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M. Autio et al.
T-cell phenotypes. They also provide a rationale for test- ing combination treatment strategies in this context. Interestingly, in preclinical studies, use of a dual therapy with PD1 and TIM3 blockades has been demonstrated to improve efficacy in comparison to single agent PD1 targeted therapy in solid tumors and acute myeloid leukemia.37-40 A recent report further shows that combin- ing checkpoint inhibitors with chimeric antigen receptor T cells might also be potent in DLBCL.41
The focus of our study was to characterize tumor-infil- trating lymphocytes in DLBCL. However, we also identi- fied a population of TIM3+CD4+CD3– cells which had a significant adverse impact on the outcome of patients in both cohorts. Considering CD4 positivity, these cells are likely to represent monocytes/macrophages, dendritic cells, or NK cells.42-44 In fact, our in silico immunophenotyp- ing utilizing CIBERSORTx suggests that tumor infiltrating monocytes/macrophages express TIM3. However, the true identity of the TIM3+CD4+CD3– cells requires further validation.
We also identified a subgroup of patients characterized by a large proportion of tumor-infiltrating cytotoxic cells and Tregs. Data suggesting that a large proportion of cyto- toxic cells in the TME translates to inferior survival contra- dict the concept that cytotoxic T cells act as killers of tumor cells. However, previous studies have also reported an inferior effect of tumor-infiltrating cytotoxic cells on the outcome in Hodgkin’s lymphoma,45-47 and also in DLBCL.48 Oudejans et al.47 speculated that in tumors with a larger proportion of cytotoxic cells, malignant cells might be more resistant to cell-mediated killing, which would explain the relatively poorer outcome of these patients. This resistance might also reflect refractoriness of the disease to standard therapies as immune cells may partly mediate the effect of chemotherapy.49 In contrast to previous studies on different lymphoma entities,45,46,50,51 higher proportion of Tregs was associated with poor sur- vival in the NLG Trial cohort but this observation could not be validated in the HEL-DLBCL cohort. The conflict- ing results may be explained by heterogeneous patient populations, cytokines produced by Tregs and the com- plex interplay between the cells in the TME. Nevertheless, further research is needed to validate our findings and to
determine the role of cytotoxic cells and Tregs in the DLBCL TME.
In conclusion, our results demonstrate a novel adverse prognostic impact of immune checkpoint expressing T cells, especially TIM3+ T cells, on the survival of DLBCL patients in response to standard immunochemotherapy. Additional research on the effect of TME and checkpoint blockage on the outcome of DLBCL is warranted. It will be interesting to test whether a subset of patients with immune checkpoint positive T cells may be more likely to respond to PD1 inhi- bition, or if combination therapies with TIM3 and/or LAG3 inhibitors can further improve the outcome.
Disclosures
SM has received honoraria and research funding from Novartis, BMS and Pfizer (not related to this study); SL has received honoraria and research funding from Roche, Novartis, Celgene, Takeda, Bayer and Janssen-Cilag (not related to this study). Other authors have no conflicts of interest to disclose.
Contributions
MA and S-KL designed and conceived the study, analyzed data, and wrote the manuscript; OB participated in designing the mIHC analyses; SM provided guidance and support; JMJ, M-LK-L, KB and HH provided samples; TP designed and per- formed mIHC data analyses; SL designed and supervised the study and wrote the manuscript. All authors have read and approved the manuscript.
Acknowledgments
We thank the DNA Sequencing and Genomics Laboratory at the Institute of Biotechnology, University of Helsinki for the Nanostring analyses. Annabrita Schoonenberg (FIMM) is thanked for performing the mIHC stainings. We thank the Digital and Molecular Pathology Unit supported by Helsinki University and Biocenter Finland. Anne Aarnio and Marika Tuukkanen are acknowledged for technical assistance.
Funding
The study was supported by grants from the Academy of Finland (to SL), Finnish Cancer Foundation (to SL, SM), Juselius Foundation (to SL, SM), University of Helsinki (to SL, SM), and Helsinki University Hospital (to SL, SM).
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