Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells

BackgroundDendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence, activation of tumor-associated DCs may improve efficacy of cancer immunotherapies. Toll-like receptor (TLR) agonists and interferons are known to promote DC maturation. However, it is unclear if DCs in human tumors respond to activation signals and which stimuli induce the optimal activation of human tumor DCs.MethodsWe first screened combinations of TLR agonists, a STING agonist and interferons (IFNs) for their ability to activate human conventional DCs (cDCs). Two combinations: TL8-506 (a TLR8 agonist)+IFN-γ and TL8-506+Poly(I:C) (a TLR3 agonist) were studied in more detail. cDC1s and cDC2s derived from cord blood stem cells, blood or patient tumor samples were stimulated with either TL8-506+IFN-γ or TL8-506+Poly(I:C). Different activation markers were analyzed by ELISA, flow cytometry, NanoString nCounter Technology or single-cell RNA-sequencing. T cell activation and migration assays were performed to assess functional consequences of cDC activation.ResultsWe show that TL8-506 synergized with IFN-γ or Poly(I:C) to induce high expression of different chemokines and cytokines including interleukin (IL)-12p70 in human cord blood and blood cDC subsets in a combination-specific manner. Importantly, both combinations induced the activation of cDC subsets in patient tumor samples ex vivo. The expression of immunostimulatory genes important for anticancer responses including CD40, IFNB1, IFNL1, IL12A and IL12B were upregulated on stimulation. Furthermore, chemokines associated with CD8$^{+}$ T cell recruitment were induced in tumor-derived cDCs in response to TL8-506 combinations. In vitro activation and migration assays confirmed that stimulated cDCs induce T cell activation and migration.ConclusionsOur data suggest that cord blood-derived and blood-derived cDCs are a good surrogate to study treatment responses in human tumor cDCs. While most cDCs in human tumors display a non-activated phenotype, TL8-506 combinations drive human tumor cDCs towards an immunostimulatory phenotype associated with Th1 responses on stimulation. Hence, TL8-506-based combinations may be promising candidates to initiate or boost antitumor responses in patients with cancer

Supplementary Table S2 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Table S2. Specification of reagents for flow cytometry (Whole Blood Assay for single cell RNA-sequencing)</p

Supplementary Figure S3 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Figure S3. Identification of an interacting B and T cells cluster by scRNA-sequencing of human whole blood treated with 0.2 μg/mL CD20-TCB using the BD Rhapsody platform.</p

Supplementary Table S3 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Table S3. Specification of reagents for flow cytometry (HUVEC staining)</p

Supplementary Table S4 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Table S4. Specification of reagents for flow cytometry (In vivo work)</p

Supplementary Figure S11 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Figure S11. Single cell RNA-sequencing of whole blood treated with 0.2 μg/mL CD20-TCB was performed using the BD Rhapsody platform. A. UMAP plots of neutrophils colored by treatment. B. UMAP plots showing IL-1β (IL1B), IL-1Ra (IL1RN), IL-6 (IL6), IP-10 (CXCL10), IL-8 (CXCL8), TNF-α (TNF), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4) gene expression within neutrophil clusters identified above. C. Violin plots showing the kinetics of gene expression distribution in neutrophils. The cytokine and cytokine receptor genes are listed on the y axis. The color code on the right side of the violin plots represents the median gene expression. The untreated condition corresponds to the pooled baseline and untreated samples. N= 4 donors (baseline, and 20 hrs) and n=2 donors (2 hrs, 4 hrs and 6 hrs).</p

Supplementary Figure S5 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Figure S5. Overview of GO Biological process pathways found enriched in A. CD4+ T cells (6 hrs), B. CD8+ T cells (6 hrs), C. monocytes (6 hrs) and D. neutrophils (4 hrs) after treatment with 0.2 μg/mL CD20-TCB.</p

Supplementary Figure S8 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Figure S8. Single cell RNA-sequencing of whole blood treated with 0.2 μg/mL CD20-TCB was performed using the BD Rhapsody platform.</p

Supplementary Table S1 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Table S1. Specification of reagents for flow cytometry (Whole Blood Assay)</p

Supplementary Figure S6 from Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies

Supplementary Figure S6. A. Bulk-rna-sequencing of neutrophils purified from whole blood 4 hrs and 20 hrs following treatment with 0.2 μg/mL CD20-TCB or 0.2 μg/mL DP47-TCB for N=4 donors. B. Heat map showing the enriched hallmark pathways in neutrophils, 4 hrs and 20 hrs following treatment with CD20-TCB or DP47-TCB. C. Heat map showing the expression of TNF-α (TNF), IL-1β (IL1B), IL-6 (IL6), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4) and IP-10 (CXCL10) genes in neutrophils, 4 hrs and 20 hrs following treatment with CD20-TCB or DP47-TCB (untargeted TCB). Volcano plots depicting the cytokine genes in neutrophils D. 4 hrs and E. 20 hrs after treatment with CD20-TCB. Each dot represents one gene. The x axis shows the logarithmic fold change and the y axis shows the negative log of the p value for each gene by comparing samples treated with CD20-TCB to samples treated with DP47-TCB (untargeted TCB).</p