Case Study

Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in peripheral blood of non-small cell lung carcinoma patients responding to atezolizumab treatment

Michael Fehlings, Suchit Jhunjhunwala, Marcin Kowanetz, William E. O’Gorman, Priti S. Hegde, Hermi Sumatoh, Boon Heng Lee, Alessandra Nardin, Etienne Becht, Susan Flynn, Marcus Ballinger, Evan W. Newell and Mahesh Yadav

Programmed death ligand-1 (PD-L1) is expressed by cancer cells and its binding to programmed cell death-1 (PD-1) results in the suppression of T-cell-mediated tumor responses. Antibody blockade of PD-1 has been shown to induce durable therapeutic responses in some patients and may be predicted by the presence of CD8+ T cell infiltrates in melanoma patients (1). Nevertheless, since most cancer patients do not experience any clinical benefit from PD-1/PD-L1 inhibition, the mechanisms of action underlying the anti-tumor T cell response needs to be better characterized in responding patients, and the mechanisms of resistance in non-responding patients (2–5). From an immunological perspective, tumor-specific antigens are proteins absent from normal human tissues, and neoantigens are a subset of these that arise from somatic mutations. Tumor neoantigens are presented on the tumor cell surface and have the potential to drive anti-tumor T cell responses. It is hypothesized that PD-1 checkpoint blockade reinvigorates these neoantigen-specific T cells resulting in tumor cell killing. However, direct evidence linking the induction of neoantigen-specific T cell responses to clinical benefit during checkpoint blockade is not clear (6-8). In this study, atezolizumab, a monoclonal antibody that binds to PD-L1 and blocks its interaction with PD-1 (fig. 1), was administered to patients with non-small cell lung cancer (NSCLC), with the aim of enhancing their tumor specific T cell responses. ImmunoScape used mass cytometry in combination with combinatorial MHC-tetramer multiplexing to screen, identify and phenotypically profile neoantigen-specific CD8+ T cells in patients with partial response or progressive disease, before and after treatment with atezolizumab.

Figure 1. Blockade of PD-L1 by the checkpoint inhibitor, atezolizumab, prevents interaction with PD-1 and reinvigorates the anti-tumor T cell response.

Treatment-related differences were not discernible by analysing the expression of  phenotypic markers across the total CD8+ T cell population. Mass cytometry coupled with highly-multiplexed combinatorial tetramer staining enabled the identification of neoantigen-specific T cells and their associated phenotypes, which had distinct properties in responders versus non-responders (fig. 2).

Patients with an objective response to treatment had an enrichment of neoantigen-reactive T cells and these cells showed a phenotype that differed from non-responding patients, specifically displaying a late differentiated effector-like phenotype similar to viral-specific CD8+ T cells targeting cytomegalovirus (CMV) or Epstein-Barr virus (EBV), associated with cytotoxic T cells capable of controlling persistent viral infections (9). Moreover, in responders, the terminally differentiated phenotype might be an indication of antigen persistence in the tumor, and such antigen can then be recognized by tumoricidal T cells reactivated by immune checkpoint inhibition. In contrast, neoantigen-specific T cells of non-responders displayed memory-like phenotypes, which may either be less effective at conferring anti-tumor effects or not able to target the tumor if the neoantigen presentation is lost.

These results show that using mass cytometry together with highly-multiplexed combinatorial tetramer staining enable identification, tracking and in-depth profiling of neoantigen-specific CD8 T cells at single cell level. Although further studies are required to confirm these results, this study suggests that detection and phenotypic profiling of neoantigen-specific T cells in the periphery might be developed to support patient selection for immune checkpoint inhibition strategies.

Figure 2. Summary figure for the study. Different phenotypic profiles of neoantigen-specific T cells found in the patients responding to atezolizumab treatment compared with the non-responding patients.


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Peripheral blood mononuclear cells (PBMC) from 14 non-small cell lung cancer (NSCLC) patients were collected pre- and post-treatment with the anti-PD-L1 antibody atezolizumab. Using whole exome and RNA sequencing we identified tumor neoantigens that are predicted to bind to major histocompatibility complex class I (MHC-I). We utilized mass cytometry, together with cellular ‘barcoding’, to phenotypically profile immune cells. A specific antibody staining panel based on lineage, descriptive and specific markers was used. In parallel, three-metal combinatorial tetramer approach was employed to screen antigen-specific CD8+ T cells in peripheral blood for 782 candidate tumor neoantigens and 71 known viral-derived control peptide epitopes across all patient samples (10).


To investigate the effects of PD-L1 blockade on overall CD8+ T cell responses during cancer immunotherapy, we performed a mass cytometry-based analysis of CD8+ T cells derived from PBMCs from NSCLC patients taken before and after treatment with atezolizumab. Samples were stained using a panel of up to 29 markers dedicated to T cell identification and profiling, including several markers of activation and co-stimulation, as well as inhibitory molecules and markers associated with T cell dysfunction. As shown in figure 3, the frequencies of most of the markers on CD8+ T cells did not significantly differ between the two groups. These results underline the challenges associated with using a broad phenotypic profiling strategy on bulk CD8+ T cells where extensive heterogeneity confounds the ability to discern differences between responding and non-responding NSCLC patients, on atezolizumab.

Figure 3. No difference in bulk CD8+ T cell phenotype in responders and non-responders before and after treatment with atezolizumab.

To gain deeper insight into the effects of atezolizumab on patient clinical outcomes, we screened, identified and analysed neoantigen-specific CD8+ T cells in PBMC from responders and non-responders using highly-multiplexed combinatorial peptide-MHC tetramer staining.

Figure 4. Neoantigen-specific CD8 T cells are enriched in patients responding to atezolizumab treatment. Total number of unique neoantigen-specific CD8+ T cells (hits) detected from a total of 782 neoantigen candidates (left). Frequencies of all neoantigen-specific CD8+ T cells detected within the responders and non-responders, pre- and post-atezolizumab treatment. Patients where baseline sample was available but no antigen-specific T cells were detected are depicted as N.D. Abbreviations: N.D., not detected; PR, responders; PD, non-responders (right).

We identified CD8 T cells reactive for 13 different neoantigens across all responders (5/8 responders) and 7 neoantigen specificities across the non-responders (3/6 non-responders) (fig. 4, left). Furthermore, there is a trend toward further enrichment of neoantigen-specific T cells post treatment in responders (fig. 4, right). In summary, these data show a trend toward a greater abundance of neoantigen-specific T cells in patients responding to atezolizumab treatment, i.e. the presence of neoantigen-specific T cell responses at baseline or their expansion post-treatment might be associated with clinical response to PD-L1 blockade.

Figure 5. Expression of phenotypic markers in responder versus non-responder groups.

Most of the phenotypic differences in neoantigen-specific CD8 T cells observed in this study were reflective of patient clinical response to atezolizumab. Among responders, neoantigen specific T cells displayed a trend toward increased expression of the markers related to a late-differentiated effector phenotype (KLRG-1, 2B4, CD57, CD161, TIGIT, and CD25). In contrast, the majority of antigen specific T cells detected in non-responders showed a trend toward higher expression of the markers related to a memory-like phenotype (CD127, CD28, CD27, and CCR7) (fig. 5). These observations suggest that the unique phenotype of neoantigen-specific T cells in responding patients and their functional similarity to CMV-reactive T cells, may be indicative of their tumoricidal effect.


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