Journal: RSC Chemical Biology

Article Title: Co-delivery of antigen and adjuvant by site-specific conjugation to dendritic cell-targeted Fab fragments potentiates T cell responses †

doi: 10.1039/d5cb00014a

Figure Lengend Snippet: Ex vivo analysis of conjugates. (A) Competitive binding assay of conjugates against parental mAb (NLDC-145). Data ( n = 3 for aDEC205 conjugates, n = 2 for non-targeting conjugates) are shown as mean fluorescence intensity ± SD normalized to highest signal intensity with a one site - fit log IC 50 least squares fit curve. (B) Schematic overview of ex vivo T cell activation assay. In short, Flt3L-BMDCs were generated and pulsed for 2 h with 10 nM conjugate. Sequentially, the BMDCs were washed and a co-culture of BMDCs and OT-I cells (1 : 5 ratio) was set-up. After 3 days, OT-I cells were analyzed using FACS and cytokines in the supernatant were assessed via ELISA. (C)–(E) Flow cytometry analysis of OT-I cells. Data ( n = 6, technical duplicates) are depicted as division index (C) and as mean fluorescence intensity normalized to positive control ± SD for CD25 (D) and 4-1BB (E). Statistical significance using one-way ANOVA with Sidak's multiple comparison correction is depicted as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P > 0.05. (F)–(H) ELISA analysis ( n = 6, technical duplicates) of IL-2 (F), TNFα (G), and IFNγ (H). Data is depicted as mean ± SD. Statistical significance using one-way ANOVA with Sidak's multiple comparison correction is depicted as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P > 0.05.

Article Snippet: Manufacturer's protocol was followed for mIL-2 (Invitrogen IL-2 Mouse Uncoated ELISA kit, 88-7024-88, ThermoFisher), mIFNγ (Invitrogen IFN gamma Mouse Uncoated ELISA kit, 88-7314-88, ThermoFisher) and mTNFα (Invitrogen mouse TNF alpha Uncoated ELISA Kit, 88-7324-88, ThermoFisher).

Techniques: Ex Vivo, Competitive Binding Assay, Fluorescence, Activation Assay, Generated, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Positive Control, Comparison

Journal: RSC Chemical Biology

Article Title: Co-delivery of antigen and adjuvant by site-specific conjugation to dendritic cell-targeted Fab fragments potentiates T cell responses †

doi: 10.1039/d5cb00014a

Figure Lengend Snippet: Ex vivo and in vivo activation assays for targeted adjuvant. (A)–(C) Flow cytometry analysis of Flt3L BMDCs pulsed overnight with 1 μM adjuvant. Data ( n = 4, 2 donors) are depicted as mean fluorescence intensity ± SD for CD40 (A), CD80 (B), and MHC II (C). Statistical significance using one-way ANOVA with Tukey's multiple comparison correction is depicted as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P > 0.05. (D) TNFα ELISA analysis of Flt3L BMDCs pulsed overnight at 1 μM adjuvant. Data ( n = 4, 2 donors) are depicted as mean ± SD. Statistical significance using one-way ANOVA with Tukey's multiple comparison correction is depicted as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P > 0.05. (E) Schematic representation of in vivo assay. In short, 8–12 week old female wildtype C57BL/6j mice received 1 × 10 6 CTV-labeled OT-I cells and the following day were injected (i.v.) with the vaccine conjugates. 3 days later, inguinal lymph nodes and spleen were harvested and analyzed using flow cytometry. (F)–(H) Flow cytometry analysis of OT-I cells. Data ( n = 3) are depicted as division index ± SEM for OT-I cells isolated from spleen (F) and inguinal lymph node (G). Representative histograms for proliferation of OT-I cells in the spleen are shown (H). Statistical significance using one-way ANOVA with Sidak's multiple comparison correction is depicted as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P > 0.05.

Article Snippet: Manufacturer's protocol was followed for mIL-2 (Invitrogen IL-2 Mouse Uncoated ELISA kit, 88-7024-88, ThermoFisher), mIFNγ (Invitrogen IFN gamma Mouse Uncoated ELISA kit, 88-7314-88, ThermoFisher) and mTNFα (Invitrogen mouse TNF alpha Uncoated ELISA Kit, 88-7324-88, ThermoFisher).

Techniques: Ex Vivo, In Vivo, Activation Assay, Adjuvant, Flow Cytometry, Fluorescence, Comparison, Enzyme-linked Immunosorbent Assay, Labeling, Injection, Isolation