Potent B Cell Leukemia-Reactive T Cells Present in Naive Donor T Cell Populations Can Be Isolated under GMP Conditions Using a Universal Stimulation and Isolation Procedure.
Authors: Jedema I. , Wolbers J. Olde , Steeneveld E. , Rasmussen A.-M. , Smit W.M. , Willemze R. , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
Treatment of patients with relapsed or persistent acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL) after allo-SCT with donor lymphocyte infusions (DLI) is frequently unsuccessful, probably due to the limited immunogenicity of these malignant B cells, and the lack of specificity of the T cells. In this study, we generated a broadly applicable stimulation and isolation procedure for the induction of leukemia-reactive T cells under good manufacturing practice (GMP) conditions. First, B-cell leukemias were modified into professional antigen presenting cells (APC). We previously demonstrated that CD40 crosslinking was required for the production of appropriate malignant B cell-APCs. CD40L expressing mouse fibroblasts were potent activators of CD40, but not GMP approved. CD40L trimers and CD40 antibodies crosslinked to plates or beads only minimally triggered CD40 on malignant B cells. As previously demonstrated, activated T cells briefly express CD40L. We first determined the dynamics and kinetics of CD40L expression on peripheral blood (PB) donor T cells after stimulation with CD3/28 Dynal T cell expansion-beads added in a ratio of 1 bead/10 PBMC. The kinetics were highly variable, with the optimal surface expression of CD40L on a median of 20% of the T cells (range 10–35%, n=6) being detectable between day 1 and 5. We next investigated the capacity of these activated T cells to stimulate malignant B cells in PB from patients with ALL or CLL containing 60–95% leukemic cells. We developed a two-step strategy using special CD3/28 Dynal isolation beads applicable not only for T cell activation, but also for T cell isolation. Using these beads, the T cells were depleted from the PB of the patient, directly irradiated and added back to the B cell cultures. After 3–7 days all B cells displayed a good APC phenotype with expression of CD80, CD86, and CD83 in the patient samples containing >5% T cells. If insufficient T cell numbers were present, donor T cells could be used as source of CD40L. Leukemia-reactive T cells could be reproducibly generated after two stimulations of fully HLA matched donor T cells with the leukemic APC under mild stimulatory conditions, followed by isolation of the responding T cells based on their production of interferon gamma (IFNg). Next, we investigated whether the leukemia-reactive T cells were the result of the successful induction of a primary immune response or that recognition of mimicry epitopes by previously activated T cells was underlying the activity. We separated unmodified donor T cells into CD45 RO+ memory cells, CD45RA+/CD27+ naive cells, and CD45RA+/CD27− effector cells by MACS CD45RO depletion, followed by FACS sorting on CD27, and stimulated these fractions with malignant APCs. Whereas high frequencies of IFNg producing T cells (5–15%) with cytotoxic activity against the primary leukemia (20–50% lysis) were induced from the naive T cell population, no leukemia-reactive T cells could be isolated from the memory or effector T cell populations (n=3). In conclusion, T cells stimulated with CD3/28 Dynal beads have a transient expression of CD40L and can be used as an alternative source of CD40L to generate good APC of malignant B cells under GMP conditions. These malignant B cell APCs were capable of inducing efficient priming of primary anti-leukemic immune responses by activating naive donor T cells.