Journal: bioRxiv

Article Title: Targeting Langerhans cells using a modular mannosylated nucleic acid-based vaccine platform

doi: 10.1101/2025.06.04.657560

Figure Lengend Snippet: Delivery of gp100 on mannosylated HJ scaffolds into moLCs for antigen presentation. A ) Reaction scheme for gp100-functionalization of Q2 and Q4 with NHS-DBCO ( 1 ), followed by SPAAC reaction to azide-modified ( pink ) Val-Cit-PAB-linked ( yellow) gp100 ( red ) ( 2 ). B ) Schematic representation of gp100-conjugated HJs employed for antigen presentation. C ) Visual representation of the antigen-presentation assay using moLCs incubated with gp100-conjugated HJs to facilitate receptor-mediated endocytosis ( I ), followed by release of gp100 in the lysosomes ( II ) and presentation of gp100 on HLA-A2 ( III ). Subsequently, moLCs were co-cultured with wildtype- or gp100 Jurkat cells to verify antigen presentation by T cell activation. D ) IL-2 concentration (pg/mL) in the co-culture media by ELISA. Results shown from four independent human donors were expressed by the mean ± SD. Statistical analysis was conducted by a one-way ANOVA followed by a Tukey’s post hoc test. E ) Antigen-presentation assay comparing HJ-3xTriMan-1xgp100 assembled with either a cleavable linker ( Val-Cit ) or a non-cleavable linker. Results shown from three independent human donors were expressed by the mean ± SD. Statistical analysis was conducted by a two-way t test. p-value * = <0.05, ** = <0.01, *** = <0.001, ****= <0.0001.

Article Snippet: Antigenic peptide gp100 154-162 (sequence: KTWGQYWQV, specificity: HLA-A*02:01, GENAXXON bioscience) was conjugated to the oligonucleotide strands in a 2-step one-pot reaction.

Techniques: Immunopeptidomics, Modification, Incubation, Cell Culture, Activation Assay, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Potentiating Cancer Immunotherapy Using an Oncolytic Virus

doi: 10.1038/mt.2010.98

Figure Lengend Snippet: Immunological features of oncolytic virus immune boosting. (a) Comparison of the numbers of DCT-specific, IFN-γ+ CD8+ T cells in the blood of tumor-bearing (TB, n = 7) and tumor-free (TF, n = 5) C57BL/6 mice at the peak of the response after VSV treatment. (b) Pooled data demonstrating the correlation between the magnitude of the anti-DCT response in the blood and survival. Data includes mice that were mock vaccinated (Ad-BHG, cross), Ad-hDCT vaccinated (open squares) or treated with the Ad-hDCT+VSV-hDCT combination (closed circles). After accounting for group, a unit increase in CD8 resulted in a 29.1% reduction in hazard of death (95% CI 7.2–29.5%), P = 0.0024, hazard ratio 0.809, 95% CI 0.705–0.928. Those three mice having the highest responses actually survived much longer than 100 days. The horizontal lines indicate the mean response achieved in tumor-free mice ± SEM (dashed lines). (c) Ad-hDCT vaccinated C57BL/6 mice bearing intracranial B16-F10 tumors were subsequently treated with PBS, VSV-GFP, or VSV-hDCT. Seven days later, tumors were collected and IFN-γ+ tumor-infiltrating lymphocytes responsive to DCT180–188 peptide were enumerated. (d) gp100-responsive CD8+ T cells were enumerated by intracellular cytokine staining (ICS) for IFN-γ demonstrating that combination therapy of TB animals induces epitope spreading. Limit of detection (LOD) is indicated. Ad, adenovirus; CI, confidence interval; DCT, dopachrome tautomerase; hDCT, human DCT; PBS, phosphate-buffered saline; IFN, interferon; VSV, vesicular stomatitis virus.

Article Snippet: The H-2K b -restricted epitope from the N protein of VSV (RGYVYQGL) and a D b -binding murine gp100 peptide (mgp100 25–33 ; EGSRNQDWL) were purchased from Biomer Technologies (Hayward, CA).

Techniques: Virus, Comparison, Staining, Saline

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