Computational protocol: Immune profiling of NF1-associated tumors reveals histologic subtype distinctions and heterogeneity: implications for immunotherapy

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Protocol publication

[…] We used a pre-clinical and clinical mRNA microarray Affymetrix database consisting of thirteen dermal neurofibromas, thirteen plexiform neurofibromas, six MPNST tumors, and thirteen MPNST cell lines and normalized their results against ten samples of normal human Schwann cells (GSE14038) to identify MHC I (HLA-A, -B, and –C), B2M, PD-L1 (CD274), and CTLA-4 gene expression []. We used Affymetrix GeneChip Human Genome U133 Plus 2.0 Array to measure gene expression. Data were processed using GeneSpring GX v7.3.1 (Agilent Technologies) with background correction and Robust Multi-array Average (RMA) normalization. Differential gene expression was predicted using Bioconductor/ limma package []. [...] To assess HLA-A/B/C, B2M, PD-L1, and CTLA-4 protein expression in human MPNST cell lines, 2.5x105 cells were harvested with Cell Dissociation Buffer Enzyme-Free PBS-based (Thermo Fisher Scientific, catalog number 13151014). The tumor cells were pelleted by centrifugation, resuspended in 100 μl buffer (PBS, 1% FBS, 1% EDTA), and blocked with 20% final volume of human Ig Fc blocking antibody (Miltyni Biotech, catalog number 130-059-901). Cell suspensions were stained with 1:100 dilutions of Phycoerythrin-conjugated anti-human HLA-A/B/C antibody (W6/32) (eBioscience, catalog number 12-9983-42), Phycoerythrin-conjugated anti-human β-2-microglobulin antibody (2M2) (Biolegend, catalog number 316306), Phycoerythrin -Cyanine 7-conjugated anti-human PD-L1 antibody (29E.2A3) (Biolegend, catalog number 329717), or Allophycocyanin-conjugated anti-human CTLA-4 antibody (L3D10) (Biolegend, catalog number 349907) for 30 minutes at 4°C. Following primary stain, samples were washed twice with buffer, resuspended in 1% paraformaldehyde fixative, and analyzed on a BD FACS LSR II using BD FACSDiVa software version 4.0 (BD Biosciences). Flow cytometry data analysis was performed using FlowJo version 7.6.5 (Tree Star). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Homo sapiens
Diseases Immune System Diseases, Neoplasms, Neurilemmoma, Neurofibroma, Neurofibroma, Plexiform