Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Anti-melanoma effect of total body irradiation and interleukin-2 treatment in adoptive T cell therapy. ( A ) Schematic drawing of the experiment. Rag1 knock-out mice were subcutaneously inoculated with B16-F10 melanoma and treated with activated Pmel-1 as a form of adoptive T cell therapy. Pmel-1 stimulated for 2 days was administered into the mice on day 5. On day 3, some mice were exposed to 4 Gy total body irradiation (TBI). The interleukin-2 (IL-2) treatment group was injected daily (intraperitoneally) with 10,000 IU IL-2 on day 5 to day 7. ( B ) Tumor growth rate measured for 100 days. Each symbol and error bar indicate the mean and standard error of the mean (s.e.m.) of the tumor size in the same group. ( C ) Tumor growth rate of each mouse is indicated. ( D ) Kaplan–Meier curves showing the survival rate of the mice. ( E ) Representative images of the surviving mice in the TBI + Pmel-1 and TBI + Pmel-1 + IL-2 groups on day 80. Arrows indicate the tumor inoculation sites. UnTx (untreated) group, n = 7 mice; Pmel-1 and TBI + Pmel-1 groups, n = 5 mice per group; Pmel-1 + IL-2 and TBI + Pmel-1 + IL-2 groups, n = 6 mice per group. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Knock-Out, Injection

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of adoptively transferred T cells in lymphoid tissues. ( A ) Schematic drawing of the experiment. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, the inguinal tumor-draining lymph nodes (TdLNs) and spleen were collected and analyzed. ( B ) Total count of the collected cells in the TdLNs and spleen. ( C ) Gating strategy of flow cytometry analysis. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the FSC-A/FSC-H and SSC-A/SSC-H plots prior to the analyses of relevant markers. ( D ) Representative flow cytometry images showing Pmel-1 in the TdLNs and spleen. ( E ) Calculated frequency (left) and cell count (right) of Pmel-1 in lymphoid tissues. Pmel-1 and Pmel-1 + IL-2 groups, n = 3 mice per group; TBI + Pmel-1 and TBI + Pmel-1 + IL-2 groups, n = 5 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Flow Cytometry, Cell Counting, Standard Deviation

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of adoptively transferred T cells in tumor tissues. ( A ) Schematic drawing of the experiment. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, the inoculated tumor tissue was dissociated into a single cell suspension and analyzed using flow cytometry. ( B ) Gating strategy of flow cytometry analysis. ( C ) Representative flow cytometry images showing tumor-infiltrating Pmel-1. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the SSC-A/SSC-H and FSC-A/FSC-H plots prior to the analyses of relevant markers. ( D ) Determined frequency of Pmel-1 in tumor tissue. ( E ) Representative flow cytometry images showing PD-1 expression on Pmel-1. ( F ) Determined frequency of PD-1 + cells within the Pmel-1 population. Pmel-1 and Pmel-1 + IL-2 groups, n = 3 mice per group; TBI + Pmel-1 and TBI + Pmel-1 + IL-2 groups, n = 4 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Suspension, Flow Cytometry, Expressing, Standard Deviation

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of the immune cell population in the spleen after adoptive T cell therapy. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, the spleen was collected and subjected to multi-parameter flow cytometry analysis. ( A ) Gating strategy of the analysis. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the SSC-A/SSC-H and FSC-A/FSC-H plots prior to the analyses of relevant markers. First, Pmel-1 (CD8b + /Thy1.1 + ) and natural killer cells (NK; NK1.1 + ) were defined among the singlets. Myeloid cells (CD11c + and/or CD11b + ) were divided into macrophages (F4/80 + ), neutrophils (Ly6G + ), and other cells (double negative) in the Ly6G/F4/80 plot. After dendritic cells (DCs; CD11c + /MHC-II + ) were excluded from the double negative subset, monocytes (Ly6C + /CD11b + ) were defined. Types 1 and 2 macrophages (M1 and M2) were gated in the CD206/MHC-II plot. ( B ) Frequency of diverse immune cell subsets in the spleen. The frequency of Pmel-1, NKs, neutrophils, macrophages, DCs, and monocytes among lineage marker-positive cells (Lin + ; positive for Thy1.1, NK1.1, CD11b, and/or CD11c) was calculated. For M1 and M2, the ratio is indicated. ( C ) Representative flow cytometry images showing conventional types 1 and 2 DCs (cDC1 and cDC2) within the DC population. ( D ) Calculated ratio between cDC1 and cDC2. Pmel-1 + IL-2 and TBI + Pmel-1 groups, n = 3 mice per group; TBI + Pmel-1 + IL-2 group, n = 4 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Flow Cytometry, Marker, Standard Deviation

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of the immune cell population in the tumor after adoptive T cell therapy. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, tumor tissue was collected and subjected to multi-parameter flow cytometry analysis. ( A ) Gating strategy of the analysis. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the SSC-A/SSC-H and FSC-A/FSC-H plots prior to the analyses of relevant markers. First, Pmel-1 (CD8b + /Thy1.1 + ) and natural killer cells (NK; NK1.1 + ) were defined among the singlets. Dendritic cells (DCs; CD11c+/MHC-II+) were defined among CD11c- and/or CD11b-expressing myeloid cells. Monocytic myeloid-derived suppressor cells (Mo-MDSCs) and polymorphonuclear MDSC (PMN-MDSC) were gated in the Ly6G/Ly6C plot. ( B ) Frequency of diverse immune cell subsets in the tumor. The frequency of Pmel-1, NKs, DCs, monocytic myeloid-derived suppressor cells (Mo-MDSCs), and polymorphonuclear MDSC (PMN-MDSC) among lineage marker-positive cells (Lin + ; positive for Thy1.1, NK1.1, CD11b, and/or CD11c) was calculated. For Pmel-1, the PD-1-positive proportion is additionally indicated. ( C) Representative flow cytometry images showing monocyte-derived DCs (MoDCs) and conventional types 1 and 2 DCs (cDC1 and cDC2) within the DC population. ( D ) The frequency of cDC1, cDC2, and MoDC is indicated. Pmel-1 + IL-2 and TBI + Pmel-1 groups, n = 3 mice per group; TBI + Pmel-1 + IL-2 group, n = 4 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Flow Cytometry, Expressing, Derivative Assay, Marker, Standard Deviation