Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop

•The TCR repertoire of Treg cells is enriched for reactivity to antigens in the TME•Tumor Treg cells use CTLA-4 to destabilize their own interactions with dendritic cells•CTLA-4 blockade causes the CD28-mediated expansion of tumor-associated Treg cells•Following CTLA-4 blockade, Treg cells continue to promote tumor immune tolerance Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients. Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients. Checkpoint blockade immunotherapy (CBI) enhances anti-tumor immune responses by inhibiting the interaction of molecules that restrain the activation and effector function of T cells, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and PD-1, with their ligands. This has allowed for long-term progression-free survival of a subset of cancer patients with otherwise dismal prognoses (Borghaei et al., 2015Borghaei H. Paz-Ares L. Horn L. Spigel D.R. Steins M. Ready N.E. Chow L.Q. Vokes E.E. Felip E. Holgado E. et al.Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer.N. Engl. J. Med. 2015; 373: 1627-1639Crossref PubMed Scopus (6181) Google Scholar; Ferris et al., 2016Ferris R.L. Blumenschein Jr., G. Fayette J. Guigay J. Colevas A.D. Licitra L. Harrington K. Kasper S. Vokes E.E. Even C. et al.Nivolumab for recurrent squamous-cell carcinoma of the head and neck.N. Engl. J. Med. 2016; 375: 1856-1867Crossref PubMed Scopus (2520) Google Scholar; Motzer et al., 2018Motzer R.J. Tannir N.M. McDermott D.F. Arén Frontera O. Melichar B. Choueiri T.K. Plimack E.R. Barthélémy P. Porta C. George S. et al.CheckMate 214 InvestigatorsNivolumab plus Ipilimumab versus Sunitinib in advanced renal-cell carcinoma.N. Engl. J. Med. 2018; 378: 1277-1290Crossref PubMed Scopus (2109) Google Scholar; Wolchok et al., 2017Wolchok J.D. Chiarion-Sileni V. Gonzalez R. Rutkowski P. Grob J.J. Cowey C.L. Lao C.D. Wagstaff J. Schadendorf D. Ferrucci P.F. et al.Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma.N. Engl. J. Med. 2017; 377: 1345-1356Crossref PubMed Scopus (1960) Google Scholar). Immune checkpoints are, however, only one component of a multi-layered system of regulatory mechanisms that synergize in the tumor microenvironment (TME) to antagonize immune-mediated elimination of malignant cells (Binnewies et al., 2018Binnewies M. Roberts E.W. Kersten K. Chan V. Fearon D.F. Merad M. Coussens L.M. Gabrilovich D.I. Ostrand-Rosenberg S. Hedrick C.C. et al.Understanding the tumor immune microenvironment (TIME) for effective therapy.Nat. Med. 2018; 24: 541-550Crossref PubMed Scopus (1651) Google Scholar). Extending the benefits of CBI to more patients will likely require concurrent targeting of additional immune-regulatory mechanisms (Sanmamed and Chen, 2018Sanmamed M.F. Chen L. A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization.Cell. 2018; 175: 313-326Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). The activities of T regulatory (Treg) cells constitute another layer of immune regulation and include modulation of antigen presenting cell (APC) function, secretion of inhibitory cytokines, and consumption of T cell survival factors (Vignali et al., 2008Vignali D.A.A. Collison L.W. Workman C.J. How regulatory T cells work.Nat. Rev. Immunol. 2008; 8: 523-532Crossref PubMed Scopus (2055) Google Scholar). Consequently, approaches to target Treg cells are an important element of efforts to improve cancer therapy (Sakaguchi et al., 2020Sakaguchi S. Mikami N. Wing J.B. Tanaka A. Ichiyama K. Ohkura N. Regulatory T Cells and Human Disease.Annu. Rev. Immunol. 2020; 38: 541-566Crossref PubMed Scopus (179) Google Scholar; Wing et al., 2019Wing J.B. Tanaka A. Sakaguchi S. Human FOXP3+ Regulatory T Cell Heterogeneity and Function in Autoimmunity and Cancer.Immunity. 2019; 50: 302-316Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The mechanisms that prevent tumor eradication in cancer patients have evolved in order to maintain immune homeostasis in healthy individuals by containing self-reactive responses and to regulate the magnitude and duration of anti-pathogen responses to prevent exaggerated, pathogenic immune activation. However, these tolerance mechanisms need to be calibrated in order to avoid over-regulation of appropriate immune responses, which would impede pathogen elimination. The dependence of Treg cells on interleukin (IL)-2 produced by activated effector T cells, which ties Treg cell activation and expansion to the magnitude of the effector response, is one mechanism through which this control is achieved in secondary lymphoid organs (SLOs) (Liu et al., 2015Liu Z. Gerner M.Y. Van Panhuys N. Levine A.G. Rudensky A.Y. Germain R.N. Immune homeostasis enforced by co-localized effector and regulatory T cells.Nature. 2015; 528: 225-230Crossref PubMed Scopus (180) Google Scholar; O’Gorman et al., 2009O’Gorman W.E. Dooms H. Thorne S.H. Kuswanto W.F. Simonds E.F. Krutzik P.O. Nolan G.P. Abbas A.K. The initial phase of an immune response functions to activate regulatory T cells.J. Immunol. 2009; 183: 332-339Crossref PubMed Scopus (116) Google Scholar). However, how Treg cell function is regulated at effector sites, and especially in the TME, is not known. The ability of Treg cells to establish tumor tolerance depends on their infiltration and local cognate antigen encounter in the TME (Bauer et al., 2014Bauer C.A. Kim E.Y. Marangoni F. Carrizosa E. Claudio N.M. Mempel T.R. Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction.J. Clin. Invest. 2014; 124: 2425-2440Crossref PubMed Scopus (144) Google Scholar). This indicates that their function needs to be continually sustained by T cell antigen receptor (TCR) signals or by other signals that they receive during antigen-dependent cellular interactions. Accordingly, Treg cells isolated from human tumors express genes that reflect cellular activation (Plitas et al., 2016Plitas G. Konopacki C. Wu K. Bos P.D. Morrow M. Putintseva E.V. Chudakov D.M. Rudensky A.Y. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.Immunity. 2016; 45: 1122-1134Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar; De Simone et al., 2016De Simone M. Arrigoni A. Rossetti G. Gruarin P. Ranzani V. Politano C. Bonnal R.J.P. Provasi E. Sarnicola M.L. Panzeri I. et al.Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.Immunity. 2016; 45: 1135-1147Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar; Zheng et al., 2017Zheng C. Zheng L. Yoo J.K. Guo H. Zhang Y. Guo X. Kang B. Hu R. Huang J.Y. Zhang Q. et al.Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.Cell. 2017; 169: 1342-1356.e16Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar) and whose expression by Treg cells depends on TCR recognition in mice (Levine et al., 2014Levine A.G. Arvey A. Jin W. Rudensky A.Y. Continuous requirement for the TCR in regulatory T cell function.Nat. Immunol. 2014; 15: 1070-1078Crossref PubMed Scopus (328) Google Scholar; Vahl et al., 2014Vahl J.C. Drees C. Heger K. Heink S. Fischer J.C. Nedjic J. Ohkura N. Morikawa H. Poeck H. Schallenberg S. et al.Continuous T cell receptor signals maintain a functional regulatory T cell pool.Immunity. 2014; 41: 722-736Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). However, whether Treg cells recognize their cognate antigens indiscriminately on a wide range of tumor-associated APC, or through interactions with a specific subset, has not been examined. Here, we used multiphoton intravital microscopy (MP-IVM) and a fluorescent reporter of nuclear factor of activated T cell (NFAT) activation to directly visualize how Treg cells receive TCR signals in the TME. We found that the preferentially self-reactive polyclonal Treg cell pool is pre-enriched for recognition of antigen in the TME. We also observed that unlike conventional CD4+ T helper (Th) cells, which were activated during stable APC contacts, tumor-infiltrating Treg cells were sustained through transient TCR signals that they received during unstable, short-lived interactions with conventional dendritic cells (cDCs), but not tumor-associated macrophages (TAMs). This contact instability was, however, not cell-intrinsic, but resulted from Treg cell-mediated and CTLA-4-dependent downregulation of CD80 and CD86 proteins on cDCs, which limited Treg cell co-stimulation via CD28 and controlled their proliferation within tumors. Disruption of this negative feedback loop through CTLA-4-targeted CBI stabilized Treg cell-cDC contacts in the TME, boosted Treg cell proliferation, and induced their local expansion in excess of that of effector T cells. Inactivating NFAT signaling in Treg cells during CTLA-4-targeted CBI unleashed its therapeutic anti-tumor efficacy, indicating that tumor-associated Treg cells continue to promote tumor tolerance even when their CTLA-4-dependent immune-suppressive function is disrupted. To examine the single cell-dynamics of Treg cell activation in tumor tissue by in vivo imaging, we selected a fusion of the NFAT1 regulatory domain and GFP (“NFAT-GFP”) as a fluorescent reporter of productive TCR engagement (Aramburu et al., 1998Aramburu J. Garcia-Cózar F. Raghavan A. Okamura H. Rao A. Hogan P.G. Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT.Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). NFAT-GFP accumulates in the nucleus of CD8+ T cells within 1–3 min after they form cognate APC contacts and redistributes to the cytoplasm with a half-life of 20 min upon contact cessation (Marangoni et al., 2013Marangoni F. Murooka T.T. Manzo T. Kim E.Y. Carrizosa E. Elpek N.M. Mempel T.R. The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells.Immunity. 2013; 38: 237-249Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We activated and transduced purified CD4+ Foxp3+ Treg cells from the SLOs of C57BL/6 mice with a bicistronic retroviral vector expressing NFAT-GFP and the fluorescent histone fusion protein H2B-RFP as a genetically encoded nuclear marker (Figure S1A). As a reference population, we used CD4+ Foxp3– Th cells (Figure S1B). Six days later, Treg, but not Th cells, expressed Foxp3 (Figure S1C) and both coordinately expressed NFAT-GFP and H2B-RFP (Figure S1D). These cells were then separately intravenously (i.v.) injected into sublethally irradiated CD11cmCherry reporter mice. Six weeks later, mice were implanted subcutaneously (s.c.) in the back with MC38 colon carcinoma expressing a blue fluorescent histone H2B-Cerulean fusion protein (hereafter “MC38 H2B-Cer”), a model in which Treg cells strongly restrain anti-tumor immunity (Arce Vargas et al., 2017Arce Vargas F. Furness A.J.S. Solomon I. Joshi K. Mekkaoui L. Lesko M.H. Miranda Rota E. Dahan R. Georgiou A. Sledzinska A. et al.Melanoma TRACERx ConsortiumRenal TRACERx ConsortiumLung TRACERx ConsortiumFc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors.Immunity. 2017; 46: 577-586Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar; Imai et al., 2007Imai H. Saio M. Nonaka K. Suwa T. Umemura N. Ouyang G.-F. Nakagawa J. Tomita H. Osada S. Sugiyama Y. et al.Depletion of CD4+CD25+ regulatory T cells enhances interleukin-2-induced antitumor immunity in a mouse model of colon adenocarcinoma.Cancer Sci. 2007; 98: 416-423Crossref PubMed Scopus (80) Google Scholar; Mahne et al., 2017Mahne A.E. Mauze S. Joyce-Shaikh B. Xia J. Bowman E.P. Beebe A.M. Cua D.J. Jain R. Dual Roles for Regulatory T-cell Depletion and Costimulatory Signaling in Agonistic GITR Targeting for Tumor Immunotherapy.Cancer Res. 2017; 77: 1108-1118Crossref PubMed Scopus (69) Google Scholar). Ex vivo TCR-activation induces loss of the SLO homing receptor CCR7 and gain of trafficking receptors for migration into inflamed tissues, enabling Treg, and likely also Th cells, to directly infiltrate tumor tissue, bypassing the requirement for antigenic priming in tdLNs (Wang et al., 2012Wang C. Lee J.H. Kim C.H. Optimal population of FoxP3+ T cells in tumors requires an antigen priming-dependent trafficking receptor switch.PLoS ONE. 2012; 7: e30793Crossref PubMed Scopus (29) Google Scholar). Accordingly, 6 days after tumor implantation, when we installed dorsal skinfold chambers (DSFCs) around tumors to allow for MP-IVM analysis (Figure 1A), adoptively transferred Foxp3+ Treg cells and Foxp3– Th cells had accumulated in tumors (Figure 1B). Four and 5 days later, Treg cells were found both in the tumor parenchyma, identified by the blue fluorescence of tumor nuclei, as well as in the surrounding stroma. Treg cells in close vicinity or direct contact with CD11c-mCherry+ APCs frequently reduced their motility and redistributed NFAT-GFP from their cytoplasm to the nucleus (Figure 1C; Video S1). However, NFAT-GFP nuclear accumulation was not accompanied by sustained migratory arrest but occurred during low speed-scanning of APC surfaces (Video S2). Treg cells also transiently maintained NFAT activity while moving away from APCs, likely reflecting delayed nuclear export of NFAT1 upon cessation of a TCR stimulus (Figure S1E), as previously reported for CD8+ effector T cells (Marangoni et al., 2013Marangoni F. Murooka T.T. Manzo T. Kim E.Y. Carrizosa E. Elpek N.M. Mempel T.R. The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells.Immunity. 2013; 38: 237-249Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In contrast to Tregs, nearly all Th cells migrated at higher speed, did not arrest, and did not accumulate NFAT-GFP in the nucleus (Figures 1D–1F; Video S1). This indicated that most Th cells did not recognize antigen, in line with the prediction that transferred Th and Treg cells would predominantly be recruited to the TME based on homing receptor expression induced during polyclonal ex vivo activation. https://www.cell.com/cms/asset/41f2afdc-92ee-42a1-be50-ee54e17d61b9/mmc1.mp4Loading ... Download .mp4 (7.08 MB) Help with .mp4 files Video S1. Polyclonal Treg cells are frequently activated in tumors, related to Figure 1Left: Visualization of productive interactions of NFAT-GFP (green) and H2B-RFP (red)-expressing polyclonal Treg (top) and Th cells (bottom) with CD11c-mCherry+ APC (magenta) within the TME (tumor cell nuclei in blue). Right: T cell tracks and NFAT SI, color coded from blue (no signaling) to red (signaling). NFAT activation is frequently observed in Treg but not in Th cells. Scale bar represents 20 μm; time is in h:min:sec. https://www.cell.com/cms/asset/27ca8568-2168-4198-bb4f-e518a865c1e0/mmc2.mp4Loading ... Download .mp4 (3.7 MB) Help with .mp4 files Video S2. An instance of polyclonal Treg cell activation within the TME, related to Figure 1An example of a polyclonal Treg cell translocating NFAT-GFP (green) to the H2B-RFP counterstained nucleus (red) after establishing contact with mCherry+ APC (magenta) in MC38 tumors expressing H2B-Cerulean (blue nuclei). Scale bar represents 20 μm; time is in h:min:sec. To quantify the rate of T cell activation, we devised an automated algorithm to measure 3D central accumulation of NFAT-GFP in T cells, which was validated against manual as well as automated measurements of nuclear accumulation through independent methods (Figures S1F–S1L). This algorithm produced an instantaneous NFAT signaling index (NFAT SI), ranging from 0 to 1, and we defined 0.5 as the threshold above which we considered NFAT to be activated. By this measure, ~40% of all observed Treg cells (compared to 6% of Th cells) were activated, both based on instantaneous NFAT SI as well as on the frequency of individual cells with an NFAT SI >0.5 during more than 20% of the time they were observed (Figures 1G and 1H). Although Treg and especially rare Th cell activation events were associated with lower migratory velocity (Figure 1I), suggesting that they occurred during APC interactions, Treg cells also maintained NFAT activity while moving away from APCs. However, the onset of NFAT activity always occurred in direct contact with an APC (Figure 1J). Considering the likely antigen-independent recruitment of ex vivo activated Treg and Th cells to tumors, these observations suggest that the polyclonal Treg cell repertoire is pre-enriched for specificity for antigens presented in the TME, whereas only rare cells among the Th cell repertoire recognize such antigens. Second, although tumor-reactive Treg cells only partially stabilize their interactions with APCs in tumor tissue, these unstable interactions trigger productive TCR signals that activate the NFAT pathway. Although NFAT nuclear accumulation in Treg cells was always initiated in contact with a CD11c-mCherry+ APC, many APC contacts did not trigger NFAT-GFP activity. This raised the possibility that only a subset of CD11c+ APCs interact with Treg cells in a productive manner. Indeed, among all mCherry+ cells in tumor tissue, the majority were CD11c+ CD64+ F4/80+ TAM, as described (Franklin et al., 2014Franklin R.A. Liao W. Sarkar A. Kim M.V. Bivona M.R. Liu K. Pamer E.G. Li M.O. The cellular and molecular origin of tumor-associated macrophages.Science. 2014; 344: 921-925Crossref PubMed Scopus (759) Google Scholar), and only a small fraction were cDCs. Among those, approximately half were CD172a+ cDC2 and fewer were XCR1+ cDC1 (Figures S2A and S2B). Because cDCs help recruit and activate tumor-infiltrating cytotoxic T lymphocytes (CTL) (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 (531) Google Scholar; Spranger et al., 2017Spranger S. Dai D. Horton B. Gajewski T.F. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy.Cancer Cell. 2017; 31: 711-723.e4Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar), we hypothesized that they may also activate Treg cells. We therefore generated zDCDTR → B6 irradiation bone marrow chimeras (BMCs), where cDCs can be acutely ablated through diphtheria toxin (DT) treatment (Meredith et al., 2012Meredith M.M. Liu K. Darrasse-Jeze G. Kamphorst A.O. Schreiber H.A. Guermonprez P. Idoyaga J. Cheong C. Yao K.-H. Niec R.E. Nussenzweig M.C. Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage.J. Exp. Med. 2012; 209: 1153-1165Crossref PubMed Scopus (355) Google Scholar). DT-treatment reduced the frequencies of XCR1-expressing cDC1 and CD172a-expressing cDC2 in the MC38 TME by 90% and 70%, respectively, relative to either vehicle-treated zDCDTR → B6 or DT-treated B6 → B6 control BMCs, while TAMs were not affected (Figures S2C and S2D). We transferred NFAT-GFP-expressing Treg cells into BMCs 3 weeks after bone marrow transfer, when the T cell compartment was not yet reconstituted, in order to enhance their engraftment. Another 3 weeks later, we implanted MC38 H2B-Cer tumors, installed DSFC, and treated animals either with DT or vehicle (Figure 2A). MP-IVM analysis of tumor-infiltrating Treg cells in vehicle-treated animals revealed a similar pattern of restrained cell motility and frequent NFAT nuclear accumulation as described above. In contrast, Treg cell migration was unconstrained and the frequency of NFAT activation events was reduced from about one third to 6%–7% in DT-treated mice (Figures 2B–2D; Video S3). Thus, local TCR-dependent Treg cell activation within the TME predominantly relies on antigen presentation by cDC.Figure 2cDCs activate Treg cells locally within the TMEShow full caption(A) Experimental protocol.(B) MP-IVM micrographs of Treg cells in MC38 tumors in zDCDTR bone marrow chimeras. Images on right show representative migratory tracks and false-color-coded NFAT SI. Scale bar, 50 μm.(C) Distribution of instantaneous Treg cell NFAT SI in the TME in presence (black, n = 933 from 15 cells) or following ablation (red, n = 824 from 28 cells) of cDC. Gate frequencies refer to NFAT SI >0.5.(D) The fraction of time in which NFAT SI >0.5 in individual Treg cells. Percentages above graphs refer to frequencies of cells in which NFAT >0.5 for greater than 20% of time. Data in (C) and (D) are pooled from 4 recordings per condition.(E) Experimental protocol.(F and G) Frequency (gated on CD45+ cells) (F) and activation markers (G) on Treg cells in the TME following ablation of cDC. n = 8 mice per group pooled from two independent experiments.(H) Experimental protocol.(I and J) Frequency of Treg cells in the TME and expression of Foxp3 (I), and of activation markers (J) after CnB depletion. For Treg cell quantification, n = 12–16 in 3 independent experiments. For marker expression analysis, n = 7–11 from two separate experiments. Bars depict medians, symbols individual mice. MFIs normalized by the mean of control groups. p values calculated by Mann-Whitney U test. Fold change as compared to control groups in parentheses.See also Figures S2 and S3 and Video S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Experimental protocol. (B) MP-IVM micrographs of Treg cells in MC38 tumors in zDCDTR bone marrow chimeras. Images on right show representative migratory tracks and false-color-coded NFAT SI. Scale bar, 50 μm. (C) Distribution of instantaneous Treg cell NFAT SI in the TME in presence (black, n = 933 from 15 cells) or following ablation (red, n = 824 from 28 cells) of cDC. Gate frequencies refer to NFAT SI >0.5. (D) The fraction of time in which NFAT SI >0.5 in individual Treg cells. Percentages above graphs refer to frequencies of cells in which NFAT >0.5 for greater than 20% of time. Data in (C) and (D) are pooled from 4 recordings per condition. (E) Experimental protocol. (F and G) Frequency (gated on CD45+ cells) (F) and activation markers (G) on Treg cells in the TME following ablation of cDC. n = 8 mice per group pooled from two independent experiments. (H) Experimental protocol. (I and J) Frequency of Treg cells in the TME and expression of Foxp3 (I), and of activation markers (J) after CnB depletion. For Treg cell quantification, n = 12–16 in 3 independent experiments. For marker expression analysis, n = 7–11 from two separate experiments. Bars depict medians, symbols individual mice. MFIs normalized by the mean of control groups. p values calculated by Mann-Whitney U test. Fold change as compared to control groups in parentheses. See also Figures S2 and S3 and Video S3. https://www.cell.com/cms/asset/e02b5ac2-e0b4-4bb8-b1a5-796b08538601/mmc3.mp4Loading ... Download .mp4 (8.53 MB) Help with .mp4 files Video S3. Activation of tumor-associated Treg cells depends on cDCs, related to Figure 2Left: Activation of polyclonal Treg cells (NFAT-GFP, green; H2B-RFP, red) in MC38 H2B-Cer tumors (blue nuclei) was recorded in zDCDTR bone marrow chimeric mice in which conventional DC (cDC) were present (top row) or ablated though DT treatment (bottom row) 48h earlier. Right: Cell tracks and NFAT SI, color coded from blue (no signaling) to red (signaling). NFAT activation in Treg cells depends on the presence of cDC. Scale bar represents 20 μm; time is in h:min:sec. To test the functional relevance of Treg cell interactions with cDCs specifically in tumor tissue, we ablated cDCs while at the same time blocking lymphocyte egress from SLOs using the functional S1P-receptor antagonist FTY720 (Figure 2E) to prevent further recruitment of Treg cells from tdLNs to tumors, as described (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 (94) Google Scholar). Treatment with FTY720 alone increased the frequency of Treg cells in tdLN, as expected, accompanied by a decrease in tumor tissue. Treg cells in the TME also decreased their expression of Foxp3 and of markers of effector Treg (eTreg) cell differentiation (Figures S2E and S2F), revealing that the tumor-infiltrating Treg cell pool is continuously replenished from SLO. However, upon concomitant ablation of cDC, tumor-associated Treg cells became even less abundant and expressed even lower levels of Foxp3 (Figure 2F), proliferated less, and further reduced their expression of activation markers, such as CTLA-4, ICOS and GITR (Figure 2G). cDC-depletion also decreased the abundance of tumor-associated Th cells, albeit less profoundly than for Treg cells (Figure S2G). Similar changes were observed in tdLN (Figures S2G–S2I), in line with prior studies on CD11c+ DC depletion (Darrasse-Jèze et al., 2009Darrasse-Jèze G. Deroubaix S. Mouquet H. Victora G.D. Eisenreich T. Yao K.H. Masilamani R.F. Dustin M.L. Rudensky A. Liu K. Nussenzweig M.C. Feedback control of regulatory T cell homeostasis by dendritic cells in vivo.J. Exp. Med. 2009; 206: 1853-1862Crossref PubMed Scopus (319) Google Scholar). Thus, despite low frequency, cDCs play a non-redundant role in local Treg cell activation to maintain their population size in the TME. To examine the relevance of the NFAT pathway for the maintenance of intratumoral Treg cells, we s.c. implanted MC38 tumors in the flanks of Foxp3creERT2 x CnBf/f mice. This allowed for tamoxifen-inducible deletion of the regulatory B subunit of the phosphatase calcineurin (CnB), which is required for TCR-induced NFAT activation (Marangoni et al., 2018Marangoni F. Zhang R. Mani V. Thelen M. Ali Akbar N.J. Warner R.D. Äijö T. Zappulli V. Martinez G.J. Turka L.A. Mempel T.R. Tumor Tolerance-Promoting Function of Regulatory T Cells Is Optimized by CD28, but Strictly Dependent on Calcineurin.J. Immunol. 2018; 200: 3647-3661Google Scholar). Following CnB deletion in Treg cells and concurrent FTY720 treatment (Figures 2H and S3A), Treg cells in tumors declined in frequency, expressed less Foxp3, proliferated less, and expressed less CTLA-4, while their ICOS levels remained unchanged (Figures 2I and 2J). CnB deletion without concurrent FTY720 treatment also diminished Treg cell proliferation and expression of markers of eTreg cell differentiation, but the decrease in Treg cell frequency did not reach statistical significance, presumably due to continued influx of eTreg cells generated in SLOs already prior to CnB deletion (Figures S3B and S3C). Thus, interruption of the CnB-NFAT signaling axis had similar effects on tumor-infiltrating Treg cells as removal of cDC, suggesting a critical role of this pathway among the signaling activities triggered by their interactions with cDC. Considering that the TCR repertoir