This analysis will be performed on FFPE tissue blocks or cut slides. This is a service provided by an NCI Core Facility. Requests for this 44 Abbreviated Title: Neoadjuvant IO for HNSCC Version Date: August 31, 2022 service will be made online through the NCI at Frederick Access System, which will then provide instructions on shipping and receiving. 5.1.3 Assessment of antigen-specific responses in TIL and antigens predicted from exome and RNA sequencing Standardized protocols developed in the NCI TIL Laboratory will be used to culture tumor infiltrating lymphocytes (TIL) form tumor fragments. Briefly, tumor will be minced into 1-2 mm fragments and plated in T cell supportive mediate supplemented with human IL-2. Following 3 weeks of culture, T cells will be sorted via negative magnetic selection and assessed for antigen specific responses by ELISpot. Samples stimulated with Ionomycin, phorbol-myristate-acetate (PMA/Iono) will be used as a positive control. Autologous B cells expanded from PBMC will be used as antigen-presenting cells. Messenger RNA (mRNA) encoding genes of interest will be in vitro transcribed (IVT) from expression vectors engineered to express genes harboring potential antigens. mRNA will be electroporated into B cells. TIL will be screened for responses against naturally processed and presented MHC class I restricted epitopes from MUC-1, CEA, or brachyury, or predicted neoepitopes. This work will be performed in the Allen Lab, NIDCD. Prediction of neoepitopes requires exome sequencing, RNA sequencing and prediction pipeline bioinformatics. This work will be perfomed by Nant. Nant will use Illumina's HiSeq and NovaSeq platforms for sequencing. All samples will be run according to clinical sequencing quality standards, as regulated by CLIA/CAP, by clinical laboratory scientists with specific certifications as Clinical Genetic Molecular Biologist Scientists Bioinformatic analysis will be performed by both Nant and NCI investigators. 5.1.4 Biomarker analysis related to wound healing. Expression analysis of genes related to wound dealing and TGF signaling may we assessed. Hypotheses related to the role of immunotherapy-induced inflammation, TGF signaling and wound healing will be studied by immunohistochemistry, immunofluorescence, and focused PCR arrays on FFPE or frozen tissues. This work will be performed in the Allen Lab, NIDCD. 5.1.5 Assessment of antigen-specific responses in peripheral T cells. PBMCs may be analyzed for tumor antigen-specific immune responses to MUC-1, CEA, and Brachyury using an intracellular cytokine staining assay. PBMCs will be stimulated in vitro with overlapping 15-mer peptide pools encoding the tumor-associated antigens listed above; control peptide pools will involve the use of human leukocyte antigen peptide as a negative control and CEFT peptide mix as a positive control. CEFT is a mixture of peptides of CMV, Epstein-Barr virus, influenza, and tetanus toxin. Post-stimulation analyses of CD4 and CD8 T cells will involve the production of IFN-γ, IL-2, TNF, and the degranulation marker CD107a. If sufficient PBMCs are available, assays may also be performed for the development of T cells to other tumor-associated antigens. A detailed description of this assay has been previously reported [21].