Early Detection and Rapid Isolation of Leukemia-Reactive Donor T Cells for Adoptive Transfer Using the IFN-γ Secretion Assay

Abstract

Purpose: The poor immunogenicity of most leukemias and the lack of specificity of the donor Tcells limit the in vivo effectiveness of conventional donor lymphocyte infusions in many patients suffering from persistent or recurrent leukemia after allogeneic stem cell transplantation. These limitations may be overcome by the adoptive transfer of in vitro generated leukemia-reactive T cells. Although the potential clinical efficacy of this approach has been shown previously, lack of reproducibility of the procedure and the inability to show persistence and survival of the transferred T cells hampered further clinical application. The purpose of this study was to develop a new, broadly applicable strategy for the efficient generation and isolation of leukemia-reactive Tcells with a better probability to survive and expand in vivo. Experimental Design: Myeloid and B-cell leukemias were modified into professional immunogenic antigen-presenting cells, and used to stimulate HLA-matched donor T cells. After two stimulations, responding donorTcells were isolated based on their secretion of IFN-g and tested for their capacity to recognize and kill the primary leukemia. Results: Using one universal stimulation and isolation protocol for various forms of leukemia, T-cell populations containing high frequencies of leukemia-reactive T cells could reproducibly be generated and early isolated under mild stimulatory conditions. Isolated Tcells still had high proliferative potential and their reactivity seemed to be restricted to cells of the patient's hematopoiesis. Conclusion: We here show a new robust procedure for the generation and isolation of leukemiareactiveTcells for adoptive transfer.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation

“…[41][42][43][44] It has been argued that ALL cells are intrinsically resistant to (alloreactive) T cells although several reports have described that activated T cells do recognize and lyse unmanipulated ALL cells. [45][46][47][48][49] An alternative explanation for the lack of a clinical effect may be found in the rapid growth characteristics of ALL cells.…”

Characterization of graft-versus-leukemia responses in patients treated for advanced chronic lymphocytic leukemia with donor lymphocyte infusions after in vitro T-cell depleted allogeneic stem cell transplantation following reduced-intensity conditioning

“…6 T cells responding to in vitro stimulation with the leukemia or the leukemic APC by the production of interferon-g (IFN-g) were sorted one cell per well and expanded. 7 In patient 6, the percentages of IFN-g-secreting T cells after 16 h of stimulation with primary CLL were 0.04% and with CLL-APC 0.22%. From patient 6, 23 proliferating clones out of 192 sorted T cells were obtained in response to primary CLL (plating efficiency 12%), and 3 of these clones exerted cytolytic activity against CLL-APC but not against primary CLL (data not shown).…”

“…7 The repetitive stimulation with CLL-APC may ultimately result in a relatively enrichment of leukemia-reactive T cells as compared to GvHDmediating T cells. Furthermore, by in vitro priming of CLLreactive donor T cells against CLL-APC T-cell anergy towards primary CLL may be minimized.…”

Human allo-reactive CD4+ T cells as strong mediators of anti-tumor immunity in NOD/scid mice engrafted with human acute lymphoblastic leukemia

“…Leukemic cells were modified into leukemic-APC, as previously described. 21 Briefly, primary leukemic cells were cultured in IMDM with 10% ABO serum, 500 IU/ml IL4 (Schering-Plough, Innishammon, Ireland) and 10 mg/ml synthetic CpG oligonucleotides (Eurogentec, Seraing, Belgium) followed by co-culturing on a monolayer of murine fibroblast cell line (Ltk-) transfected with human CD40-ligand (provided by Dr C van Kooten, LUMC, the Netherlands). Phenotypical analysis of in vitro generated leukemic-APC was performed as CD4 þ T cells as mediators of anti-tumor immunity S Stevanović et al described for leukemic cells directly ex vivo.…”

“…It is known that antigen-presenting cells (APC) are required for efficient induction of anti-tumor immunity in vivo, and that APC acquire a mature phenotype on interaction with T-helper cells. [8][9][10]19 In vitro, in the presence of a variety of soluble factors, various types of leukemic cells, including CML, 20,21 acute myeloid leukemia, [21][22][23] ALL 21,22 and chronic lymphocytic leukemia, 21,24 cells have been shown to differentiate into leukemic-APC displaying increased expression of HLA-class II, adhesion and costimulatory molecules and an enhanced capacity to stimulate allogeneic T cell responses. [20][21][22][23][24] On the basis of these findings, it can be speculated that the efficacy of DLI depends on the capacity of leukemic cells to become leukemic-APC in vivo.…”