Figure 3.
Evaluation of in vitro immune response in primary human T cells and cytotoxic effect of CTLs on cancer cells. (A) Isolation of T cells and MoDCs from the peripheral blood of the same healthy donor. After treatment, MoDCs were cocultured with T cells for 5 days. (B) DC activation marker CD80 was analyzed on CD11c+ MoDCs after treatment with different hBCMA-mRNA cohorts along with irrelevant mRNA or mock-loaded LNP. (C-D) For the T-cell proliferation assay, naïve T cells were prestained with CFSE and cocultured for 5 days with pulsed MoDCs; then the percentage of CFSE-stained CD8+ T cells and CD4+ T cells was determined by flow cytometry. (E-F) The hBCMA-specific CD8+ T-cell percentage was assessed by hBCMA-specific tetramer staining using flow cytometry. The quantification of hBCMA-specific CD8+ T-cell percentage was determined after treatment with different hBCMA-mRNA LNPs, irrelevant mRNA, or mock-loaded LNP. (G-J) ELISpot analysis was performed with the treated MoDCs and T cells coculture to quantify the spot-forming CD8+ T cells for both TNF-α and IFN-γ, dual staining of the same well treated with control and hBCMA-mRNA cohorts. (K-L) CTLs were generated from the 5-day coculture of CD8+ T cells and treated MoDCs from the patient peripheral blood, and their cytotoxicity was evaluated against same patient bone marrow-derived CD138+ MM cells. For flow analysis, CD138+ MM cells were first gated with CD138-APC–positive population followed by MM cell death percentage evaluated by PI staining with respective quantifications. (M-N) CTLs generated from the 5-day cocultures of CD8+ T cells and treated MoDCs isolated from the peripheral blood of healthy donors and the cytotoxicity of CTLs were evaluated against bone marrow–derived CD138+ MM cells and CD138− stromal cells from patients with MM. (O) The cytotoxicity of these isolated CTLs was determined against CFSE-stained U266-WT and U266-BMCA-KO cell lines. The data are shown as mean ± SD from 3 independent experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. IFN-γ, interferon gamma; PI, propidium iodide; SFC, spot-forming cells; SSC, side scatter; TNF-α, tumor necrosis factor α.

Evaluation of in vitro immune response in primary human T cells and cytotoxic effect of CTLs on cancer cells. (A) Isolation of T cells and MoDCs from the peripheral blood of the same healthy donor. After treatment, MoDCs were cocultured with T cells for 5 days. (B) DC activation marker CD80 was analyzed on CD11c+ MoDCs after treatment with different hBCMA-mRNA cohorts along with irrelevant mRNA or mock-loaded LNP. (C-D) For the T-cell proliferation assay, naïve T cells were prestained with CFSE and cocultured for 5 days with pulsed MoDCs; then the percentage of CFSE-stained CD8+ T cells and CD4+ T cells was determined by flow cytometry. (E-F) The hBCMA-specific CD8+ T-cell percentage was assessed by hBCMA-specific tetramer staining using flow cytometry. The quantification of hBCMA-specific CD8+ T-cell percentage was determined after treatment with different hBCMA-mRNA LNPs, irrelevant mRNA, or mock-loaded LNP. (G-J) ELISpot analysis was performed with the treated MoDCs and T cells coculture to quantify the spot-forming CD8+ T cells for both TNF-α and IFN-γ, dual staining of the same well treated with control and hBCMA-mRNA cohorts. (K-L) CTLs were generated from the 5-day coculture of CD8+ T cells and treated MoDCs from the patient peripheral blood, and their cytotoxicity was evaluated against same patient bone marrow-derived CD138+ MM cells. For flow analysis, CD138+ MM cells were first gated with CD138-APC–positive population followed by MM cell death percentage evaluated by PI staining with respective quantifications. (M-N) CTLs generated from the 5-day cocultures of CD8+ T cells and treated MoDCs isolated from the peripheral blood of healthy donors and the cytotoxicity of CTLs were evaluated against bone marrow–derived CD138+ MM cells and CD138− stromal cells from patients with MM. (O) The cytotoxicity of these isolated CTLs was determined against CFSE-stained U266-WT and U266-BMCA-KO cell lines. The data are shown as mean ± SD from 3 independent experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. IFN-γ, interferon gamma; PI, propidium iodide; SFC, spot-forming cells; SSC, side scatter; TNF-α, tumor necrosis factor α.

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