Publications
Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in peripheral blood of non-small cell lung carcinoma patients responding to atezolizumab treatment
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.
Dimensionality reduction for visualizing single-cell data using UMAP
Advances in single-cell technologies have enabled high-resolution dissection of tissue composition. Several tools for dimensionality reduction are available to analyze the large number of parameters generated in single-cell studies. Recently, a nonlinear dimensionality-reduction technique, uniform manifold approximation and projection (UMAP), was developed for the analysis of any type of high-dimensional data. Here we apply it to biological data, using three well-characterized mass cytometry and single-cell RNA sequencing datasets. Comparing the performance of UMAP with five other tools, we find that UMAP provides the fastest run times, highest reproducibility and the most meaningful organization of cell clusters. The work highlights the use of UMAP for improved visualization and interpretation of single-cell data.
Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates
Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types1,2,3,4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control2,4,7,8,9,10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein–Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39− CD8+ TILs. Furthermore, frequencies of CD39 expression among CD8+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.
Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice
Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. The use of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8+ T cells in human blood samples. The present study shows that this approach can also be used to rapidly assess the magnitude of influenza-specific CD8+ T cell epitope dominance across lymph nodes and lungs in a murine model of a highly pathological influenza infection. Moreover, we show feasibility of extending this approach to include concurrent identification of virus-specific CD4+ T cells. By using a double coding approach, we probed for five influenza-specific MHCI-peptide complexes as well as one influenza-specific MHCII-peptide complex in the presence of irrelevant control peptides and show that this approach is capable of tracking antigen-specific T cells across individual lymph nodes and lungs. The simultaneous staining with 26 surface maker molecules further facilitated an in-depth characterization of T cells reacting with influenza epitopes and revealed tissue specific phenotypic differences between CD4+ T cells targeting the same pathogenic epitope. In conclusion, this approach provides the possibility for a rapid and comprehensive analysis of antigen-specific CD8+ and CD4+ T cells in different disease settings that might be advantageous for subsequent vaccine formulation strategies.
Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based Heterogeneity in Phenotype and Frequency
Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra-epithelial (ie)ILC1-like cells that represent a broader category of NK cells in mucosal and non-mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals and tissues. It also provides a global, comprehensive, and detailed description of ILC heterogeneity in humans across patients and tissues.