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

Abstract

Background: There is strong evidence that immunotherapy-mediated tumor rejection can be driven by tumor-specific CD8+ T cells reinvigorated to recognize neoantigens derived from tumor somatic mutations. Thus, the frequencies or characteristics of tumor-reactive, mutation-specific CD8+ T cells could be used as biomarkers of an anti-tumor response. However, such neoantigen-specific T cells are difficult to reliably identify due to their low frequency in peripheral blood and wide range of potential epitope specificities.

Methods: 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 sequencing and RNA sequencing we identified tumor neoantigens that are predicted to bind to major histocompatibility complex class I (MHC-I) and utilized mass cytometry, together with cellular 'barcoding', to profile immune cells from patients with objective response to therapy (n = 8) and those with progressive disease (n = 6). In parallel, a highly-multiplexed combinatorial tetramer staining was used 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.

Results: No significant treatment- or response associated phenotypic difference were measured in bulk CD8+ T cells. Multiplexed peptide-MHC multimer staining detected 20 different neoantigen-specific T cell populations, as well as T cells specific for viral control antigens. Not only were neoantigen-specific T cells more frequently detected in responding patients, their phenotypes were also almost entirely distinct. Neoantigen-specific T cells from responder patients typically showed a differentiated effector phenotype, most like Cytomegalovirus (CMV) and some types of Epstein-Barr virus (EBV)-specific CD8+ T cells. In contrast, more memory-like phenotypic profiles were observed for neoantigen-specific CD8+ T cells from patients with progressive disease.

Conclusion: This study demonstrates that neoantigen-specific T cells can be detected in peripheral blood in non-small cell lung cancer (NSCLC) patients during anti-PD-L1 therapy. Patients with an objective response had an enrichment of neoantigen-reactive T cells and these cells showed a phenotype that differed from patients without a response. These findings suggest the ex vivo identification, characterization, and longitudinal follow-up of rare tumor-specific differentiated effector neoantigen-specific T cells may be useful in predicting response to checkpoint blockade.

Trial registration: POPLAR trial NCT01903993 .

Keywords: Atezolizumab; Immunotherapy; NSCLC; Tumor neoantigen-specific T cells.

Conflict of interest statement

M.Y., S.J., B.O., M.B., S.F. and P.H. are employees and stockholders of Genentech/Roche.

A.N., M.F., H.S. and B.H.L. are employees and stockholders of immunoSCAPE Pte Ltd. A.N. and E. N. are stockholders and Board Directors of immunoSCAPE Pte Ltd.

Figures

Fig. 1

No difference in bulk CD8+ T cells phenotype at baseline or following treatment between atezolizumab responders and non-responders. a Frequencies of CD8+ T cells positive for all assessed marker molecules at baseline. b Frequencies of major CD8+ T cell subsets (naïve: CD45RO-,CCR7+; central memory: CD45RO+,CCR7+; effector memory: CD45RO+,CCR7-; effector: CD45RO-,CCR7-; and activated cells: CD38+/CCR7-) at baseline and on atezolizumab treatment. Each dot represents a patient. c Representative t-SNE map visualizing CD8+ T cells from one responder and one non-responder at baseline and on atezolizumab treatment with related plots showing relative position of cells expressing CD45RO, CCR7 and CD38. d Frequencies of CD8+ T cells positive for all analyzed markers at baseline and on atezolizumab treatment. Data shown from responders (green, n = 6) and non-responders (blue, n = 3)

Fig. 2

Neoantigen-specific T cells are enriched in patients responding to atezolizumab treatment. a Schematic overview of the multiplexed tetramer staining approach and corresponding example of identification of triple positive neoantigen and virus-specific T cells from a representative responder patient at baseline levels (cycle 1 day 1) in two staining configurations. Screening for antigen-specific CD8+ T cells was performed by using a mass cytometry-based multiplex triple coding tetramer staining approach assessing 153 candidate antigens, 126 neoantigens, and 30 cancer-unrelated control antigens for this patient. Each peptide-MHC was labelled with a unique combination of three heavy metal-streptavidin labels. b Same patient before (detection threshold 0.007%) and post atezolizumab treatment (detection threshold 0.009%). T cells specific for one neoantigen and two viral epitopes were identified based on the detection criteria set (see also Methods). t-SNE plots are based on the expression of all phenotypic markers. Relative expression levels of CCR7 and CD45RO are shown. c Total number of unique neoantigen-specific CD8+ T cells (hits) detected from a total of 782 neoantigen candidates within the responders (n = 8 patients) and non-responders (n = 6 patients) groups. d Frequencies of all neoantigen-specific CD8+ T cells detected within the responders (13 neoantigens) and non-responders (7 neoantigens) group pre- and post- atezolizumab treatment. The frequencies of T cells specific for neoantigens ranged from as low as 0.01% to as high as 0.65% of total CD8+ T cells. For patients where baseline sample was available but no antigen-specific T cells were detected are shown as N.D. Abbreviations: N.D., not detected; PR, responders; PD, non-responders

Fig. 3

Neoantigen-specific T cells in atezolizumab responder patients show a more differentiated effector phenotype. a Heatmap representing frequency of antigen-specific CD8+ T cells positive for all phenotypic markers analyzed. Results for all neoantigen-specific and virus-specific CD8+ T cells detected in individual patients are shown, grouped by responders and non-responders. Markers are ordered based on unsupervised hierarchical clustering. Numbers in brackets correspond to unique neoantigens detected in each patient. b The first two components obtained from PCA of percentages of neoantigen-specific T cells for each marker are plotted for each hit (left). Boxplots show the trends toward a higher number of neoantigen-specific T cells positive for CD27, CD28, CD127, and CCR7 in the non-responder group and 2B4, KLRG-1, CD57, CD161, TIGIT, and CD25 in the responder group, respectively (Wilcoxon signed rank test). c Biaxial dot plots showing an example of neoantigen-specific T cells displaying an activated phenotype with co-expression of PD-1 and CD39. t-SNE plots are based on the expression of all phenotypic markers. Relative expression levels of CCR7 and CD45RO are shown. Data shown from Patient 4 (red, neoantigen-specific T cells; blue, EBV-specific T cells; grey, bulk CD8+ T cells)

Fig. 4

Neoantigen-specific T cells in atezolizumab responders are skewed towards a late differentiated CMV-like phenotype. a PCA of all neoantigen-and virus-specific CD8+ T cells hits identified in this study. PCA is based on phenotypic profiling (percent of antigen-specific CD8+ T cells positive for the markers shown in Fig. 3a). The distribution pattern of all hits across the first two principal components allows for an annotation of three distinct clusters. b The majority of the neoantigen-specific T cells from the responder group are located within Cluster 1 and 3, whereas most of the neoantigen-specific T cells from non-responder patients are detected in Cluster 2. CMV-specific T cells were mostly found in Cluster 1, EBV- and influenza-specific T cells mapped within Cluster 2 and 3. Labels are according to patient response and virus-specificity. c Pie chart summarizing the data shown in 4B: top, number of neoantigen hits; bottom, number of viral hits for each PCA cluster. d Graphical representation of the most differentially expressed markers of all virus-specific CD8+ T cells in the three PCA clusters; Bubble size is proportional to mean frequencies of all virus-specific CD8+ T cells positive for the indicated marker in any given cluster