Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of A/J mice (n = 4–8/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from either WT C57BL/6 (WTB6) or B6.IL12p40–/– donors. The indicated recipients of WT allografts were treated with 200 μg anti–IL-12 p40 mAb i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant. The following day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (B) Groups of A/J mice (n = 4/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from WT C57BL/6 or B6.IL12p35–/– donors. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1. On day 2 after transplant, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05 as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 5–6/group) received C57BL/6 cardiac isografts or A/J cardiac allografts subjected to 0.5 or 8 hours of CIS. Grafts were harvested on day 2 after transplant, total RNA was isolated, and expression of the indicated cytokine mRNA was tested by qPCR. Results shown indicate relative expression vs. expression in naive hearts of A/J mice. **P < 0.01, as determined by the Mann-Whitney nonparametric test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Injection, Staining, Flow Cytometry, MANN-WHITNEY, Isolation, Expressing

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts or C57BL/6 isografts subjected to 0.5 or 8 hours of CIS. The indicated recipients of 8 hours of CIS allografts were treated with 200 μg control rat IgG or anti-CD4 mAb on days –3, –2, and –1 prior to transplant. On day 2 after transplant, serum and graft lysates were prepared and quantities of p40 homodimers and IL-12 p70 heterodimers were tested by ELISA. *P < 0.05, as determined by the Mann-Whitney nonparametric test. (B) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS and the indicated recipients were injected with 200 μg recombinant p40 homodimers i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant; the next day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and numbers of proliferating memory CD8+ T cells infiltrating the grafts. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 4–7/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS, and on days 0 and 1, were treated with 200 μg control rat IgG or 250 μg CTLA-4Ig i.p. with or without 200 μg recombinant p40 homodimers i.p. Allograft survival was monitored by daily abdominal palpation and rejection confirmed by laparotomy. ***P < 0.001 vs. all other groups, as determined using the Log-rank/Mantel-Cox test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Injection, Recombinant, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes

doi: 10.1038/s41467-023-38541-2

Figure Lengend Snippet: a A schematic demonstrated the principles of the BRET competition assay using IL12p40 as an example. Schematic created with Biorender.com. IL23R is shown in orange, IL12Rβ1 in purple, IL23p19 in blue, IL12p40 in green and the presence of a TAMRA fluorophore by a pink star. The competitive inhibition of the binding of 300 pM IL23-TMR by increasing concentrations of ( b ) endogenously produced IL-12p40 containing proteins and ( c ) synthetic IL-23 receptor antagonists with previously generated IL-23 competition data for comparison. d The combined data from b and c plotted without error bars. In b and c , data are mean ± SEM from 5 independent experiments conducted in either duplicate or triplicate. Details on absolute number of data points included for each concentration are provided in the associated Source Data file. K i values obtained in the 5 independent experiments are shown in Table .

Article Snippet: Recombinant HEK293T produced IL23p19-Fc (Sinobiological; Cat #: 13062-H02H) and IL-12 (Sinobiological; Cat #: CT050-HNAH), CHO produced IL12p40 (Peprotech; Cat #: 200-12p40), and BTI-Tn-5B1-4 produced IL12p80 (Merck; Cat #: SRP3075) were purchased from commercial suppliers as lyophilised solids which were re-constituted in PBS (Merck) with 1 mg/ml BSA (Merck).

Techniques: Competitive Binding Assay, Inhibition, Binding Assay, Produced, Generated, Concentration Assay

Journal: Nature Communications

Article Title: Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes

doi: 10.1038/s41467-023-38541-2

Figure Lengend Snippet: a The displacement of 75 nM P630-TMR by increasing concentrations of various IL-23 receptor binders. b The displacement of either 75 nM P630-TMR or 300 pM IL23-TMR by a single concentration of several antagonists. Data in a are mean ± SEM from three (TEEEQQLY), four (P630, IL12p40) or five (IL-23, IL12p80) independent experiments performed in triplicate. Data in b are mean ± SEM from five independent experiments performed in triplicate apart from 200 nM IL23p19 (4 experiments) and 500 nM IL12p40 with P630-TMR (4 experiments). In b statistical analysis (two-tailed, non-paired, t -test) was performed on the mean values obtained in each independent experiment. ** indicates a statistical significance of p < 0.01. *** indicates a statistical significance of p < 0.001. Absolute p values in b were 0.000001 (IL-12p40), 0.000002 (IL12p80), 0.0069 (IL12) and 0.74 (IL23p19). Source data are provided as a Source Data file.

Article Snippet: Recombinant HEK293T produced IL23p19-Fc (Sinobiological; Cat #: 13062-H02H) and IL-12 (Sinobiological; Cat #: CT050-HNAH), CHO produced IL12p40 (Peprotech; Cat #: 200-12p40), and BTI-Tn-5B1-4 produced IL12p80 (Merck; Cat #: SRP3075) were purchased from commercial suppliers as lyophilised solids which were re-constituted in PBS (Merck) with 1 mg/ml BSA (Merck).

Techniques: Concentration Assay, Two Tailed Test