Phycoerythrin

For the adoptive transfer of B cells into μMT mice, splenic B cells from naïve WT or BAFFR^-/- were isolated by negative selection enrichment (STEMCELL Technologies, Vancouver, BC, Canada). Highly purified B cells were obtained by both WT and BAFFR^-/- mice, as 98% of B cells purified from naïve BAFFR^-/- mice were T1 B cells. 2 × 10⁷ purified total B cells from WT mice and T1 B cells from BAFFR^-/- mice or PBS as vehicle control were transferred i.v. into μMT recipients 18 h before immunization. The hapten 4-hydroxy-3-nitro-phenacetyl (NP) was conjugated to anti-CD180 (NP-αCD180) as previously reported [27 (link)]. The adoptively transferred mice were inoculated i.v. with 200 μl of PBS containing 100 μg/mouse of NP-αCD180. For direct immunizations of WT and BAFFR^-/- mice, mice were inoculated i.v. with 50 μg/mouse of NP-αCD180. ELISA and ELISPOT assays to detect NP-IgG in the serum and NP-IgG ASCs in the spleens were performed as previously described [27 (link)]. To detect NP-specific cell subsets by flow cytometry NP-PE was prepared by conjugation of NPOsu (Biosearch Technologies) to phycoerythrin (Sigma-Aldrich, St. Louis, MO) as described [27 (link)].

In order to characterize in details neoantigen recognition by neoepitope-specific T-cells, we established neoepitope-specific T-cell clones. For CD8⁺ T-cells, presensitized T-cells that showed neoantigen-specific reactivity in ELISPOT assays were restimulated by mutated peptide-pulsed T-APC and IFN-γ-producing T-cells were labeled using IFN-γ-capture reagent (Miltenyi Biotec) and sorted by flow-cytometry as described [86 (link)]. For CD4⁺ T-cells, presensitized T-cells were similarly restimulated in the presence of monensin (Sigma) and phycoerythrin (PE)-labeled anti-CD154 monoclonal antibody (mAb) as described [87 (link)] and CD154-expressing cells were sorted. Isolated T-cells were polyclonally expanded by PHA stimulation in the presence of allogeneic irradiated PBMC, IL-2 and IL-7. Purity and clonality of T-cells were tested by low-resolution TCR spectratyping using Vβ subtype-specific antibodies (Beckman Coulter). For some oligoclonal T-cell cultures containing different Vβ-expressing T-cells, cells were sorted again based on Vβ expression. Reactivity of neoepitope-specific T-cell clones were tested by ELISA and/or intracellular cytokine staining [88 (link)].

IEC-6 cell apoptosis was evaluated utilizing flow cytometric techniques. Once harvested, these cells were suspended in a solution of 500 μL binding buffer. A staining solution was prepared using 5 μL each of phycoerythrin (PE) and fluorescein 5-isothiocyanate (FITC), both sourced from Sigma-Aldrich. The cell suspension was subsequently treated with this stain for a duration of 15 min under ambient conditions. Following this, apoptosis levels and fluorescence properties of the H9c2 cells were critically examined using a BD FACSVerse flow cytometer based in the USA.