Kim Margolin

Supplementary Figure S2 shows the genomic variants detected by WES and statistical analaysis of WES data.

Supplementary Figure S3 shows the annotation of cells evaluated by NanoString.

Supplementary Figure S1 shows the processing and review of sample processing for WES, RNAseq, and MIBI.

Supplementary Figure S4 show the comparison of cell types by MIBI across sample/response groups.

Supplementary Figure S5 shows the expression of protein markers by cell type subsets in MIBI data.

Supplementary Figure S6 shows the TCR/BCR repertoire metrics in RNAseq data from biopsies from patients nonresponsive to combination therapy.

Supplementary Figure S7 shows the evaluation of TLS signatures in RNAseq and MIBI samples.

Supplementary Methods contains additional methods details with a data dictionary for the supplementary tables.

Supplementary Tables containing sample and patient metadata, statistical results, annotation of cell types detected by MIBI, and manually reviewed variants in genes of interest.

In the phase 2 trial SWOG S1616 (NCT03033576), patients with advanced melanoma with primary resistance to anti-PD-1/L1 therapies had improved outcomes on the combination of the anti-CTLA-4 antibody ipilimumab with continued anti-PD-1 with nivolumab, over ipilimumab alone. Baseline biopsies from patients responsive to combination therapy had increased transcriptomic expression of complement by myeloid cells, interferon pathways by endothelial cells, and oxidative phosphorylation and lipid metabolism by melanoma cells. Using spatial proteomics, some on-therapy biopsies from patients responding to combination therapy had networks of activated CD8 T cells nearby melanoma cells, while others had T cells and myeloid cells, reflective of different timepoints in a dynamic antitumor response. Conversely, biopsies from patients progressing on combination immunotherapy displayed impaired T-cell infiltration adjacent to plasma cells. Our results define cellular neighborhoods and transcriptomes in melanoma biopsies when reversing resistance to anti-PD-1 with the addition of anti-CTLA4, and plasma cell sheets in non-responding biopsies.