PDF file - 381K, Results of Melanoma Specific Mutation Panel.

Supplementary Figure Legends 1-3 from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Gating strategy for CD8+ T-cell subsets. CM: Central memory; EM: Effector memory; TEMRA: Effector memory expressing CD45RA

Raw scanned data from ELISpot plate scanning reader (AID ELISpot Reader Classic); data was then processed using the AID ELISpot software to standardise and analyse the spot counts using intensity and size measurements, to provide the final data presented in Supplementary Figure 2.

Populations of IFNγ high and IFNγ low in response to tumour/peptide stimulation in MLTC at day 34 in patients (A) D05 and (B) D14

XLS file - 50K, Confirmation panel assay design.

Supplementary Table 1 from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

PDF file - 270K, Mutation List used in confirmation phase.

Whole -exome alignment of PABPC3 compared to the PABPC1 sequence (A): PABPC3 sequencing reads alignment with PABPC1 and PABPC3 reference sequence. (B): PABPC3 dinucleotide change resulting in R -->Q amino acid change in the protein sequence.

Total raw dataset of assessment of potential neoepitope sequences through epitope prediction programs for D05, D14, D41, A02 and A06.

PDF file - 210K, Results of matching/ duplicate samples.

Raw data from the initial peptide screen across MLTC assessed by IFNγ ELISpot. The yellow line delineates 30 spots and the red line marks 40 spots. Each bar represents wells assessed in triplicate, averaged and the average background measurement subtracted. The top measurement was capped at 200 spots.

Cell line information and Sanger sequence confirmed mutations in each cell line.

Supplementary Methods from A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation ( GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX ). We identify three other significantly mutated genes ( TP53 , RPL5 and CENPE ).

By 4000 years ago, people had introduced maize to the southwestern United States; full agriculture was established quickly in the lowland deserts but delayed in the temperate highlands for 2000 years. We test if the earliest upland maize was adapted for early flowering, a characteristic of modern temperate maize. We sequenced fifteen 1900-year-old maize cobs from Turkey Pen Shelter in the temperate Southwest. Indirectly validated genomic models predicted that Turkey Pen maize was marginally adapted with respect to flowering, as well as short, tillering, and segregating for yellow kernel color. Temperate adaptation drove modern population differentiation and was selected in situ from ancient standing variation. Validated prediction of polygenic traits improves our understanding of ancient phenotypes and the dynamics of environmental adaptation.

Advancements in high-resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13829 peptides were identified; 83-87% of these were 8-11mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA-type binding prediction for 10078 9/10mer peptides assigned 88-95% to a patient-specific HLA subtype, revealing a disparity in strength of predicted binding. HLA-B*27-specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.