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• W3189614948 abstract "•CXCR6 is critical for sustained tumor control mediatedby CD8+ cytotoxic T cells (CTLs)•CXCR6 optimizes CTL interactions with the CCR7+ DC3 state of conventional DCs•DC3s trans-present IL-15 to TCF-1neg effector-like CTLs to sustain their survival in the TME•DC3s densely cluster in T cell-rich perivascular niches of the tumor stroma Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses. Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses. Successful clearance of viral infections by the immune system depends on CD8+ T cells that recognize intracellular pathogen-derived antigens. Clonal expansion of naive cells in lymphoid tissues produces short-lived effector cells that eliminate virally infected cells and produce interferon gamma (IFN-γ) to amplify the response, as well as precursors for different subsets of memory cells that persist after the infection has been cleared (Kaech and Cui, 2012Kaech S.M. Cui W. Transcriptional control of effector and memory CD8+ T cell differentiation.Nat. Rev. Immunol. 2012; 12: 749-761Crossref PubMed Scopus (929) Google Scholar). In contrast, failure to clear viruses leads to chronic infections and persistent, yet hypofunctional cytotoxic T lymphocyte (CTL) responses characterized by a gradual decline in proliferative capacity, cytokine-secretion, and cytotoxic function of individual cells. This adapted response pattern, often referred to as T cell exhaustion, may serve to avoid immune-pathological damage to host tissues that would result from continued high-level immune activation (Hashimoto et al., 2018Hashimoto M. Kamphorst A.O. Im S.J. Kissick H.T. Pillai R.N. Ramalingam S.S. Araki K. Ahmed R. CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions.Annu. Rev. Med. 2018; 69: 301-318Crossref PubMed Scopus (289) Google Scholar; Speiser et al., 2014Speiser D.E. Utzschneider D.T. Oberle S.G. Münz C. Romero P. Zehn D. T cell differentiation in chronic infection and cancer: functional adaptation or exhaustion?.Nat. Rev. Immunol. 2014; 14: 768-774Crossref PubMed Scopus (202) Google Scholar). However, even exhausted CTL responses continue to limit viral replication (Jin et al., 1999Jin X. Bauer D.E. Tuttleton S.E. Lewin S. Gettie A. Blanchard J. Irwin C.E. Safrit J.T. Mittler J. Weinberger L. et al.Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques.J. Exp. Med. 1999; 189: 991-998Crossref PubMed Scopus (1213) Google Scholar; Schmitz et al., 1999Schmitz J.E. Kuroda M.J. Santra S. Sasseville V.G. Simon M.A. Lifton M.A. Racz P. Tenner-Racz K. Dalesandro M. Scallon B.J. et al.Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.Science. 1999; 283: 857-860Crossref PubMed Scopus (1939) Google Scholar). Many features of this equilibrium state between viruses and the immune system are replicated during immune responses against established tumors. Here, CTLs that recognize mutational tumor neoantigens also exert varying levels of immune control but, similar to CTLs in chronic viral infection, adopt a hypofunctional state. Recent studies have revealed the heterogeneity and dynamics of the hypofunctional CTL populations observed in chronic viral infection and cancer (He et al., 2016He R. Hou S. Liu C. Zhang A. Bai Q. Han M. Yang Y. Wei G. Shen T. Yang X. et al.Follicular CXCR5- expressing CD8(+) T cells curtail chronic viral infection.Nature. 2016; 537: 412-428Crossref PubMed Scopus (366) Google Scholar; Im et al., 2016Im S.J. Hashimoto M. Gerner M.Y. Lee J. Kissick H.T. Burger M.C. Shan Q. Hale J.S. Lee J. Nasti T.H. et al.Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy.Nature. 2016; 537: 417-421Crossref PubMed Scopus (909) Google Scholar; Leong et al., 2016Leong Y.A. Chen Y. Ong H.S. Wu D. Man K. Deleage C. Minnich M. Meckiff B.J. Wei Y. Hou Z. et al.CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.Nat. Immunol. 2016; 17: 1187-1196Crossref PubMed Scopus (283) Google Scholar; Sade-Feldman et al., 2018Sade-Feldman M. Yizhak K. Bjorgaard S.L. Ray J.P. de Boer C.G. Jenkins R.W. Lieb D.J. Chen J.H. Frederick D.T. Barzily-Rokni M. et al.Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.Cell. 2018; 175: 998-1013.e20Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar; Snell et al., 2018Snell L.M. MacLeod B.L. Law J.C. Osokine I. Elsaesser H.J. Hezaveh K. Dickson R.J. Gavin M.A. Guidos C.J. McGaha T.L. Brooks D.G. CD8+ T Cell Priming in Established Chronic Viral Infection Preferentially Directs Differentiation of Memory-like Cells for Sustained Immunity.Immunity. 2018; 49: 678-694.e5Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar; Utzschneider et al., 2016Utzschneider D.T. Charmoy M. Chennupati V. Pousse L. Ferreira D.P. Calderon-Copete S. Danilo M. Alfei F. Hofmann M. Wieland D. et al.T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections.Immunity. 2016; 45: 415-427Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar; Wu et al., 2016Wu T. Ji Y. Ashley Moseman E. Xu H.C. Manglani M. Kirby M. Anderson S.M. Handon R. Kenyon E. Elkahloun A. et al.The TCF1-Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness.Sci. Immunol. 2016; 1: eaai8593Crossref PubMed Scopus (251) Google Scholar). These include stem-like CTLs that express the HMG box transcription factor TCF-1 and the SLAM family member Slamf6/Ly108, possess the capacity for self-renewal, and are found primarily in lymphoid tissues but in smaller numbers also at immunological effector sites such as tumors (Miller et al., 2019Miller B.C. Sen D.R. Al Abosy R. Bi K. Virkud Y.V. LaFleur M.W. Yates K.B. Lako A. Felt K. Naik G.S. et al.Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.Nat. Immunol. 2019; 20: 326-336Crossref PubMed Scopus (620) Google Scholar; Siddiqui et al., 2019Siddiqui I. Schaeuble K. Chennupati V. Fuertes Marraco S.A. Calderon-Copete S. Pais Ferreira D. Carmona S.J. Scarpellino L. Gfeller D. Pradervand S. et al.Intratumoral Tcf1+PD-1+CD8+ T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy.Immunity. 2019; 50: 195-211.e10Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). TCF-1pos CTLs continually give rise to TCF-1neg effector-like cells that acquire cytotoxic function, but also upregulate inhibitory receptors, such as TIM-3, predicted to attenuate their effector activity. TCF-1neg CTLs include cells with a continuum of differentiation states ranging from highly proliferative and functional to irreversibly hypofunctional. Highly proliferative TCF-1neg CTLs referred to as transitory CTLs express the chemokine receptor CX3CR1 and mediate antiviral control during chronic viral infection. Terminally differentiated TCF-1neg CTLs, on the other hand, are characterized by expression of CD101 and stable epigenetic repression of effector genes (Hudson et al., 2019Hudson W.H. Gensheimer J. Hashimoto M. Wieland A. Valanparambil R.M. Li P. Lin J.-X. Konieczny B.T. Im S.J. Freeman G.J. et al.Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1+ Stem-like CD8+ T Cells during Chronic Infection.Immunity. 2019; 51: 1043-1058.e4Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar; Li et al., 2019Li H. van der Leun A.M. Yofe I. Lubling Y. Gelbard-Solodkin D. van Akkooi A.C.J. van den Braber M. Rozeman E.A. Haanen J.B.A.G. Blank C.U. et al.Dysfunctional CD8 T Cells Form a Proliferative, Dynamically Regulated Compartment within Human Melanoma.Cell. 2019; 176: 775-789.e18Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar; Philip et al., 2017Philip M. Fairchild L. Sun L. Horste E.L. Camara S. Shakiba M. Scott A.C. Viale A. Lauer P. Merghoub T. et al.Chromatin states define tumour-specific T cell dysfunction and reprogramming.Nature. 2017; 545: 452-456Crossref PubMed Scopus (443) Google Scholar; Zander et al., 2019Zander R. Schauder D. Xin G. Nguyen C. Wu X. Zajac A. Cui W. CD4+ T Cell Help Is Required for the Formation of a Cytolytic CD8+ T Cell Subset that Protects against Chronic Infection and Cancer.Immunity. 2019; 51: 1028-1042.e4Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). DCs not only initiate anti-tumor responses in tumor-draining lymph nodes (tdLNs) but also support and regulate T cell functions in the tumor microenvironment (TME) (Gerhard et al., 2021Gerhard G.M. Bill R. Messemaker M. Klein A.M. Pittet M.J. Tumor-infiltrating dendritic cell states are conserved across solid human cancers.J. Exp. Med. 2021; 218: e20200264Crossref PubMed Google Scholar; Wculek et al., 2020Wculek S.K. Cueto F.J. Mujal A.M. Melero I. Krummel M.F. Sancho D. Dendritic cells in cancer immunology and immunotherapy.Nat. Rev. Immunol. 2020; 20: 7-24Crossref PubMed Scopus (772) Google Scholar). Developmental studies have identified two subsets of conventional DCs named cDC1s and cDC2s as well as plasmacytoid DCs (pDCs) as lineages distinct from monocytes, monocyte-derived DCs, and macrophages (Murphy et al., 2016Murphy T.L. Grajales-Reyes G.E. Wu X. Tussiwand R. Briseño C.G. Iwata A. Kretzer N.M. Durai V. Murphy K.M. Transcriptional Control of Dendritic Cell Development.Annu. Rev. Immunol. 2016; 34: 93-119Crossref PubMed Scopus (254) Google Scholar). cDC1s are more efficient at cross-presenting tumor cell-derived antigen to CTLs (Broz et al., 2014Broz M.L. Binnewies M. Boldajipour B. Nelson A.E. Pollack J.L. Erle D.J. Barczak A. Rosenblum M.D. Daud A. Barber D.L. et al.Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity.Cancer Cell. 2014; 26: 638-652Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), whereas cDC2s may be more relevant for CD4+ T cell activation (Binnewies et al., 2019Binnewies M. Mujal A.M. Pollack J.L. Combes A.J. Hardison E.A. Barry K.C. Tsui J. Ruhland M.K. Kersten K. Abushawish M.A. et al.Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity.Cell. 2019; 177: 556-571.e16Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). We recently identified an intratumoral DC state characterized by co-expression of IL12b, Fascin1, and the chemokine receptor gene CCR7, which we initially classified as cDC1s (Garris et al., 2018Garris C.S. Arlauckas S.P. Kohler R.H. Trefny M.P. Garren S. Piot C. Engblom C. Pfirschke C. Siwicki M. Gungabeesoon J. et al.Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12.Immunity. 2018; 49: 1148-1161.e7Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Reanalysis of these and additional mouse and human data led us to re-classify these cells as a discrete DC state we named DC3 (Gerhard et al., 2021Gerhard G.M. Bill R. Messemaker M. Klein A.M. Pittet M.J. Tumor-infiltrating dendritic cell states are conserved across solid human cancers.J. Exp. Med. 2021; 218: e20200264Crossref PubMed Google Scholar; Zilionis et al., 2019Zilionis R. Engblom C. Pfirschke C. Savova V. Zemmour D. Saatcioglu H.D. Krishnan I. Maroni G. Meyerovitz C.V. Kerwin C.M. et al.Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.Immunity. 2019; 50: 1317-1334.e10Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Others then reported on similar cell states in mouse and human tumors, to which they referred as LAMP3+ DC (Zhang et al., 2019Zhang Q. He Y. Luo N. Patel S.J. Han Y. Gao R. Modak M. Carotta S. Haslinger C. Kind D. et al.Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma.Cell. 2019; 179: 829-845.e20Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar), mregDC (Maier et al., 2020Maier B. Leader A.M. Chen S.T. Tung N. Chang C. LeBerichel J. Chudnovskiy A. Maskey S. Walker L. Finnigan J.P. et al.A conserved dendritic-cell regulatory program limits antitumour immunity.Nature. 2020; 580: 257-262Crossref PubMed Scopus (205) Google Scholar), or Ccl22+ cDC1s (Zhang et al., 2020Zhang L. Li Z. Skrzypczynska K.M. Fang Q. Zhang W. O’Brien S.A. He Y. Wang L. Zhang Q. Kim A. et al.Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.Cell. 2020; 181: 442-459.e29Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The respective roles of cDC1s, cDC2s and DC3s in intratumoral CTL activation and specifically how these cells support the differentiation of stem- to effector-like and to terminally differentiated CTLs requires further study. An unanswered question is how CTLs at various stages of differentiation navigate the TME in order to orchestrate their crosstalk with different DC subsets and ultimately to engage with their malignant target cells. Considering their well-established roles in lymphoid tissues, chemokines and their receptors are likely central orchestrators of this process. Inflammatory chemokine receptors such as CXCR3, CCR5, and CCR4 are generally assumed to be important for the recruitment of blood-borne T cells to tumor tissue, although this has only in some cases been directly demonstrated, e.g., for CXCR3 (Mikucki et al., 2015Mikucki M.E. Fisher D.T. Matsuzaki J. Skitzki J.J. Gaulin N.B. Muhitch J.B. Ku A.W. Frelinger J.G. Odunsi K. Gajewski T.F. et al.Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints.Nat. Commun. 2015; 6: 7458Crossref PubMed Scopus (291) Google Scholar). In addition, CXCR3 guides the local positioning of T cells in both lymphoid and non-lymphoid tissues (Ariotti et al., 2015Ariotti S. Beltman J.B. Borsje R. Hoekstra M.E. Halford W.P. Haanen J.B.A.G. de Boer R.J. Schumacher T.N.M. Subtle CXCR3-Dependent Chemotaxis of CTLs within Infected Tissue Allows Efficient Target Localization.J. Immunol. 2015; 195: 5285-5295Crossref PubMed Scopus (44) Google Scholar; Groom et al., 2012Groom J.R. Richmond J. Murooka T.T. Sorensen E.W. Sung J.H. Bankert K. von Andrian U.H. Moon J.J. Mempel T.R. Luster A.D. CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation.Immunity. 2012; 37: 1091-1103Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Expression of the CXCR3 ligand CXCL9 specifically by cDC is required for the efficacy of anti-PD-1 cancer immune checkpoint therapy through mechanisms unrelated to T cell trafficking from tdLNs to tumor tissue, hinting at a role for organizing local cDC interactions with tumor-infiltrating CXCR3+ T cells (Chow et al., 2019Chow M.T. Ozga A.J. Servis R.L. Frederick D.T. Lo J.A. Fisher D.E. Freeman G.J. Boland G.M. Luster A.D. Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy.Immunity. 2019; 50: 1498-1512.e5Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). However, the full spectrum of chemokine receptors and their ligands expressed in the TME by both immune and non-immune cells, but in particular by CTL subsets, has not been systematically explored. Here, we generated a comprehensive account of all chemokine and chemokine receptor genes expressed by all cells of the TME in mouse models of immunogenic cancer in order to provide a road map for the systematic exploration of their roles in organizing cellular interactions. We identified CXCR6 as the most highly expressed chemokine receptor in tumor-infiltrating CTLs and DC3s as the cell state most highly expressing its ligand CXCL16. Using multiphoton intravital microscopy (MP-IVM), we found that CXCR6 optimizes the positioning of TCF-1neg CTLs in perivascular clusters of DC3s in the tumor stroma and uncovered its critical role in rescuing the proliferative transitory CTL subset from activation-induced cell death (AICD) through exposure to trans-presented interleukin-15 (IL-15) cytokine, which was critical to sustain their population size and anti-tumor function. In order to explore chemokine receptors expressed by tumor-infiltrating CTLs, we used the immunogenic mouse melanoma model D4M.3A-pOVA (Di Pilato et al., 2019Di Pilato M. Kim E.Y. Cadilha B.L. Prüßmann J.N. Nasrallah M.N. Seruggia D. Usmani S.M. Misale S. Zappulli V. Carrizosa E. et al.Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.Nature. 2019; 570: 112-116Crossref PubMed Scopus (111) Google Scholar). Tumor single-cell suspensions were enriched for immune cells and all single-cell transcriptomes annotated to cell states (see STAR Methods). We detected three main cell clusters containing T and natural killer (NK) cell, myeloid cell, and non-immune cell states, as well as three minor clusters classified as pDC, B cell, and mast cell states (Figures 1A and S1A). Comparisons to published single-cell RNA sequencing (scRNA-seq) datasets revealed that T and NK cell states resembled those in MC38 mouse colorectal tumors, and CD8 T cell states resembled those in ovalbumin (OVA)-expressing B16.F10 mouse melanoma and in spleens of lymphocytic choriomeningitis virus (LCMV)-infected mice (Figure S1B) (Miller et al., 2019Miller B.C. Sen D.R. Al Abosy R. Bi K. Virkud Y.V. LaFleur M.W. Yates K.B. Lako A. Felt K. Naik G.S. et al.Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.Nat. Immunol. 2019; 20: 326-336Crossref PubMed Scopus (620) Google Scholar; Zhang et al., 2020Zhang L. Li Z. Skrzypczynska K.M. Fang Q. Zhang W. O’Brien S.A. He Y. Wang L. Zhang Q. Kim A. et al.Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.Cell. 2020; 181: 442-459.e29Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). DC states resembled those in KP1.9 mouse lung and in MC38 tumors (Figure S1C), mirroring DC state conservation observed across human solid cancers (Gerhard et al., 2021Gerhard G.M. Bill R. Messemaker M. Klein A.M. Pittet M.J. Tumor-infiltrating dendritic cell states are conserved across solid human cancers.J. Exp. Med. 2021; 218: e20200264Crossref PubMed Google Scholar; Maier et al., 2020Maier B. Leader A.M. Chen S.T. Tung N. Chang C. LeBerichel J. Chudnovskiy A. Maskey S. Walker L. Finnigan J.P. et al.A conserved dendritic-cell regulatory program limits antitumour immunity.Nature. 2020; 580: 257-262Crossref PubMed Scopus (205) Google Scholar; Zhang et al., 2020Zhang L. Li Z. Skrzypczynska K.M. Fang Q. Zhang W. O’Brien S.A. He Y. Wang L. Zhang Q. Kim A. et al.Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.Cell. 2020; 181: 442-459.e29Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar; Zilionis et al., 2019Zilionis R. Engblom C. Pfirschke C. Savova V. Zemmour D. Saatcioglu H.D. Krishnan I. Maroni G. Meyerovitz C.V. Kerwin C.M. et al.Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.Immunity. 2019; 50: 1317-1334.e10Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Cell state annotation was further validated by marker genes (Figure S1D) and distinct cell state-enriched gene expression revealing known marker genes (Figure S1E; Table S1A).Figure S1Cell states identified by scRNA-seq analysis of melanoma tissue, related to Figure 1Show full caption(A) Numbers of single-cell transcriptomes analyzed for each identified cell state in each experimental replicate.(B and C) Heatmaps showing the reciprocal similarity score for cell states identified in the T/NK cluster (B) and the myeloid cluster (C) in this study (Di Pilato), in Miller B. et al., 2019 (Miller and Miller LCMV), Zhang Q. et al., 2019 (Zhang), Maier et al., 2020 (Maier), and Zilionins R. et al., 2019 (Zilionis). The score was calculated using the probability estimates returned by the Linear Support Vector Machine classifier applied to log2-transformed data. The Di Pilato-CD8 T E state has internal heterogeneity because it has reciprocal similarity with both the CD8 T E proliferative state and CD8 T E state. Although we identified the subset of the CD8 T E state that is most similar to the CD8 T E proliferative state (marked by Mki67 on UMAP see Figure S1D), we decided not to annotate the CD8 T E proliferative state for the purpose of this paper.(D) Single-cell expression of the indicated genes.(E) Expression of cell state-enriched genes (see Table S1A for numerical data underlying the heatmap).(F) PD-1 expression of OT-I CTLs in D4M.3A-pOVA tumors 8 and 22 days following adoptive transfer into tumor-bearing mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Numbers of single-cell transcriptomes analyzed for each identified cell state in each experimental replicate. (B and C) Heatmaps showing the reciprocal similarity score for cell states identified in the T/NK cluster (B) and the myeloid cluster (C) in this study (Di Pilato), in Miller B. et al., 2019 (Miller and Miller LCMV), Zhang Q. et al., 2019 (Zhang), Maier et al., 2020 (Maier), and Zilionins R. et al., 2019 (Zilionis). The score was calculated using the probability estimates returned by the Linear Support Vector Machine classifier applied to log2-transformed data. The Di Pilato-CD8 T E state has internal heterogeneity because it has reciprocal similarity with both the CD8 T E proliferative state and CD8 T E state. Although we identified the subset of the CD8 T E state that is most similar to the CD8 T E proliferative state (marked by Mki67 on UMAP see Figure S1D), we decided not to annotate the CD8 T E proliferative state for the purpose of this paper. (D) Single-cell expression of the indicated genes. (E) Expression of cell state-enriched genes (see Table S1A for numerical data underlying the heatmap). (F) PD-1 expression of OT-I CTLs in D4M.3A-pOVA tumors 8 and 22 days following adoptive transfer into tumor-bearing mice. The T/NK cell cluster contained an NK cell state (NK), a CD4+ T cell state containing both regulatory and helper T cells (CD4 T R/H), as well as two CD8+ T cell states annotated as effector-like (CD8 T E) and memory-like (CD8 T M). CD8 T E expressed the cytotoxic effector gene Gzmb and Havcr2 (encoding TIM-3), while CD8 T M expressed the memory gene Nsg2 (Best et al., 2013Best J.A. Blair D.A. Knell J. Yang E. Mayya V. Doedens A. Dustin M.L. Goldrath A.W. Immunological Genome Project ConsortiumTranscriptional insights into the CD8(+) T cell response to infection and memory T cell formation.Nat. Immunol. 2013; 14: 404-412Crossref PubMed Scopus (215) Google Scholar) and Tcf7, which encodes TCF-1 expressed by naive as well as stem-like CTLs (Utzschneider et al., 2016Utzschneider D.T. Charmoy M. Chennupati V. Pousse L. Ferreira D.P. Calderon-Copete S. Danilo M. Alfei F. Hofmann M. Wieland D. et al.T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections.Immunity. 2016; 45: 415-427Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar) (Figure 1B). The by far most highly expressed chemokine receptor gene in both CD8 T cell states was Cxcr6, followed by Cx3cr1 in the CD8 T E state, and by Cxcr4, Cxcr3, Ccr7, and lower amounts of Cxcr5 in the CD8 T M state (Figures 1C and 1D; Table S1B). Cxcr6 was also present, but much less abundant, in some NK and CD4 T R/H cell states. When validating gene expression at the protein level by flow cytometry, we used the T cell activation marker PD-1 (Honda et al., 2014Honda T. Egen J.G. Lämmermann T. Kastenmüller W. Torabi-Parizi P. Germain R.N. Tuning of antigen sensitivity by T cell receptor-dependent negative feedback controls T cell effector function in inflamed tissues.Immunity. 2014; 40: 235-247Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) to focus our analysis on tumor-reactive CTLs and exclude bystander CTLs with other, for instance, anti-viral reactivities (Rosato et al., 2019Rosato P.C. Wijeyesinghe S. Stolley J.M. Nelson C.E. Davis R.L. Manlove L.S. Pennell C.A. Blazar B.R. Chen C.C. Geller M.A. et al.Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy.Nat. Commun. 2019; 10: 567Crossref PubMed Scopus (111) Google Scholar; Scheper et al., 2019Scheper W. Kelderman S. Fanchi L.F. Linnemann C. Bendle G. de Rooij M.A.J. Hirt C. Mezzadra R. Slagter M. Dijkstra K. et al.Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers.Nat. Med. 2019; 25: 89-94Crossref PubMed Scopus (261) Google Scholar; Simoni et al., 2018Simoni Y. Becht E. Fehlings M. Loh C.Y. Koo S.-L. Teng K.W.W. Yeong J.P.S. Nahar R. Zhang T. Kared H. et al.Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates.Nature. 2018; 557: 575-579Crossref PubMed Scopus (603) Google Scholar). While TCF-1neg effector-like cells expressed PD-1 almost uniformly, only a fraction of TIM-3– TCF-1pos stem-like CTLs, which gradually declined over time, expressed this receptor (Figures 1E and 1F). When we transferred CD8-depleted mice with congenic, highly purified CD44low CD62Lhi naive CD8+ T cells that require prior activation to enter the TME, all of their tumor-infiltrating progeny, both TCF-1pos and TCF-1neg, expressed PD-1 (Figure 1G), similarly to adoptively transferred clonal populations of TCR transgenic OT-I cells recognizing the tumor cell-expressed SIINFEKL neoepitope (Figure S1F). Hence, PD-1 expression identifies tumor-reactive CTLs. CXCR6 was also the by far most highly expressed chemokine receptor protein on CTLs on day 18 of tumor growth, mirroring our transcriptional analysis (Figures 1H and 1I). Three discrete populations with negative/low, intermediate, and high expression were apparent among PD-1+ stem-like CTLs, while PD-1+ effector-like CTLs uniformly expressed CXCR6 at the highest level. Considering the lineage relationship between TCF-1pos and TCF-1neg cells (Siddiqui et al., 2019Siddiqui I. Schaeuble K. Chennupati V. Fuertes Marraco S.A. Calderon-Copete S. Pais Ferreira D. Carmona S.J. Scarpellino L. Gfeller D. Pradervand S. et al.Intratumoral Tcf1+PD-1+CD8+ T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy.Immunity. 2019; 50: 195-211.e10Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar; Utzschneider et al., 2016Utzschneider D.T. Charmoy M. Chennupati V. Pousse L. Ferreira D.P. Calderon-Copete S. Danilo M. Alfei F. Hofmann M. Wieland D. et al.T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections.Immunity. 2016; 45: 415-427Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar), this pattern suggests that full CXCR6 upregulation immediately precedes or accompanies loss of TCF-1 expression in tumor-reactive PD-1+ CTLs. In contrast, PD-1– CTLs, both TCF-1pos and TCF-1neg, were either CXCR6-low/negative or -intermediate but rarely high. PD-1+ TCF-1neg CTLs also upregulated CX3CR1, CCR5, and CCR2 but downregulated CXCR3. Generally, inflammatory chemokine receptors were more highly expressed by PD-1+ than PD-1– CTLs, indicating that they were induced or sustained in the TME through TCR activation (Figures 1H and 1I). Again, CXCR3 formed an exception and was most highly expressed by PD-1– TCF-1pos bystander CT" @default.
• W3189614948 created "2021-08-16" @default.
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• W3189614948 date "2021-08-01" @default.
• W3189614948 modified "2023-10-17" @default.
• W3189614948 title "CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment" @default.
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