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A) Generation of monocyte-derived DCs from head and neck squamous cell carcinoma (HNSCC) patients. Plot on left shows representative flow cytometric staining of freshly isolated monocytes; right plot shows DC-like phenotype after 3 days of culture in medium containing GM-CSF and IL-4. Graph on right shows expression of key differentiation markers, symbols show results from different patient samples, bars show means. B) Co-incubation of HNSCC patient DCs with allogeneic iNKT cells from healthy donors results in formation of tightly-adhered iNKT-DC conjugates. Plot on left shows representative flow cytometric staining; graph on right shows percent of DCs in conjugates, symbols show results from different donors, bars show means. C) Conjugation of HNSCC patient DCs with iNKT cells leads to increased expression of key T cell activating ligands. Paired symbols show results for DCs alone vs. iNKT-DC conjugates from the same patient; points below dotted line did not have positive staining. P values by Wilcoxon matched pairs t-test. D) CFSE-labeled T cells from blood of <t>HPV16</t> + HNSCC patients were cultured in the indicated conditions in the presence or absence of recombinant HPV E7 protein. Graphs show percent of CD4 or CD8 T cells that had undergone cell division after 10 days. Pairings show +/− antigen conditions from the same experiment; P values calculated by Wilcoxon match pairs signed rank test. E) Plots showing flow cytometric analysis of CD8 + T cells from one representative experiment out of seven. T cells cultured with E7 antigen are shown in filled histograms (top percentages); cultures without E7 antigen shown by dotted lines (bottom percentages). F) Tetramer staining of T cells from HLA-A2 + patients. Plots on left show staining for CTV and HPV E7 peptide-loaded HLA-A2 tetramer after gating on live cells expressing CD3 and CD8β. Graph on right shows data from 3 HLA-A2 + patients on the percent tetramer positive of proliferated CD8 + T cells after 10 days of culture with DCs alone or iNKT-DCs in the absence (light symbols) or presence (dark symbols) of recombinant E7 antigen. P values by paired t-test.
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A) Generation of monocyte-derived DCs from head and neck squamous cell carcinoma (HNSCC) patients. Plot on left shows representative flow cytometric staining of freshly isolated monocytes; right plot shows DC-like phenotype after 3 days of culture in medium containing GM-CSF and IL-4. Graph on right shows expression of key differentiation markers, symbols show results from different patient samples, bars show means. B) Co-incubation of HNSCC patient DCs with allogeneic iNKT cells from healthy donors results in formation of tightly-adhered iNKT-DC conjugates. Plot on left shows representative flow cytometric staining; graph on right shows percent of DCs in conjugates, symbols show results from different donors, bars show means. C) Conjugation of HNSCC patient DCs with iNKT cells leads to increased expression of key T cell activating ligands. Paired symbols show results for DCs alone vs. iNKT-DC conjugates from the same patient; points below dotted line did not have positive staining. P values by Wilcoxon matched pairs t-test. D) CFSE-labeled T cells from blood of <t>HPV16</t> + HNSCC patients were cultured in the indicated conditions in the presence or absence of recombinant HPV E7 protein. Graphs show percent of CD4 or CD8 T cells that had undergone cell division after 10 days. Pairings show +/− antigen conditions from the same experiment; P values calculated by Wilcoxon match pairs signed rank test. E) Plots showing flow cytometric analysis of CD8 + T cells from one representative experiment out of seven. T cells cultured with E7 antigen are shown in filled histograms (top percentages); cultures without E7 antigen shown by dotted lines (bottom percentages). F) Tetramer staining of T cells from HLA-A2 + patients. Plots on left show staining for CTV and HPV E7 peptide-loaded HLA-A2 tetramer after gating on live cells expressing CD3 and CD8β. Graph on right shows data from 3 HLA-A2 + patients on the percent tetramer positive of proliferated CD8 + T cells after 10 days of culture with DCs alone or iNKT-DCs in the absence (light symbols) or presence (dark symbols) of recombinant E7 antigen. P values by paired t-test.
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A) Generation of monocyte-derived DCs from head and neck squamous cell carcinoma (HNSCC) patients. Plot on left shows representative flow cytometric staining of freshly isolated monocytes; right plot shows DC-like phenotype after 3 days of culture in medium containing GM-CSF and IL-4. Graph on right shows expression of key differentiation markers, symbols show results from different patient samples, bars show means. B) Co-incubation of HNSCC patient DCs with allogeneic iNKT cells from healthy donors results in formation of tightly-adhered iNKT-DC conjugates. Plot on left shows representative flow cytometric staining; graph on right shows percent of DCs in conjugates, symbols show results from different donors, bars show means. C) Conjugation of HNSCC patient DCs with iNKT cells leads to increased expression of key T cell activating ligands. Paired symbols show results for DCs alone vs. iNKT-DC conjugates from the same patient; points below dotted line did not have positive staining. P values by Wilcoxon matched pairs t-test. D) CFSE-labeled T cells from blood of <t>HPV16</t> + HNSCC patients were cultured in the indicated conditions in the presence or absence of recombinant HPV E7 protein. Graphs show percent of CD4 or CD8 T cells that had undergone cell division after 10 days. Pairings show +/− antigen conditions from the same experiment; P values calculated by Wilcoxon match pairs signed rank test. E) Plots showing flow cytometric analysis of CD8 + T cells from one representative experiment out of seven. T cells cultured with E7 antigen are shown in filled histograms (top percentages); cultures without E7 antigen shown by dotted lines (bottom percentages). F) Tetramer staining of T cells from HLA-A2 + patients. Plots on left show staining for CTV and HPV E7 peptide-loaded HLA-A2 tetramer after gating on live cells expressing CD3 and CD8β. Graph on right shows data from 3 HLA-A2 + patients on the percent tetramer positive of proliferated CD8 + T cells after 10 days of culture with DCs alone or iNKT-DCs in the absence (light symbols) or presence (dark symbols) of recombinant E7 antigen. P values by paired t-test.
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A) Generation of monocyte-derived DCs from head and neck squamous cell carcinoma (HNSCC) patients. Plot on left shows representative flow cytometric staining of freshly isolated monocytes; right plot shows DC-like phenotype after 3 days of culture in medium containing GM-CSF and IL-4. Graph on right shows expression of key differentiation markers, symbols show results from different patient samples, bars show means. B) Co-incubation of HNSCC patient DCs with allogeneic iNKT cells from healthy donors results in formation of tightly-adhered iNKT-DC conjugates. Plot on left shows representative flow cytometric staining; graph on right shows percent of DCs in conjugates, symbols show results from different donors, bars show means. C) Conjugation of HNSCC patient DCs with iNKT cells leads to increased expression of key T cell activating ligands. Paired symbols show results for DCs alone vs. iNKT-DC conjugates from the same patient; points below dotted line did not have positive staining. P values by Wilcoxon matched pairs t-test. D) CFSE-labeled T cells from blood of HPV16 + HNSCC patients were cultured in the indicated conditions in the presence or absence of recombinant HPV E7 protein. Graphs show percent of CD4 or CD8 T cells that had undergone cell division after 10 days. Pairings show +/− antigen conditions from the same experiment; P values calculated by Wilcoxon match pairs signed rank test. E) Plots showing flow cytometric analysis of CD8 + T cells from one representative experiment out of seven. T cells cultured with E7 antigen are shown in filled histograms (top percentages); cultures without E7 antigen shown by dotted lines (bottom percentages). F) Tetramer staining of T cells from HLA-A2 + patients. Plots on left show staining for CTV and HPV E7 peptide-loaded HLA-A2 tetramer after gating on live cells expressing CD3 and CD8β. Graph on right shows data from 3 HLA-A2 + patients on the percent tetramer positive of proliferated CD8 + T cells after 10 days of culture with DCs alone or iNKT-DCs in the absence (light symbols) or presence (dark symbols) of recombinant E7 antigen. P values by paired t-test.

Journal: Cancer immunology research

Article Title: CD8 + T cell antitumor immunity via human iNKT-DC conjugates

doi: 10.1158/2326-6066.CIR-25-0454

Figure Lengend Snippet: A) Generation of monocyte-derived DCs from head and neck squamous cell carcinoma (HNSCC) patients. Plot on left shows representative flow cytometric staining of freshly isolated monocytes; right plot shows DC-like phenotype after 3 days of culture in medium containing GM-CSF and IL-4. Graph on right shows expression of key differentiation markers, symbols show results from different patient samples, bars show means. B) Co-incubation of HNSCC patient DCs with allogeneic iNKT cells from healthy donors results in formation of tightly-adhered iNKT-DC conjugates. Plot on left shows representative flow cytometric staining; graph on right shows percent of DCs in conjugates, symbols show results from different donors, bars show means. C) Conjugation of HNSCC patient DCs with iNKT cells leads to increased expression of key T cell activating ligands. Paired symbols show results for DCs alone vs. iNKT-DC conjugates from the same patient; points below dotted line did not have positive staining. P values by Wilcoxon matched pairs t-test. D) CFSE-labeled T cells from blood of HPV16 + HNSCC patients were cultured in the indicated conditions in the presence or absence of recombinant HPV E7 protein. Graphs show percent of CD4 or CD8 T cells that had undergone cell division after 10 days. Pairings show +/− antigen conditions from the same experiment; P values calculated by Wilcoxon match pairs signed rank test. E) Plots showing flow cytometric analysis of CD8 + T cells from one representative experiment out of seven. T cells cultured with E7 antigen are shown in filled histograms (top percentages); cultures without E7 antigen shown by dotted lines (bottom percentages). F) Tetramer staining of T cells from HLA-A2 + patients. Plots on left show staining for CTV and HPV E7 peptide-loaded HLA-A2 tetramer after gating on live cells expressing CD3 and CD8β. Graph on right shows data from 3 HLA-A2 + patients on the percent tetramer positive of proliferated CD8 + T cells after 10 days of culture with DCs alone or iNKT-DCs in the absence (light symbols) or presence (dark symbols) of recombinant E7 antigen. P values by paired t-test.

Article Snippet: Antigen preparations used were inactivated EBV (equivalent to 2.5U EBV/1000 DC), or 5ng/ml dialyzed recombinant HPV16 E7 protein (Sino Biological Cat# 40965-V07E).

Techniques: Activation Assay, Derivative Assay, Staining, Isolation, Expressing, Incubation, Conjugation Assay, Labeling, Cell Culture, Recombinant