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Vaccine overcomes checkpoint inhibitor limitation
strated significantly decreased leukemia burden compared to that of controls as detected from the GFP signal via flow cytometry (Figure 3I and J).
Peripheral blood T cells were obtained from animals on day 14 after tumor challenge. Consistent with our previ- ous findings, intracellular flow cytometric analysis demonstrated a 4-fold increase in circulating tumor-specif- ic CD8+ T cells after treatment with the fusion vaccine and checkpoint blockade compared to the numbers in controls detected following ex vivo stimulation with autologous tumor lysate (Figure 3K and L).
H
Treatment with a dendritic cell/acute myeloid leukemia fusion vaccine and checkpoint inhibition
is protective upon re-challenge with acute myeloid leukemia
Having demonstrated that animals treated with the com- bination of vaccination and checkpoint blockade develop leukemia-specific immunity with a memory phenotype and durable disease response, we subsequently assessed whether combination vaccine and checkpoint inhibitor therapy provided long-term protection from disease re- challenge.
IJ
KL
Figure 3. Increase in tumor-specific T cells following combination treatment with the fusion vaccine and PD1/TIM3/RGMb blockade. C57BL/6J mice were treated as described in Figure 2. (A, B) On day 14 peripheral blood was collected and CD8+ T cells were assessed for intracellular interferon-gamma (IFNγ) expression using multichannel flow cytometry following exposure to autologous tumor lysate for 3 days. Results are presented as a summary of data from the five mice in each group (**P<0.05) (A) and representative dot plots (B). (C-E) In a similar independent experiment on day 17 after tumor challenge, the mice underwent bioluminescence imaging analysis (C). The mice were then euthanized; splenocytes were harvested and assessed for IFNγ expression using multichannel flow cytometry following expo- sure to autologous tumor lysate. Results are presented as a summary of data from the five mice in each group (***P<0.05) (D) and as representative dot plots (E). (F) Spleen-derived CD8+ T cells were also assessed for frequency of tumor antigen-specific T cells with multichannel flow cytometry using pentamer analysis. TIB-49 acute myeloid leukemia (AML) cells were confirmed to express survivin using intracellular flow cytometric analysis; unstained cells and cells incubated with an appro- priate isotype control were used as negative controls. Binding of APC-labeled multimeric MHC/survivin peptide complexes to T-cell receptors was examined to deter- mine the frequency of the survivin-specific T cells. (G, H) The frequency of survivin-specific T cells is presented in gated CD8+ T cells as a summary of data from the five mice in each group (***P<0.05) (G) and as representative dot plots (H). The frequency of cytomegalovirus-specific T cells was analyzed as a control. C57BL/6J mice were inoculated by tail vein injection with 200x103 primary syngeneic mutant IDH2 AML cells that were stably transduced with green fluorescence protein (GFP). Syngeneic DC/mIDH2 AML fusion cells were generated. The mice were then treated with either vaccine alone, anti-PD1/TIM3/RGMb or a combination of anti- PD1/TIM3/RGMb and the fusion vaccine. Some mice were treated with an appropriate isotype control as a negative control. (I, J) Disease burden was assessed in spleens of animals at day 36 after tumor challenge by detection of the GFP signal via flow cytometric analysis. Results are presented as a summary of data from the five mice in each group (***P<0.05) (I) and as representative histograms (J). On day 14 peripheral blood was collected and CD8+ T cells were assessed for intracel- lular IFNγ expression using multichannel flow cytometry following exposure to autologous tumor lysate for three days. Results are presented as (K) a summary of 5 analyzed mice (n=5; P<0.05) and (L) as representative dot plots.
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