Page 112 - 2021_05-Haematologica-web

D. Stroopinsky et al.
transcriptome studies. Further details are provided in the Online Supplementary Methods.
Analysis of single-cell RNA sequencing data
RNA sequencing data were analyzed using standard statistical algorithms after quality control filtering and alignment to the ref- erence genome (mm10) to generate raw counts for transcripts from each cell type. The preprocessed raw count data were log normal- ized using the Seurat R package (version 3.0) for unsupervised and supervised analyses.22 The single-cell RNA sequencing data from samples from the control, vaccine and combination treatment groups were merged using Find Integration Anchors and integra- tion functions in Seurat to generate an integrated matrix of normal- ized data.23 The distribution of various cell-specific markers in a transcriptome profile was determined using the feature plot func- tion in the Seurat R package.24 Further details are provided in the Online Supplementary Methods.
Results
Treatment with checkpoint inhibitors alone is ineffective at preventing disease progression
The effect of checkpoint inhibition alone on AML engraftment, progression and survival was interrogated in an immunocompetent murine AML model using the TIB- 49 murine AML cell line. These leukemia cells originated spontaneously in a C57BL/6 mouse and grow aggressively in syngeneic models. As a means of monitoring disease bur- den, TIB-49 AML cells were genetically manipulated to express luciferase and mCherry (ImTIB-49) via lentiviral transduction and selection. C57BL/6J mice underwent retro-orbital inoculation with 50×103 ImTIB-49 AML cells. The mice were then treated with six doses of isotype con- trol, anti-PD1, anti-TIM3, anti-RGMb or a combination of
A
all three monoclonal antibodies starting 3 days after tumor challenge. The treatment doses were administered intraperitoneally every 3 days. Animals were monitored for disease bulk, by serial bioluminescence imaging, and for survival. Untreated animals rapidly developed AML with symptomatic disease requiring euthanasia. Treatment with single-agent anti-PD1, anti-TIM3 or anti-RGMb did not affect AML progression as compared to isotope control, with all animals euthanized by day 53 after tumor challenge whereas the combination of the three monoclonal antibod- ies modestly delayed the onset of demonstrable leukemia with mildly improved survival (Figure 1A and B).
Checkpoint inhibition in combination with dendritic cell/acute myeloid leukemia fusion vaccine leads to prolonged survival
We subsequently assessed the effect of checkpoint inhi- bition in conjunction with DC/AML fusion vaccination. Syngeneic DC/TIB-49 fusion cells were generated as described and confirmed to co-express both DC and tumor markers (Figure 2A). C57BL/6J mice underwent retro-orbital inoculation with 50×103 lmTIB cells. 24 h after tumor challenge, cohorts of mice were treated with a single dose of 100×103 syngeneic fusion cells, 6 doses of anti-PD1/TIM3/RGMb monoclonal antibodies adminis- tered IP every 3 days, or the combination of the fusion vaccine and either isotype control or anti- PD1/TIM3/RGMb monoclonal antibodies. Serial quantifi- cation of disease burden was assessed via biolumines- cence imaging analysis beginning approximately 1 month after tumor challenge. All control mice rapidly demon- strated evidence of AML engraftment, progressive disease by day 29 and required euthanasia by day 36 after the ini- tial tumor challenge (Figure 2B). Mice treated with anti- PD1/TIM3/RGMb monoclonal antibodies alone demon-
B
Figure 1. Checkpoint blockade does not significantly affect acute myeloid leukemia engraftment in vivo. C57BL/6J mice were retro- orbitally inoculated with 50x103 syngeneic TIB-49 acute myeloid leukemia cells that were stably transduced with luciferase/mCherry. The mice were then treated with six doses of 200 μg anti-PD1, anti-TIM3, or anti-RGMb or a combination (combo) of the three monoclonal antibodies using intraperitoneal injections every 3 days. Some mice were treated with appropriate isotype control as a negative control. To determine the progression of the leukemia, (A) bioluminescence imaging of each group of mice (n=5) was performed on days 17 and 21 after inoculation with the tumor and (B) the mice were followed for survival for 90 days. The results are shown in a Kaplan-Meier curve.
1332
haematologica | 2021; 106(5)