Background
Currently, patients with high-risk neuroblastoma are treated with an intense multimodal regimen including chemotherapy, leading to severe side effects. Still, many patients relapse, which indicates the need for new treatment options. Immunotherapy represents a promising solution. However, neuroblastoma tumors exploit many immunosuppressive mechanisms to evade immune cell recognition. Targeting these immunosuppressive pathways may enhance tumor killing and increase patient survival.
Materials and Methods
To unveil T cell dynamics, (dys)functionality and immunosuppressive mechanisms, tumors from 3 patients at diagnosis, after chemotherapy and at relapse were dissociated into single cells and enriched for CD45+CD3+ T cells. Single-cell RNA and single-cell αβ and T-cell receptor sequencing were performed in parallel with the 10x platform. Data were analyzed in R (Seurat package) and validated with high-dimensional spectral flow cytometry.
Results
In tumors, clonally expanded tumor-infiltrating T lymphocytes are expected to be largely responsible for the anti-tumor response. Preliminary TCR sequencing data from matched tumor and blood before and after chemotherapy of n=3 high-risk neuroblastoma patients, revealed the presence of clonally expanded T cells in the tumor samples at diagnosis. Some of these expanded clones at diagnosis persisted throughout the course of chemotherapy and were found in the resected tumor as expanded clones. Many of the expanded clones in the tumors could be traced back in the blood of the same patients (as non-expanded clones), possibly suggesting T cell recirculation between the blood stream and the tumor site. Moreover, the same expanded clones present in the tumor were found in the adjacent normal adrenal gland tissue, suggesting that tumor-reactive T cells may reside in tumor-adjacent tissues, and that these tissues may serve as reservoir for tumor-reactive cells. Expanded clones were particularly enriched in the CD8+ central memory and terminally differentiated effector memory (TEMRA) subsets, suggesting tumor-reactivity of the CD8 T cell compartment. Functionally, the top expanded clones displayed high levels of the immune checkpoint molecule LAG-3 and of the immunosuppressive cytokine TGF-β, both at transcriptional and protein level, compared to non-expanded clones. Expression of these markers may be indicative of a dysfunctional/exhausted phenotype in the expanded tumor-reactive clones. Further in-depth characterization of expanded clones, and analysis of an additional n=7 patients, is currently ongoing.
Conclusions
These preliminary results give first insights into the dynamics of tumor-reactive T cells in neuroblastoma throughout the course of therapy. Our understanding of the dysfunctionality mechanisms specifically in the hyperexpanded, likely tumor-reactive, clones will enable us to target them, e.g. by immune checkpoint inhibition, to promote tumor killing. These findings will be at the base for the generation of new immunotherapies to implement in clinic with the final goal to increase survival of high-risk neuroblastoma patients.
E. Zappa: None. A. Boltjes: None. N. Hiddink Verberne: None. J.J. Molenaar: None. J. Wienke: None.