PMC8733210_01
Peripheral blood (PB) CD16+ myeloid dendritic cell (mDC) enrichment of a patient with psoriasis and cutaneous squamous cell carcinoma (cSCC). (A) Protocol of intratumoral injection of autologous DC-enriched PB plus anti-PD-L1 antibody combined with local radiation. (B) Population of mDC and plasmacytoid DC (pDC) in PB before versus after DC enrichment. Blood cells were first gated on FSC-H versus SSC-H, single cells were gated on FSC-A versus FSC-H, and CD45+ leukocytes were gated for DC analyses in Lin- (CD3-CD14-CD19-CD20-CD56-) and HLA-DR+ cells. CD11c+ and CD123+ were then respectively used to identify mDCs and pDCs, with subsequent analyses of CD1c+, CD16+, and CD141+ mDC subpopulations. (C) Dimensionality reduction in multiparametric flow cytometry for pDCs, CD1c+ mDCs, CD16+ mDCs, and CD141+ mDCs in Lin-HLA-DR+ DC-enriched PB, assessed with a t-distributed stochastic neighbor embedding (t-SNE)-based algorithm. (D) Pooled data for frequencies of CD123+ pDCs and CD11c+ mDCs in the Lin-HLA-DR+ myeloid cells assessed through flow cytometry. ***P <0.001 using an unpaired t test. (E) Pooled data for frequencies of CD1c+, CD16+, or CD141+ cells in the Lin-HLA-DR+CD11c+mDC population of DC-enriched PB assessed using flow cytometry. ***P <0.001 using one-way analysis of variance with Tukey's multiple comparison test.
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The therapeutic schedule and regimens are presented in  Figure 1A . The blood DC compartment was identified as Lin-HLA-DR+ and increased approximately fourfold compared with the PBMC sample without leukapheresis ( Figure 1B ). Moreover, most DC subsets in the DC-enriched PB were CD11c+ mDCs (approximately 80%) instead of plasmacytoid DCs (pDCs; approximately 1%) ( Figures 1B, C , representative data;  Figure 1D , pooled data for the three replicates). In these DC-enriched PB samples, CD16+ mDCs were the major subset in both the overall DC population ( Figure 1C ) and mDC subpopulation, with approximately 80% of the subpopulation being CD16+ and only approximately 15% and less than 1% being CD1c+ and CD141+, respectively ( Figure 1B , representative data;  Figure 1E , pooled data for the three replicates).
Therapeutic effect of intratumoral immunotherapy combined with local radiation. (A) Primary lesion conditions assessed (a) pre-TRT status, (b) after the first TRT course, (c) after the second TRT course, and (d) after the whole TRT period. (B) The tumor mass shrank with remarkable SUVmax reduction of 42% on FDG positron emission tomography (PET)-Scan. Tumor sizes before any treatment (left panel) and after the complete TRT period (right panel) were assessed using PET.
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TRT ( Figure 2A ); the right inguinal mass only measured 5.0 cm x 4.1 cm in the axial dimension and 5 cm in craniocaudal distance compared with pre-TRT imaging. To ascertain the therapeutic effect on the progressive tumor, a PET scan was performed to analyze the tumor size before and after TRT, which indicated a dramatic postintervention change in FDG avidity over the right inguinal mass and right external iliac regions. There was a remarkable SUVmax reduction (approximately 42%) on FDG PET-Scan despite relatively low-dose DCs were available ( Figure 2B ).
TRT results in enrichment of local T-cell-related pathways and systemic antitumor immunity through Metascape analysis. (A) T-cell-related pathways were enriched in the gene ontology biological processes for tumors with TRT versus no treatment by P-values. (B) The upregulated pathways were composed of differentially expressed genes, presented as a network of enriched terms for peripheral blood with TRT versus no treatment. (C) Top 20 clusters of both innate and adaptive immunity-associated pathways were enriched in the gene ontology biology processes for PB with TRT versus no treatment by P-values.
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T-cell-related pathways, with over 10 highly significantly enriched pathways (P <0.00001) identified in postintervention biopsies. These pathways included the following: leukocyte chemotaxis, positive regulation of T-cell proliferation, T-cell-mediated immunity, alpha-beta T-cell activation, interferon-gamma production, lymphocyte chemotaxis, alpha-beta T-cell differentiation, regulation of T-cell-mediated immunity, T-cell differentiation involved in immune response, and T-helper cell differentiation. These data indicated that T-cell-mediated immunity was locally induced after intratumoral injection with CD16+ mDC-enriched cells and anti-PD-L1 therapy ( Figure 3A ). The network is visualized using Cytoscape5, where the activation of both innate and adaptive immunity was significantly enriched in postintervention PB ( Figure 3B , colored by cluster pathway;  Figure 3C , list of top 20 enriched pathways with highly significant P-values (P <0.00001) for PB with TRT versus no treatment).