Fig 2: In vitro CpG stimulation generates T cells with a signature phenotype which is maintained post ACT. (A) Design of proteomic analysis of T cells; vehicle or CpG-expanded T cells were collected on day 7 of culture (n=4 mice/group), subjected to protein extraction and purification, and then analyzed using LC-MS/MS. (B) Principle component analysis of vehicle or CpG-generated pmel-1 T cell proteomes. (C) Volcano plot comparing protein expression between vehicle and CpG-expanded T cells. (D) Top 5 GO: molecular functions enriched from proteins expressed more in CpG-generated pmel-1 T cells compared with vehicle-generated cells (using https://toppgene.cchmc.org/enrichment.jsp). (E) Representative flow cytometry histogram (left) and biological replicates (right) of extracellular expression markers from day 7 of cell culture of vehicle or CpG-treated pmel-1. (F) MFI of ICOS (top) or CD39 (bottom) of donor pmel-1 cells in the blood on day 4 or day 12 post ACT. (G) Percentage of human OCSCC TIL expressing IL-2Rα and ICOS, but not CD39 after expansion with CpG. (H) Diagram of CpG-generated T cell characteristics. Statistics: (C) significance threshold was set using two-sided t-test adjusted p value (<0.05 FDR) and a S0 parameter of 0.1. (E) Two-sample t-test, (F) Mann-Whitney U test. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ACT, adoptive T cell transfer; CoA, coenzyme A; FDR, false discovery rate; GO, gene ontology; ICOS, inducible T cell Ccstimulator; IL, interleukin; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MFI, mean fluorescence intensity; ns, not significant; OCSCC, oral cavity squamous cell carcinoma; TBI, total body irradiation; TIL, tumor infiltrating lymphocytes.

Fig 3: Colocalization of Cx3Cr1‐GFP with IBA1 and flow cytometric analysis of APCs. (A–C) Baseline expression of Cx3Cr1‐GFP (A) and IBA1 (B) by microglia in the CA3 region of the hippocampus 24 h after FS at P15. (D–F) Expression of Cx3Cr1‐GFP (D) or IBA1 (E) by microglia in the CA3 region of the hippocampus after FS at P15. (C, F) Cx3Cr1 and IBA1 are highly colocalized in both conditions. Scale bar = 50 μm. (G) A cytoflurogram of Cx3Cr1‐GFP with RFP‐labeled IBA1 cells 24 h after FS. In P15 control animals, the thresholded Manders’ coefficients for the fraction of Cx3Cr1‐GFP overlapping IBA1 (tM1) and for the fraction of IBA1 overlapping Cx3Cr1‐GFP (tM2) were 0.49 and 0.68, respectively. The Pearson correlation coefficient (Rr) was 0.67. In mice that experienced FS at P15, tM1 = 0.67, tM2 = 0.77, and Rr = 0.78. (H) Flow cytometric analysis of brain‐resident microglia versus brain‐infiltrating macrophage/monocytes 24 h after KA‐SE. The frequencies of microglia (red) and infiltrating macrophage/monocytes (blue) are shown for PBS controls (left column) and KA‐SE (right column). A combination of markers CD39/CD45/Ly6C was used to distinguish these two cell types. Microglia express high levels of CD39, but low levels of Ly6C and CD45 (CD39hi CD45lowLy6Clow), while macrophage/monocytes are CD39int CD45hiLy6Chi. Microglia and macrophage/monocytes were gated on forward scatter/side scatter (FSC/SSC) appropriate for antigen presenting cells that were live and CD11b+/ CD11c−/ CD3−/Ly6G−/ B220−.

Fig 4: Schematic view of the proposed mechanism of action of EVT-701 as modulator of the immune checkpoints PD-L1 and CD39 membrane levels.

Fig 5: EVT-701 decreases the immune checkpoint expression to boost the immune response and increase the therapeutic benefit. (a) Volcano plot showing GSEA results in the GSE97631 data set containing the transcriptomic profiles of residual AML in PDXs AraC or vehicle-treated. Immune modulation-related gene signatures are in purple. In the middle, a plot showing AraC vs. PBS-treated PDXs differentially expressed gene sets with their normalized enrichment scores (NES). On the right, the enrichment plot of the hallmark Interferon-γ gene signature compatible with PD-L1 increases after AraC in vivo. (b) Enrichment plots of the hallmark Interferon-γ gene signature (left), KEGG purine metabolism-containing CD39 (middle) and Reactome PD-1 signaling (right) in the short group survival of the TCGA cohort. (c) Plot showing CD39- and PD-L1-related signatures in the short group of the TCGA cohort with their normalized enrichment scores (NES). (d) FACS assessment of the modulation of inducible PD-L1 and CD39 membrane expression on THP1 and MOLM13 cells by EVT-701, AraC or a combination of both (n > 10). (e) Enrichment plot of the hallmark Interferon-γ gene signature in C1498-resistant vs. -sensitive cell transcriptomes. (f) FACs assessment of the modulation of inducible and CD39 membrane expression on C1498 (n = 7) and L1210 (n > 10) by EVT-701, AraC or a combination of both at 72 h. (g) Effect of EVT-701, AraC or a combination of both on the NAD+/NADH ratio in L1210 at 72 h (n = 4). (h) Effect of exogenous NAD+ on the modulation of PD-L1 and CD39 membrane levels by EVT-701 or with IACS (n = 6). * is p < 0.05, ** is p < 0.01, *** is p < 0.005, **** is p < 0.001, ns is statistically non-significant.