Single-cell RNA sequencing unveils the shared and the distinct cytotoxic hallmarks of human TCRVδ1 and TCRVδ2 γδ T lymphocytes Gabriele Pizzolatoa,b,c,d,e,f,g,h,i,j,1, Hannah Kaminskik,l,1, Marie Tosolinia,b,c,d,e,f,g, Don-Marc Franchinia,b,c,d,e,f,g, Fréderic Ponta,g, Fréderic Martinsm,n, Carine Vallea,b,c,d,e,f,g, Delphine Labourdetten,o, Sarah Cadota,b,c,d,e,f,g, Anne Quillet-Marya,b,c,d,e,f,g, Mary Poupota,b,c,d,e,f,g, Camille Laurenta,b,c,d,e,f,g, Loic Ysebaerta,b,c,d,e,f,g, Serena Meravigliai,j, Francesco Dielii,j, Pierre Mervillek,l, Pierre Milpiedp, Julie Déchanet-Mervillek, and Jean-Jacques Fourniéa,b,c,d,e,f,g,2 aCentre de Recherches en Cancérologie de Toulouse, INSERM UMR1037, 31100 Toulouse, France; bToulouse University, 31000 Toulouse, France; cERL 5294 CNRS, 31024 Toulouse, France; dInstitut Universitaire du Cancer-Oncopole de Toulouse, 31100 Toulouse, France; eLaboratoire d’Excellence ‘TOUCAN’, Toulouse, France; fProgramme Hospitalo, Universitaire en Cancérologie CAPTOR, 31059 Toulouse, France; gInstitut Carnot Lymphome CALYM, 69495 Lyon-Pierre Bénite, France; hHumanitas University, 20089 Rozzano (MI), Italy; iDepartment of Biopathology and Medical Biotechnologies, University of Palermo, 90133 Palermo, Italy; jCentral Laboratory of Advanced Diagnosis and Biomedical Research, University of Palermo, 90133 Palermo, Italy; kUniversity of Bordeaux, CNRS, ImmunoConcEpT, UMR 5164, F-33076 Bordeaux, France; lService de Néphrologie et Transplantation Rénale, Centre Hospitalo-Universitaire de Bordeaux, 33000 Bordeaux, France; mInstitut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, 31432 Toulouse, France; nPlateforme GeT, Genotoul, 31100 Toulouse, France; oLaboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, Université de Toulouse, CNRS, INRA, INSA, 31077 Toulouse, France; and pAix Marseille University, CNRS, INSERM, Centre d’ Immunologie de Marseille-Luminy, 13007 Marseille, France Edited by Willi K. Born, National Jewish Health, Denver, CO, and accepted by Editorial Board Member Philippa Marrack April 26, 2019 (received for review November 13, 2018) γδ T lymphocytes represent ∼1% of human peripheral blood studies currently focus on the cytotoxic CD8 T lymphocytes, mononuclear cells and even more cells in most tissues of verte- which represent the main target of immune checkpoint blockade brates. Although they have important anticancer functions, most therapies, and are readily detected by their scRNA-seq profile. current single-cell RNA sequencing (scRNA-seq) studies do not iden- Other subsets of cytolytic T cells are also critical in this thera- tify γδ T lymphocytes because their transcriptomes at the single-cell peutic perspective; however, currently they have never been char- level are unknown. Here we show that high-resolution clustering of acterized by scRNA-seq and are therefore missing from all current large scRNA-seq datasets and a combination of gene signatures tumor microenvironments mapped by these technologies. allow the specific detection of human γδ Tlymphocytesandiden- Human γδ T lymphocytes represent a peculiar lymphoid cell tification of their T cell receptor (TCR)Vδ1 and TCRVδ2subsetsin subset displaying hallmarks of both innate and adaptive immu- large datasets from complex cell mixtures. In t-distributed stochastic nity, and reacting to microbial pathogens and malignancies (5). − − neighbor embedding plots from blood and tumor samples, the few These CD4 CD8 T cells express a somatically rearranged γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1andTCRVδ2subsetsformcloseyet Significance distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation None of the single-cell RNA sequencing (scRNA-seq) studies genes. Similar pseudotime maturation trajectories of TCRVδ1and published so far convincingly identified human γδ T lympho- TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets cytes despite their anticancer functions. To address this, we an unattended pool of terminally differentiated effector memory cells here report scRNA-seq of γδ T lymphocytes purified from hu- with preserved proliferative capacity, a finding confirmed by in vitro man blood, and a signature identifying γδ T cells. The single-cell proliferation assays. Overall, the single-cell transcriptomes of thou- + transcriptomes of TCRVδ1 and TCRVδ2 lymphocytes are inter- sands of individual γδ T lymphocytes from different CMV and mediates resembing those of NK cells and T CD8, respectively, CMV− donors reflect cytotoxic maturation stages driven by the im- they reflect their respective maturation stage and their pseudo- munological history of donors. This landmark study establishes the time maturation trajectory unveils terminally differentiated cells rationale for identification, subtyping, and deep characterization of with mitotic capacity. In human cancers, the γδ TIL, mostly human γδ T lymphocytes in further scRNA-seq studies of complex expressing TCRVδ1, appear fewer in tumors than in blood and not tissues in physiological and disease conditions. correlated to αβ TIL abundance. This report is a landmark resource for future studies of γδ T lymphocytes at the single-cell level. γδ T lymphocyte | transcriptome | single-cell RNA-sequencing | human immunology | cancer Author contributions: C.L., S.M., F.D., J.D.-M., and J.-J.F. designed research; G.P., H.K., D.-M.F., F.M., C.V., D.L., S.C., A.Q.-M., M.P., P. Merville, and P. Milpied performed research; ingle-cell level mRNA-sequencing (scRNA-seq) of hetero- M.T., F.P., L.Y., P. Merville, P. Milpied, J.D.-M., and J.-J.F. contributed new reagents/ana- lytic tools; G.P., H.K., M.T., F.P., S.C., A.Q.-M., P. Merville, P. Milpied, and J.D.-M. analyzed Sgeneous cell populations has become the reference tool for data; and S.M., F.D., P. Milpied, J.D.-M., and J.-J.F. wrote the paper. establishing cellular lineages and composition of tissues from the The authors declare no conflict of interest. human body (1). In addition, the development of novel and This article is a PNAS Direct Submission. W.K.B. is a guest editor invited by the open-source computational tools for processing scRNA-seq Editorial Board. datasets enables delineation of both broad and subtle differ- Published under the PNAS license. ences in rare cell subsets present in complex mixtures, such as Data deposition: The scRNASeq data were deposited in the NCBI Gene Expression Omni- peripheral blood mononuclear cells (PBMC) (2, 3). Such de- bus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE128223). velopments are expected to build more knowledge about the 1G.P. and H.K. contributed equally to this work. cellular composition and states composing tumors. Recent 2To whom correspondence may be addressed. Email: [email protected]. scRNA-seq analyses in melanoma and colorectal cancer evi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. denced the various tumor microenvironments and exhaustion 1073/pnas.1818488116/-/DCSupplemental. patterns of the tumor-infiltrating lymphocytes (4). Most of such Published online May 22, 2019. 11906–11915 | PNAS | June 11, 2019 | vol. 116 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1818488116 Downloaded by guest on September 25, 2021 T cell receptor (TCR), as well as cytotoxicity receptors more γδ TILs positively correlates with good outcome (27). Although typically expressed by NK cells. The diversity of γ-andδ-encoded encouraging, such results suffered of poor learning from too few TCR relies on VJ rearrangements of the TRG locus at 7p14 and (only two) γδ T cell transcriptomes, however, as CIBERSORT VDJ rearrangements of the TRD locus at 14q11.2, respectively. In identifies erroneously most of CD8 T, NK cells, and γδ Tlym- the human genome, the TRD locus contains three variable genes phocytes (28). This problem reflects the massive gene multi- (TRDV1–3), three diversity genes (TRDD1–3), four joining genes collinearity of transcriptomes from these three closely related cell (TRDJ1–4) and one constant TRDC gene, which rearrange to en- types (29), suggesting that deeper learning from many more γδ codeaTCRδ chain. The TRG locus that rearrange to encode a T cell transcriptomes is necessary. In addition to unfaithfully iden- TCRγ chain contains 14 variable genes, of which only 6 are func- tifying γδ T cells as a whole, determining their subsets defined by tional (TRGV2–5, TRGV8,andTRGV9), five joining genes (TRGJ1, cell surface TCR and stage of maturation is currently out of reach J2, JP, JP1, JP2), and two constant genes (TRGC1, TRGC2). De- for the same reasons. Thus, a decisive milestone would be the spite this low number of TCR-encoding gene segments, the human straightforward identification of γδ T lymphocytes from scRNA-seq TCR repertoire of γδ T cells is almost as diversified as that of αβ T data. Such an achievement could allow us to determine their lymphocytes. In addition, the repertoire of γδ TCR expressed at the presence, their TCR, maturation stage, and activation/exhaustion cell surface of γδ T cells is biased according to their tissue locali- status in the tumor microenvironment of a large panel of human zation. TRGC1 encodes for the γ-constant region of the cell surface cancers. Nevertheless, in this aim it remains necessary to identify γδ TCRVγ9Vδ2 expressed by the most abundant γδ T lymphocytes in T lymphocytes from nonmalignant reference tissue samples, such