Journal: Scientific Reports

Article Title: Targeting of specific neuronal types in the non-human primate brain by using a murine CD25-specific recombinant immunotoxin

doi: 10.1038/s41598-026-39662-6

Figure Lengend Snippet: Production of mCD25-specific recombinant monoclonal antibodies. ( a ) Strategy for screening rabbit monoclonal antibodies that specifically react to mCD25 by ISAAC technology. A rabbit was immunized with purified mCD25/His-tag protein, and IgG + cells were isolated from peripheral blood lymphocytes, arrayed on the microarray chip, and cultured to trap secreted IgG. The chip was blocked with hCD25/His-tag protein, and then labelled with biotinylated mCD25/His-tag protein, followed by addition of Cy3-conjugated streptavidin. The cells producing mCD25-specific antibodies were visualized under a fluorescence microscope and collected from individual wells. The mRNA samples were extracted from single cell populations and subjected to reverse transcription (RT), and cDNA fragments encoding V H and V L regions were amplified by PCR, which were then cloned into the expression vectors containing rabbit immunoglobulin Y and K chains. The recombinant antibodies were produced in cultured cells and secreted into the medium. ( b ) Binding property of an mCD25-specific recombinant antibody to target proteins. Microplates were coated with either mCD25/His-tag or hCD25/His-tag, and then various concentrations of RMAb-52 were added to the wells. The binding of antibody was detected using a secondary antibody conjugated to alkaline phosphatase. ( c ) Competitive binding of antigen for the mCD25-specific recombinant antibody. RMAb-52 solution was incubated with various concentrations of mCD25/His-tag. Microplates were coated with mCD25/His-tag, and then the incubated mixture at equilibrium was added to the wells. The binding of free antibody was detected using a secondary antibody conjugated to alkaline phosphatase. The K D value was determined by using Scatchard plots. Data are expressed as mean ± SEM of four independent experiments. Individual data are overlaid in ( c ).

Article Snippet: The chip was incubated with 10 μg/mL hCD25/His-tag (Sino Biological Inc., Cat#10165-H08H) for 30 min as a blocking step, and then with 10 μg/mL biotinylated mCD25/His-tag for 30 min, followed by addition of Cy3-conjugated streptavidin (Sigma-Aldrich, Cat#S6402) for 30 min.

Techniques: Recombinant, Bioprocessing, Purification, Isolation, Microarray, Cell Culture, Fluorescence, Microscopy, Single Cell, Reverse Transcription, Amplification, Clone Assay, Expressing, Produced, Binding Assay, Incubation

Journal: Scientific Reports

Article Title: Targeting of specific neuronal types in the non-human primate brain by using a murine CD25-specific recombinant immunotoxin

doi: 10.1038/s41598-026-39662-6

Figure Lengend Snippet: Properties of anti-mCD25-PE38 ITX. ( a ) Binding property of the recombinant ITX for target proteins. Microplates were coated with either mCD25/His-tag or hCD25/His-tag, and then various concentrations of ITX protein were added to the wells. The binding of ITX was detected using a PE38-specific monoclonal antibody and a mouse IgG-specific secondary antibody conjugated to horseradish peroxidase. ( b ) Competitive binding of antigen for the recombinant ITX. ITX solution was incubated with various concentrations of mCD25/His-tag protein. Microplates were coated with mCD25/His-tag, and then the incubated mixture at equilibrium was added to the wells. The binding of free ITX was detected using a PE38-specific monoclonal antibody and a mouse IgG-specific secondary antibody conjugated to horseradish peroxidase. The K D value was determined using Scatchard plots. ( c ) Cytotoxic activity of the recombinant ITX toward HT-2 and EL-4 cells expressing mCD25 and hCD25, respectively. Cells were incubated with various concentrations of the ITX. Viable cell number was evaluated by a cell viability assay using WST reagent. Relative ratios to the average of control cell number without the ITX were calculated. Data are expressed as mean ± SEM of four independent experiments. Individual data are overlaid in ( b ).

Article Snippet: The chip was incubated with 10 μg/mL hCD25/His-tag (Sino Biological Inc., Cat#10165-H08H) for 30 min as a blocking step, and then with 10 μg/mL biotinylated mCD25/His-tag for 30 min, followed by addition of Cy3-conjugated streptavidin (Sigma-Aldrich, Cat#S6402) for 30 min.

Techniques: Binding Assay, Recombinant, Incubation, Activity Assay, Expressing, Viability Assay, Control

Journal: Cell

Article Title: Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses

doi: 10.1016/j.cell.2025.11.023

Figure Lengend Snippet: (A) Violin plots of Il2ra and Gpr183 in DC clusters from . (B) Proportion of Il2ra - and Gpr183 -expressing cells from RNA-sequencing data. (C) Surface expression of CD25 on DC subsets, represented as fold-change over baseline expression (0 hours, h). (D) Soluble CD25 produced in vitro . DCs isolated post-injection of eLNP or PBS. (E) Soluble CD25 produced in vivo from dLN extracts post eLNP injection. (F) Representative flow cytometry of Tfh and GC B cells. (G) Tfh and GC B cell frequency and absolute numbers, as detailed in F . (H) Representative microscopy images of dLN. The dashed line represents a T-B border. Scale bar, 25 μm. (I) Magnified T-B border from H . Scale bar, 15 μm. (J) qPCR analysis in dLN extracts after eLNP injection. Data is displayed as fold change versus naïve mice. (K) Cell migration in response to lipid extracts from dLNs of mice injected with PBS or eLNP. Dotted line denotes average migration to 7α,25-HC. (L) Representative flow cytometry of Tfh cells. (M) Quantification of Tfh cell frequency and number from L . Mice received 20 μg of eGFP mRNA-LNP-DiI ( C ), eLNP or PBS ( D-E and K ), 3 μg of Spikevax ( F-G ), 30 μg of HA mRNA-LNP ( H-I ), or 30 μg of RBD mRNA-LNP ( J and L-M ). ( C and J ) Two-way ANOVA was performed with Tukey’s correction for multiple comparisons. Only sample groups were compared versus all time points. ( D and E ) Kruskal-Wallis test was performed with Dunn’s correction for multiple comparisons. ( G, K, and M ) An unpaired two-tailed Mann-Whitney U test was conducted. In all experiments, n = 2-12 mice per group from 2-3 independent experiments.

Article Snippet: C57BL6/J, Cd11c-cre -GFP (C57BL/6J-Tg( Itgax-cre ,-EGFP)4097Ach/J), Ifnar flox/flox (B6(Cg)- Ifnar1 tm1.1Ees /J), Tlr2 −/− (B6.129- Tlr2 tm1Kir /J), Tlr3 −/− (B6;129S1- Tlr3 tm1Flv /J), B6129SF2/J, Tlr4 −/− (B6(Cg)- Tlr4 tm1.2Karp /J), Tlr7 −/− (B6.129S1- Tlr7 tm1Flv /J), Myd88 −/− (B6.129P2(SJL)- Myd88 tm1.1Defr /J), Mavs −/− (B6;129- Mavs tm1Zjc /J), Rigi −/− (C57BL/6NJ- Rigi em1(IMPC)J /Mmjax), and STING GT (C57BL/6J- Sting1 gt /J), Il2ra flox/flox (B6(129S4)- Il2ra tm1c(EUCOMM)Wtsi /TrmaJ), Cd11c-cre (B6.Cg.Tg( Itgax - cre )1-1Reiz/J) were purchased from The Jackson Laboratory.

Techniques: Expressing, RNA Sequencing, Produced, In Vitro, Isolation, Injection, In Vivo, Flow Cytometry, Microscopy, Migration, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses

doi: 10.1016/j.cell.2025.11.023

Figure Lengend Snippet: (A) Experimental design of panels B-F . (B and C) CD25 (B) and Cxcr5 (C) expression in DiI − eGFP − , DiI + eGFP − , and DiI + eGFP + cDC2s, 24 hours post-immunization. (D) Representative LNP binding/uptake (DiI + ) and mRNA-encoding protein expression (eGFP + ) in cDC1s, cDC2s, or LCs from dLN. (E) Quantification of D . Color represents the portion of cells that are DiI + eGFP + (green), DiI + eGFP − (red), and DiI − eGFP − (blue). (F) Frequency of DiI + eGFP + cDC1s and cDC2s. (G) Quantification of binding/uptake and expression of eGFP mRNA-LNP-DiI in human cells in vitro . In ( B-F ), mice were injected with 20 μg of eGFP mRNA-LNP-DiI. For ( B-G ), n = 6 mice/donors per time point from two independent experiments. In ( B ) and ( C ), a one-tailed paired Wilcoxon test was performed. In ( F ), an unpaired Mann-Whitney U test was performed with a two-stage Benjamini, Krieger, and Yekutieli FDR of 1% to correct for multiple comparisons.

Article Snippet: C57BL6/J, Cd11c-cre -GFP (C57BL/6J-Tg( Itgax-cre ,-EGFP)4097Ach/J), Ifnar flox/flox (B6(Cg)- Ifnar1 tm1.1Ees /J), Tlr2 −/− (B6.129- Tlr2 tm1Kir /J), Tlr3 −/− (B6;129S1- Tlr3 tm1Flv /J), B6129SF2/J, Tlr4 −/− (B6(Cg)- Tlr4 tm1.2Karp /J), Tlr7 −/− (B6.129S1- Tlr7 tm1Flv /J), Myd88 −/− (B6.129P2(SJL)- Myd88 tm1.1Defr /J), Mavs −/− (B6;129- Mavs tm1Zjc /J), Rigi −/− (C57BL/6NJ- Rigi em1(IMPC)J /Mmjax), and STING GT (C57BL/6J- Sting1 gt /J), Il2ra flox/flox (B6(129S4)- Il2ra tm1c(EUCOMM)Wtsi /TrmaJ), Cd11c-cre (B6.Cg.Tg( Itgax - cre )1-1Reiz/J) were purchased from The Jackson Laboratory.

Techniques: Expressing, Binding Assay, In Vitro, Injection, One-tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses

doi: 10.1016/j.cell.2025.11.023

Figure Lengend Snippet: (A) Violin plots of Il2ra and Gpr183 in DC clusters from . (B) Proportion of Il2ra - and Gpr183 -expressing cells from RNA-sequencing data. (C) Surface expression of CD25 on DC subsets, represented as fold-change over baseline expression (0 hours, h). (D) Soluble CD25 produced in vitro . DCs isolated post-injection of eLNP or PBS. (E) Soluble CD25 produced in vivo from dLN extracts post eLNP injection. (F) Representative flow cytometry of Tfh and GC B cells. (G) Tfh and GC B cell frequency and absolute numbers, as detailed in F . (H) Representative microscopy images of dLN. The dashed line represents a T-B border. Scale bar, 25 μm. (I) Magnified T-B border from H . Scale bar, 15 μm. (J) qPCR analysis in dLN extracts after eLNP injection. Data is displayed as fold change versus naïve mice. (K) Cell migration in response to lipid extracts from dLNs of mice injected with PBS or eLNP. Dotted line denotes average migration to 7α,25-HC. (L) Representative flow cytometry of Tfh cells. (M) Quantification of Tfh cell frequency and number from L . Mice received 20 μg of eGFP mRNA-LNP-DiI ( C ), eLNP or PBS ( D-E and K ), 3 μg of Spikevax ( F-G ), 30 μg of HA mRNA-LNP ( H-I ), or 30 μg of RBD mRNA-LNP ( J and L-M ). ( C and J ) Two-way ANOVA was performed with Tukey’s correction for multiple comparisons. Only sample groups were compared versus all time points. ( D and E ) Kruskal-Wallis test was performed with Dunn’s correction for multiple comparisons. ( G, K, and M ) An unpaired two-tailed Mann-Whitney U test was conducted. In all experiments, n = 2-12 mice per group from 2-3 independent experiments.

Article Snippet: Il2ra flox/flox mice : B6(129S4)- Il2ra tm1c(EUCOMM)Wtsi /TrmaJ , The Jackson Laboratory , Cat#033093.

Techniques: Expressing, RNA Sequencing, Produced, In Vitro, Isolation, Injection, In Vivo, Flow Cytometry, Microscopy, Migration, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: Distinct components of mRNA vaccines cooperate to instruct efficient germinal center responses

doi: 10.1016/j.cell.2025.11.023

Figure Lengend Snippet: (A) Experimental design of panels B-F . (B and C) CD25 (B) and Cxcr5 (C) expression in DiI − eGFP − , DiI + eGFP − , and DiI + eGFP + cDC2s, 24 hours post-immunization. (D) Representative LNP binding/uptake (DiI + ) and mRNA-encoding protein expression (eGFP + ) in cDC1s, cDC2s, or LCs from dLN. (E) Quantification of D . Color represents the portion of cells that are DiI + eGFP + (green), DiI + eGFP − (red), and DiI − eGFP − (blue). (F) Frequency of DiI + eGFP + cDC1s and cDC2s. (G) Quantification of binding/uptake and expression of eGFP mRNA-LNP-DiI in human cells in vitro . In ( B-F ), mice were injected with 20 μg of eGFP mRNA-LNP-DiI. For ( B-G ), n = 6 mice/donors per time point from two independent experiments. In ( B ) and ( C ), a one-tailed paired Wilcoxon test was performed. In ( F ), an unpaired Mann-Whitney U test was performed with a two-stage Benjamini, Krieger, and Yekutieli FDR of 1% to correct for multiple comparisons.

Article Snippet: Il2ra flox/flox mice : B6(129S4)- Il2ra tm1c(EUCOMM)Wtsi /TrmaJ , The Jackson Laboratory , Cat#033093.

Techniques: Expressing, Binding Assay, In Vitro, Injection, One-tailed Test, MANN-WHITNEY