Vigorous Priming of Minor Histocompatibility Antigen Specific T Cells by Frequent High Dose Peptide Vaccination May Lead to Prolonged T Cell Receptor Downregulation, Impaired T Cell Functionality, and Promotion of Tumor Escape.
Authors: Jedema Inge , van Dreunen Linda , van Loenen Marleen M. , Heemskerk Mirjam , Willemze Roel , Falkenburg J.H. Frederik 
Published: April 2020

Abstract
Allogeneic stem cell transplantation can be successfully applied in the treatment of hematological malignancies and relies on the graft versus leukemia (GVL) effect mediated by donor T cells directed against minor histocompatibility antigens (mHag) selectively expressed on malignant hematopoietic cells of the patient. However, due to insufficient in-vivo priming of donor T cells the GVL response may not be adequately initiated or amplified. Vaccination strategies using immunogenic peptides derived from hematopoeisis specific mHag like HA-1 may form a strategy to initiate or boost the in-vivo GVL response. However, it has been reported that repetitive vaccination with HLA-class I binding 9-mer peptides can lead to the induction of T cell anergy. We hypothesized that repetitive strong priming of the mHag specific T cells may also lead to prolonged downregulation of the T cell receptor (TCR) resulting in inability of the T cells to subsequently attack tumor cells expressing the mHag, allowing tumor escape despite the presence of potentially effective T cells. We tested this hypothesis in an in-vitro model using CFSE-labeled HA-1+ CD34+ chronic myeloid leukemia (CML) cells as target/stimulator cells, and HA-1 specific T cells as effector cells. In previous studies we have demonstrated the resistance of a small population of quiescent CML stem cells to all high avidity T cells, allowing the subsequent outgrowth of malignant progeny from this population. To mimick a peptide vaccination strategy, we loaded CD34+ CML cells from an HA-1+ patient with various concentrations (E-12-E-6M) of the 9-mer HA-1 peptide, and investigated the direct and residual functional cytotoxic capacity of an HA-1 specific CD8+ T cell clone. In accordance with our previous results, we observed complete deletion of all proliferating CML precursor cells after 24–48 hours of exposure to the CTLs, whereas a small subpopulation of quiescent CD34+ cells was resistant to T cell attack. The exogenous peptide loading resulted in more rapid lysis and also attack of part of the quiescent stem cell population. However, in the next days malignant progeny was formed from the quiescent stem cell population in the conditions of high peptide stimulation despite the continuous presence of the T cells, suggesting impaired residual cytotoxic function of these T cells. Therefore, we analyzed the level of TCR downregulation after exposure to the HA-1 positive CML CD34+ cells in the absence or presence of E-12-E-6 M HA-1 peptide loaded to the target cells. We observed strong dose-dependent TCR downregulation as measured by specific tetramer staining (20%–78% decrease in fluorescence intensity after 24 hours of exposure to targets loaded with 0-E-6M HA-1 peptide). At high peptide concentrations it took 6–9 days before proper functional TCR expression could be again demonstrated. In conclusion, we here demonstrate that high affinity T cells show a prolonged TCR downregulation after vigorous stimulation by peptide loaded target cells. In this period the T cells showed a dramatic loss of function and allowed the outgrowth of a leukemic subpopulation expressing the HA-1 antigen. Milder vaccination strategies using longer peptides requiring uptake and processing by the target cells may lead to expression of more physiological levels of the mHag and less vigorous priming of the mHag specific T cells, thereby preserving their functional capacity and responsiveness.