Modulation der Immunreaktion auf Tumorgewebe mittels unterschiedlich glykosylierter tumorspezifischer IgG
Hämatologie, Onkologie
Zusammenfassung der Projektergebnisse
We established a murine D4M-3A melanoma cell line, that was transduced with two copies of the SIINFEKL peptide gene sequence (antigen of OT-I T cells) into a blue fluorescent nuclear reporter H2B-Cerulean, to utilize OT-I T cells as tumor antigen-specific cytotoxic T cells (Tu-Ag spec CTL) and at the same time make the tumor cell line suitable for in vivo imaging studies. In our D4M-3A N4cer mouse tumor model in vitro activated T cell receptor (TCR-) transgenic OT-I T cells injected one week after tumor inoculation do not control tumor growth but show signs of exhaustion. Adding anti-PD-1 treatment at the same time leads to immediate tumor rejection while starting the treatment 2 weeks after tumor inoculation provides only temporary tumor control. Under this condition PD-1 inhibition elicited peaking IFNg production in treated OT-I not earlier than after one week of treatment. Isolating CTL within the tumor tissue for one week by FTY720 takes away their capacity to exert a productive therapeutic response. TCF-1 expression was preserved in a fraction of OT-I in our isolation experiment, showing that besides TCF-1 expression other factors contribute to successful anti-PD-1 therapy by tumoral T cells. Interestingly, in vitro IL-12 primed OT-I downregulate TCF-1 (TCF-1low OT-I) and, injected into tumor bearing mice, to our surprise, induce tumor control, though, dependent on the endogenous CD8 T cell pool. The same amount of TCF-1 positive OT-I (TCF-1high OT-I) do not control the tumor without PD-1 inhibition. These findings indicate that there might be interplay of fully functional TCF-1 positive and, probably IL-12 primed, TCF-1 negative CTL to provide control over tumor growth during immune checkpoint targeting therapy. We also observed that TCF-1high OT-I in competition with TCF-1low OT-I show a weaker infiltration of the tumor. A recent publication, unfortunately covering several aspects of our observations, presented evidence corroborating the requirement of TCF-1 for CTL in the tumor tissue to respond to PD-1 inhibition. Publicly available gene expression analysis showed upregulation of certain genes in TCF-1 negative tumor infiltrating CTL and let us to investigate gene expression on the protein level in our mouse model. Preliminary studies already revealed a profound role of at least one of the proteins tested in the conversion of TCF-1high to TCF-1low OT-I cells, and their ability to control tumor growth. How this protein regulates effector functions of TCF-1-expressing and nonexpressing CTL is under current investigation and in the highly competitive field of immunotherapy research the identity of the protein can therefore not be disclosed at this point. I also analyzed OT-I CTL behavior in the tumor tissue on the single cell level using two-photon intravital microscopy. While we could repeat findings that CD8 T cells show different migrational behavior depending on T cell receptor (TCR-) stimulation and their spatial and temporal location in the tumor tissue we were not able to assign a clear phenotype to CTL that are undergoing exhaustion. Taking the previously mentioned findings into account it could reflect CTLs having different functional stages of exhaustion in tumor tissue. We also investigated the nuclear translocation of ERK in TCR-transgenic CTL. In the ovalbumin expressing thymoma EG-7, which is rejected by injecting OT-I CTL, we observed signaling events, mostly in proximity to CD11c positive or tumor cells. In the D4M-3A N4cer tumor model OT-I cells did not show similar degrees of ERK-signaling, possibly reflecting changes in the cellular signaling pathways during chronic antigen exposure and development of exhaustion. Because of the diverging angles of the results obtained from the first and second part of the project we aim for publishing both in separate works. It is planned to publish the first, non-imaging part as a shared first authorship after completion of the missing functional data to explain the role of the identified protein for TCF-1 conversion and tumor control by CTL.
Projektbezogene Publikationen (Auswahl)
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Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy. Nature, 2019
Di Pilato M, Kim EY, Cadilha B, Pruessmann JN, Nasrallah MN, Seruggia D, Misale S, Zappulli V, Usmani SM, Carrizosa E, Mani V, Ligorio M, Warner RD, Medoff BD, Marangoni F, Villani AC, and Mempel TR
