(A) Schematic representation of the repeat biopsy program and sample analysis. Tumor specimens (and corresponding PDXs when available) collected at the time of resistance to ICIs and before treatment with immune checkpoint inhibitors along with germline DNA were analyzed using whole exome sequencing. For select samples with sufficient material, RNA sequencing and quantitative immunofluorescence were also performed. (B) Pie-chart illustrating the types of therapies received by patients in this study. (C) Swimmer’s plot indicating time of response, resistance to ICIs, and length of time on therapy for individual patients.

Bar graphs showing the (A) somatic mutation burden (B) neoantigen burden and (C) mutational signatures in pre-immunotherapy specimens (P, P1), immunotherapy resistant specimens (IR) and xenografts (X).

Oncoprint generated from whole exome sequencing data from 13 cases. Only genes with alterations are shown. Mutated genes are listed vertically in order of frequency of somatic single nucleotide mutations or copy number alterations in the pre-immunotherapy cases. A case with a hypermutator phenotype (#17) sample was excluded from this analysis. Pre-immunotherapy specimens are shown on the left and immunotherapy resistant tumors on the right. *Of note, sample 26IR1 was collected from a site (adrenal metastasis) that responded to a short course of ICI, but progressed during a 2-month delay of treatment when steroids were administered for cerebral edema associated with new intracranial disease (sample 26IR3) and pneumonitis. This site subsequently regressed with re-introduction of ICI.

(A) Treatment timeline for case #23. After receiving palliative thoracic irradiation and 2 lines of standard chemotherapy, the patient initiated trial therapy with anti-PD-L1 andanti-CTLA-4 agents. First on-trial imaging assessment demonstrated partial response. Imaging after 4 months of therapy showed a new celiac mass with sustained response at known sites of disease. After resection of this mass, this patient continued trial therapy, completing the prescribed one-year course, during which the patient had no further progression of disease. (B, C) Hematoxylin and eosin, CD8 and PD-L1 immunohistochemical staining of the tumors before treatment and at acquired resistance to anti-PD-L1 and anti-CTLA-4 blockade. (D) Copy number variation (CNV) analysis of paired tumor specimens and a PDX derived from the immunotherapy resistant tumor specimen revealed acquired homozygous loss of B2M at the time of acquired resistance to anti-PD-L1 and anti-CTLA-4 blockade. (E, F) Multiplex quantitative immunofluorescence (QIF) of B2M in pre-immunotherapy and immunotherapy resistant tumor tissues from case #23. (E) Bar graph depicting the levels of B2M in whole tumor sections from pre-immunotherapy and immunotherapy resistant samples measured using multiplex quantitative immunofluorescence and quantified with AQUA^® software. Numbers in the individual bars represent total fields of view analyzed. Statistical significance was calculated using the Mann-Whitney test. ****p<0.0001. AU, arbitrary units. (F) Multiplex immunofluorescence image representing one field-of-view (FOV), showing the expression of B2M (purple) specifically in the tumor compartment represented by cytokeratin positive epithelial cells (green) and nuclear staining with DAPI (blue) respectively.

(A) Western blot analysis showing the absence of B2M protein expression in a patient derived xenograft (PDX) from case #23 generated at the time of acquired resistance to anti-PD-L1 and anti-CTLA-4 blockade. Also shown is B2M protein expression in 4 PDXs established from other patients with tumors resistant to PD-1 pathway blockade (cases #26, 3, 7, and 8). (B) Flow cytometry analysis for expression of B2M on PDX samples from cases #26, 3, 23, 7, and 8. (C) Flow cytometry analysis of PDXs derived from cases #23, #26, #8 and #7 for B2M and HLA-I cell surface expression following intratumoral injection of either PBS or IFNγ. Each flow plot represents an independent mouse tumor used for these studies.

(A) Western blot analysis of B2m expression in UN-SCC680AJ lung cancer cells transfected with a plasmid expressing Cas9 and a sgRNA targeting B2m (sgB2m polyclonal). Additionally, single cell sorting identified clonal populations where B2m expression was absent (sgB2m monoclonal). (B) 5×10⁵ UN-SCC680AJ cells from the monoclonal B2m null population and vector control cells were injected into the dorsal flanks of A/J mice subcutaneously. Tumors were allowed to grow to approximately 30 mm³ before administration of 100 μg of anti-PD-1 or isotype control via intraperitoneal injection every 3 days. Tumors were measured every 3 days with a caliper, and tumor volumes were calculated. Data are presented as the mean tumor volume ± SEM. The size of the tumors in the B2m wild-type and B2m knock-out lines treated with anti-PD1 were compared at the indicated time-points using a T-test. ***p<0.001, ****p<0.0001. Additionally, cytotoxicity assays were performed to assess the percentage of viable target cells (Empty vector (EV) UNSCC680AJ or sgB2m UNSCC680AJ) after co-culturing the cell lines in vitro with EV UNSCC680AJ tumor-infiltrating CD8 T cells (C) or T cells from the spleen (D). Data represent n=2 mice, with error bars denoting standard error of the mean.

Ratio of normalized RNA sequencing-derived read counts of T cell inhibitory and activating receptors (A), ligands (B), and effector molecules (C) in immunotherapy resistant specimens (n=7) compared to pre-immunotherapy specimens (n=4). (D–F) Quantitative immunofluorescence analysis showing the levels of the immune inhibitory molecule LAG-3 (D), the T cell exhaustion marker PD-1 (E), T-cell activation effector molecule Granzyme B (GZB) (F) and T-cell proliferation marker Ki-67 (G) in CD3 positive tumor infiltrating lymphocytes (TILs) in paired pre- and post-immunotherapy tumor samples as quantified using the AQUA^® software. Numbers in the individual bars represent total fields of view analyzed. Statistical significance was calculated using the Mann-Whitney test. P1- pre-immunotherapy and IR- immunotherapy resistant. ****p<0.0001, ***p<0.001 **p<0.01, *p<0.05.