Abstract 50P
Background
Anti-PD-1 therapy is one of the most promising cancer therapies, but the efficacy is limited in EGFR TKI-resistant NSCLC. Tr1 cells are FoxP3+ regulatory T cells secreting IL-10 and TGF-β, playing a pro-tumor role. However, the effect of Tr1 cells on the immune escape and anti-PD-1 efficacy in TKI-resistant NSCLC is unknown.
Methods
We employed five 24-color flow cytometry panels on PBMC from 53 NSCLC patients by Cytek Aurora for preliminary exploration. To validate findings in the tumor microenvironment, we established humanized NOG-EXL (hGM-CSF/hIL-3 NOG) mice using CD34+ hematopoietic stem cells (Charles River Laboratories). The subcutaneous xenografts of osimertinib-sensitive (H1975) and resistant cell lines (H1975-OR) were established and treated with osimertinib (p.o., 5mg/kg, q.d.) for 28 days. At the endpoint, tumors were collected, dissociated into single cells, and were enriched for immune cells by microbeads (Miltenyi Biotech). We performed scRNA-seq for the mixed tumor and immune cells with BD Rhapsody Single-Cell Analysis System.
Results
Tr1 cells are the only population in PBMC expressing a different level of PD-1 among all groups. There is an equivalent level of PD-1 on Tr1 cells in 1st or 3rd generation of TKI-resistant (21.16±2.01%; n=20) and EGFR MT (21.16±2.04%; n=17) groups, but significantly higher than that in EGFR WT patients (13.96±1.42%; n=15; all P<0.01). Moreover, the proportion of Tr1 cells in CD4+ T cells is highest in the TKI-resistant group (63.65±6.12%), followed by EGFR MT (36.13±4.58%; P<0.01) and EGFR WT (45.68±6.12%; P<0.001). scRNA-seq for humanized tumors showed the immune response and positive regulation of T cell activation (GO pathway) were inhibited in Tr1 cells from H1975-OR tumors. The interaction of macrophages with Tr1 cells was upregulated in H1975-OR tumors, especially via SPP1-CD44. SPP1 has been considered to suppress T cell activation and anti-PD-1 immunotherapy efficacy.
Conclusions
Tr1 cells in EGFR TKI-resistant hosts showed higher expression levels of PD-1 and more negative regulation of anti-tumor immunity, which may be attributed to additional SPP1 secreted by macrophages. The anti-PD-1 therapy in EGFR TKI-resistant NSCLC might activate more Tr1 cells than in EGFR WT NSCLC, resulting in poor efficacy.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Shanghai Innovative Collaboration Project (No. 2020CXJQ02).
Disclosure
All authors have declared no conflicts of interest.