resulted in induction of CD40 in 9 out of 10 lines. Subsequent ligation of CD40 by culturing CpG-activated Leiden cells on fibroblasts expressing human CD40 ligand resulted in the induction of CD80 or CD86 in 7 of these 10 cell lines. To study the immune stimulatory properties of these Leiden ALL-APC, allogeneic HLA-identical T cells were first activated in vitro by coculturing these cells with either unmodified Leiden cells or with the corresponding Leiden ALL-APC for 3 days, and subsequently infused into groups of 6 leukemic NOD/scid mice. While T cells cocultured with unmodified Leiden cells did not expand in vivo, T cells cocultured with Leiden ALL-APC expanded after infusion in 5 out of 6 animals. This expansion coincided with a 20–75% decrease in leukemic cell numbers in the blood. In conclusion, the novel serum-free culturing system enables long-term culture and manipulation of a significant fraction of primary human ALL. These Leiden cell lines can be modified into ALL-APC that display adequate antigen presenting function, preventing the induction of T cell anergy as demonstrated in vivo in the NOD/scid mouse model.