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Tumor-associated macrophages in PTL
than TAMs, nor other TAM phenotypes correlated with survival. The findings highlight the specific roles of TAMs, TILs and PD-1-PD-L1 axis in regulating survival and ther- apy resistance in PTL.
mIHC is a novel technology enabling multi-parametric readout from a single tissue section. In our study, the simultaneous use of multiple markers is important in many ways. Firstly, while PD-L1 was found to be expressed both in TAMs and B cells including lymphoma cells, the prognostic impact of PD-L1 positivity was restricted to TAMs. Thus, the use of just one marker would not be able to detect the survival association. Secondly, the spatial relationships between TILs, TAMs and lymphoma cells are retained in our experimental strat- egy, allowing for a more precise appreciation of their bio- logical interactions. Thirdly, since mIHC was performed on all evaluable PTL tissue areas on the TMA, thereby providing an overall snapshot of the PTL microenviron- ment, we can avoid a bias of earlier observations focusing only on hot spot areas of immune cell counts using single marker immunohistochemistry. However, it should be noted that while the overall infiltration of PD-L1+ TAMs and PD-1+ TILs had a significant impact on survival, their functional statuses remain to be explored. Combining our panel with other multiplex panels for immunoregulatory molecules, such as FoxP3, LAG-3 or IDO-1 and IDO-2, may be useful in the evaluation of response to immunotherapy.
As described in a recent review article by Xu-Monette et al., the PD-L1 expression in the tumor microenvironment has not been previously well defined in B-cell lymphomas, and association with survival has not been demonstrat- ed.18 PD-1 is a protein, which is classically upregulated upon activation of T lymphocytes. Interaction between PD-1 and PD-L1 was previously thought to induce immune tolerance by leading T lymphocytes to apoptosis.26 Further studies have, however, revealed that the expression of PD-L1 on tumor cells can lead to immune escape, to T-cell exhaustion and a state of non- responsiveness, further enabling immune escape of the tumor cells.27-29 Moreover, in addition to binding to PD-1,
PD-L1 and PD-L2 can also bind to CD80/B7-1 (PD-L1)30,31
and RGMb (PD-L2),32 indicating that the PD-1 – PD-L1
pathway is much more complex than previously anticipat- ed.18
In addition to PD-L1, macrophages express PD-1.33,34 Recently, Gordon and coworkers showed that PD-1 expression by TAMs inhibits phagocytosis and tumor immunity.35 In addition, they demonstrated that blockade of PD-1 – PD-L1 interaction increases macrophage phago- cytosis, reduces tumor growth and lengthens survival in mouse models of colon cancer, suggesting the PD-1 – PD- L1 pathway has a significant role in TAM function and tumor survival.
Based on our findings, we suggest that the PD-1 - PD-L1 signaling between TAMs and TILs has clinical relevance in PTL. As PD-1 engagement on T cells to its ligands has been linked to decreased anti-tumor immunity, and early experience on PD-1 blockade in PTL has shown promising results,36 the association of high PD-L1+ TAM and PD-1+ T- cell count with favorable outcome in response to immunochemotherapy seems paradoxical. Yet, the inter- action of PD-L1+ TAMs and PD-1+ T cells might modify the tumor microenvironment in PTL, or otherwise pro- mote an anti-tumor immune response following immunochemotherapy.
In conclusion, we argue that high PD-L1+ TAM and PD-1+ T-cell counts correlate with each other and with favorable outcome in patients with PTL. Higher PD- L1+CD68+ TAM scores seem to protect the patients from progression and death, and identify a group of patients with favorable prognosis. Interestingly, apart from PD-L1+CD68+ TAMs, no association was found between other PD-L1+ cells or PD-L1– TAMs and survival. Together, the data demonstrate that the PD-1 - PD-L1 axis in PTL affects the survival of patients with PTL.
Acknowledgments
We thank Drs. Petri Auvinen and Lars Paulin (Institute of Biotechnology, University of Helsinki), Finland for the Nanostring analyses. Anne Aarnio and Marika Tuukkanen are acknowledged for technical assistance.
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