Abstract
Adoptive transfer of antigen-specific T cells is a potentially curative strategy for patients with solid tumors and leukemia. Most clinical trials of adoptive T cell therapy have used cytotoxic CD8+ T cells recognizing MHC class I-restricted tumor antigens. Despite overwhelming evidence suggesting the fundamental influence of CD4+ T cells on the immune system, clinical experience with tumor-specific CD4+ Th cells is almost non-existent. Unlike most other tissues, bone marrow-derived cells constitutively express MHC class II and CD4+ T cells play crucial role in mediating the curative GVL effect after allogeneic SCT and donor lymphocyte infusion (DLI). Furthermore, experimental evidences suggest that MHC class II-restricted antigenic targets recognized by CD4+ T cells exist in both solid cancers and in hematological malignancies. Therefore adoptive immunotherapy using CD4+ T cells in the setting of leukemia might be especially relevant.
The goal of this study is to establish a simplified non-individualized protocol of generating LAA-reactive CD4+ T cells from patients and normal donors for adoptive immunotherapy directed against common leukemia-associated antigens (LAA) expressed in acute myeloid leukemias (AML) and myelodysplastic syndrome (MDS). We isolated naïve and memory CD4+ T cells from 3 normal donors and stimulated with twice at weekly interval with autologous monocytes pulsed with libraries of overlapping 15-amino acid length peptides (pepmixes) derived from WT-1, MAGE A3 and A4, PRAME and SSX2 antigens. At the end of the experiment CD4+ T cells were evaluated for reactivity against each LAA by analyzing their ability to specifically release cytokines (IL-2, TNF-α, and IFNγ) using flow cytometry. LAA-specific cells were found in either naïve or memory-derived CD4+ T cells upon stimulation with relevant pepmixes in all donors tested. However specific cytokine production could not be demonstrated when the same T cells were exposed to LAA-transduced autologous targets (LCL and T cells), raising the possibility that the majority of pepmix-reactive cells recognized epitopes that were not naturally processed. Therefore, as an alternative strategy to induce LAA-specific cells capable of targeting only therapeutically-relevant epitopes, we used autologous dendritic cells (DCs) transduced with a lentiviral vector encoding MAGE A3 antigen. Autologous CD4+ T cells were stimulated with MAGE A3 or mock-transduced DCs at an interval of 7-10 days and tested for their antigen-specific cytokine secretion. At the end of the culture we observed that Th cells expanded in presence of MAGE A3-expressing DCs and contained a significant number of cells possessing specific reactive against MAGE A3 pepmix (Figure), but not to unrelated antigenic targets, suggesting induction of LAA-reactivity against naturally-processed MAGE A3 epitopes.
In summary, we demonstrate the feasibility of generating specific anti-tumor CD4+ T cells using autologous DCs engineered to express a full-length tumor antigen. This approach allows for selective expansion of polyclonal Th cells recognizing only naturally processed MHC class II-restricted epitopes. Therefore, this strategy circumvents the limitation inherent to usage of overlapping peptide libraries that might induce the expansion of high-avidity T cells specific to epitopes that are irrelevant to in vivo recognition of tumor targets. Furthermore, this approach does not rely on a particular pre-defined MHC class II restriction element, thus it is applicable to majority of donors or patients irrespective of their MHC haplotype.
Disclosures:
No relevant conflicts of interest to declare.
Splenic Stroma-Educated Regulatory Dendritic Cells Induce Apoptosis of Activated CD4 T Cells via Fas Ligand-Enhanced IFN-γ and Nitric Oxide
