PMC9500393_01
Identification of an expanded T cell clone following radiation and anti-PD-L1 therapy. (A) Expression of PD-L1 within the metastatic tumor biopsy by immunohistochemistry. (B) PET scan of the patient before and (C) one year of anti-PD-L1 therapy. (D) Treatment timeline of the patient in relation to TCRbeta clonality (inverted Shannon entropy), percent overlap of all TCRbeta sequences detected in the tumor and the blood, and an alluvial plot tracking the frequency of the top 100 TCRbeta sequences shared by at least 2 samples across all collection time points.
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II study evaluating the efficacy and safety of atezolizumab, an anti-PD-L1 inhibitor, in patients with locally advanced or metastatic NSCLC (NCT01846416). Immunohistochemistry of her right buttock tumor biopsy confirmed 100% of her cancer cells expressed PD-L1 (C223 DAKO monoclonal antibody,  Figure 1A ). Repeat imaging prior to her enrollment in the clinical trial revealed further progression of her cancer with nodal and lymphangitic spread ( Figure 1B ). Figure 1C ). Four years later, an enlarging right upper lobe lesion within the lung was identified and treated with empiric SBRT since the site was inaccessible for a biopsy. Pre-treatment metastatic skin lesions showed greater TCRbeta diversity than the post-treatment blood samples ( Figure 1D ).
Phenotypic characterization and antigen specificity of the dominant T cell clone. (A) Differential gene expression from targeted scRNAseq comparing the dominant ("Top TCR") to non-dominant clones. Significance was measured by T test. (B) Construction of TCRalphabeta clone. (C) Interferon-gamma elispot in relation to mean fluorescence intensity (MFI) of neopeptide generated tetramers. Dashed lines define threshold for reactive peptides (show in red) (D) Chromium release assay of candidate neopeptides. Mean with standard deviations are plotted along with the level of significance from T tests. (E) Lollipop plots showing the location of non-synonymous mutations within the protein amino acid sequence of candidate neoantigens. (F) T-Scan results showing the fold enrichment of each protein fragment in the T-Scan peptidome library. Each point represents an individual protein fragment. Enriched protein fragments with a common epitope are highlighted and labeled. ns: P > 0.05, *P <= 0.05, **P <= 0.01, ***P <= 0.001.
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T cell clone that was present in the tumor and expanded in the blood after anti-PD-L1 therapy, we next characterized its phenotype and identified its antigenic specificity. Targeted single-cell RNA sequencing using a 23 gene panel, confirmed the T cell to be CD8+ and we identified increased differential expression of IFNG and reduced expression of IL13, PDCD1, and CTLA4 ( Figure 2A ) compared with other T cells derived from the tumor. We cloned the dominant alphabeta TCR ( Figure 2B ) and transduced it into donor CD8+ human T cells and screened it for reactivity against the top 165 HLA-A2 restricted neopeptides. Additionally, we constructed HLA-A2 specific tetramers using these 165 neopeptides by UV mediated peptide exchange and screened for affinity to the dominant clone ( Figure 2C ). This combined analysis identified 6 candidate neopeptides which we validated using a chromium release assay ( Figure 2D ). Figure 2E ). Finally, to exclude the possibility that the dominant T cell clone also reacts to a self-antigen, we performed a T-Scan analysis against the entire viral and human peptidome. TCR suggesting the dominant T cell clone does not recognize a self-antigen ( Figure 2F ). Taken together, these results suggest that the expanded T cell clone was an activated, effector CD8+ T cell with reactivity against tumor-derived neopeptides.