Lsr 2

Samples were processed into single cell suspensions and stained in FACS buffer (PBS + 2% FBS + 0.1% sodium Azide) with antibody for 45 minutes on ice followed by secondary (APC- or PE-conjugated streptavidin) binding for 20 minutes. Antibodies used were CD8-BUV395 (RPA-T8, BD Biosciences), CD4-Bv605 (RPA-T4, BD Biosciences), CD3-APC (SP34–2, BD Biosciences), FGFR4-PE (4FR6D3, Biolegend), CD19-PE (HIB19, Biolegend), PDL1-APC (MIH1, BD Bioscience), PDL2-Bv421 (MIH18, BD Biosciences). Surface CAR was detected using biotinylated CD19 with Fc-tag (Acro Bio) or biotinylated FGFR4-Fc (Sino Bio). For evaluation of FGFR4 binders identified from phage screening, 500 nM VH or Fab proteins were incubated with cells at 4 °C for 30 minutes followed by PE conjugated anti-human Fc antibody (Miltenyi Biotec, 130–101-576) for 30 minutes at 4 °C. Cells were then analyzed by flow cytometry using BD LSR II (San Jose, CA). Tumor cell surface antigen was quantified using Quanti-Brite beads (BD Biosciences) according to the manufacturer’s instructions. Quantibrite beads and corresponding tumor cells stained for FGFR4 antigen were acquired on a BD LSRII and analysed using Graphpad Prism. Data were analyzed by FlowJo V10.

Flow cytometry for surface marker analysis was performed using similar procedures as cell sorting (see “Cell preparation”) and analyzed on a BD Cytoflex or BD LSRII. Fluorochromeconjugated antibodies are listed in the Key Resources Table. Cell cycle analysis was performed as follows: (1) stain MUTZ-3 cells with CD14-PE-Cy7 (Coulter A22331) and CD34-FITC (BD Pharmingen 348053) antibodies; (2) spin and permeabilize using Phosphoflow Perm 2 solution (BD 347692 diluted 10× in H₂O); (3) wash with PBS 2% FBS; (4) stain with Ki67 Alexa Fluor 647 (BD Pharmingen 561126); (4) wash; (5) resuspend in Cytofix buffer (BD 554655 diluted 4× in PBS) with DAPI at 1 μg / ml; (5) analysis on an BD LSRII analyzer within 30 minutes. Data was analyzed using FlowJo software (Tree Star, Inc.).

To investigate the efficiency of nanofiber uptake, mice were euthanized at designated time points after intraperitoneal immunization with 100 μL 2 mM OVAQ11-TAMRA nanofibers. Two mL of Hank’s balanced salt solution (HBSS) was injected to the intraperitoneal space, and about 1.2-1.5 mL was subsequently withdrawn. Cells were collected by centrifugation and washed once with flow buffer (PBS containing 2% fetal bovine serum). After blocking the Fc receptor with 2.4G2 antibody, cells were stained for F4/80, MHCII, CD11c, CD11b, CD40, CD80, and CD86. Cells were washed and resuspended in flow buffer with 1 μg/mL DAPI. Flow cytometry was performed on a BD LSRII and analyzed using FlowJo software. The percentage of TAMRA-positive cells was calculated for both macrophages (gated as F4/80⁺CD11b⁺) and dendritic cells (DCs, gated as F4/80⁻CD11c⁺MHCII^high). To investigate the presentation of Eα peptide, mice were immunized with 100 μL 2 mM EαQ11 nanofibers intraperitoneally. IP lavage cells were collected and stained as described above. The Eα:I-A^b complexes were stained using Y-Ae antibodies (eBioscience, San Diego, CA, USA). Cells were washed and re-suspended in flow buffer containing 1 μg/mL DAPI. Flow cytometry was performed on a BD LSRII and analyzed by FlowJo software.