Mitochondrial metabolism sustains CD8+ T cell migration for an efficient infiltration into solid tumors.

The ability of CD8+ T cells to infiltrate solid tumors and reach cancer cells is associated with improved patient survival and responses to immunotherapy. Thus, identifying the factors controlling T cell migration in tumors is critical, so that strategies to intervene on these targets can be developed. Although interstitial motility is a highly energy-demanding process, the metabolic requirements of CD8+ T cells migrating in a 3D environment remain unclear. Here, we demonstrate that the tricarboxylic acid (TCA) cycle is the main metabolic pathway sustaining human CD8+ T cell motility in 3D collagen gels and tumor slices while glycolysis plays a more minor role. Using pharmacological and genetic approaches, we report that CD8+ T cell migration depends on the mitochondrial oxidation of glucose and glutamine, but not fatty acids, and both ATP and ROS produced by mitochondria are required for T cells to migrate. Pharmacological interventions to increase mitochondrial activity improve CD8+ T cell intratumoral migration and CAR T cell recruitment into tumor islets leading to better control of tumor growth in human xenograft models. Our study highlights the rationale of targeting mitochondrial metabolism to enhance the migration and antitumor efficacy of CAR T cells in treating solid tumors.

A biology-driven deep generative model for cell-type annotation in cytometry.

Cytometry enables precise single-cell phenotyping within heterogeneous populations. These cell types are traditionally annotated via manual gating, but this method lacks reproducibility and sensitivity to batch effect. Also, the most recent cytometers-spectral flow or mass cytometers-create rich and high-dimensional data whose analysis via manual gating becomes challenging and time-consuming. To tackle these limitations, we introduce Scyan https://github.com/MICS-Lab/scyan, a Single-cell Cytometry Annotation Network that automatically annotates cell types using only prior expert knowledge about the cytometry panel. For this, it uses a normalizing flow-a type of deep generative model-that maps protein expressions into a biologically relevant latent space. We demonstrate that Scyan significantly outperforms the related state-of-the-art models on multiple public datasets while being faster and interpretable. In addition, Scyan overcomes several complementary tasks, such as batch-effect correction, debarcoding and population discovery. Overall, this model accelerates and eases cell population characterization, quantification and discovery in cytometry.

Dynamic CD8+ T Cell Cooperation with Macrophages and Monocytes for Successful Cancer Immunotherapy.

The essential roles endorsed by macrophages and monocytes are well established in response to infections, where they contribute to launching the differentiation of specific T-lymphocytes for long-term protection. This knowledge is the result of dynamic studies that can inspire the cancer field, particularly now that cancer immunotherapies elicit some tumor regression. Indeed, immune responses to cancer have mainly been studied after tumors have escaped immune attacks. In particular, the suppressive functions of macrophages were revealed in this context, introducing an obvious bias across the literature. In this review, we will focus on the ways inwhich monocytes and macrophages cooperate with T-lymphocytes, leading to successful immune responses. We will bring together the preclinical studies that have revealed the existence of such positive cooperation in the cancer field, and we will place particular emphasis on proposing the underlying mechanisms. Finally, we will give some perspectives to decipher the functional roles of such T-cell and myeloid cell interactions in the frame of human cancer immunotherapy.

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse.

BACKGROUND: Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase. METHODS: We designed a mouse model of tumor relapse in which mice transplanted with E7+ TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases. RESULTS: We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival. CONCLUSIONS: Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse.

Preclinical murine tumor models: a structural and functional perspective.

The goal of this review is to pinpoint the specific features, including the weaknesses, of various tumor models, and to discuss the reasons why treatments that are efficient in murine tumor models often do not work in clinics. In a detailed comparison of transplanted and spontaneous tumor models, we focus on structure-function relationships in the tumor microenvironment. For instance, the architecture of the vascular tree, which depends on whether tumor cells have gone through epithelial-mesenchymal transition, is determinant for the extension of the spontaneous necrosis, and for the intratumoral localization of the immune infiltrate. Another key point is the model-dependent abundance of TGFß in the tumor, which controls the variable susceptibility of different tumor models to treatments. Grounded in a historical perspective, this review provides a rationale for checking factors that will be key for the transition between preclinical murine models and clinical applications.

Blockade of ß-Adrenergic Receptors Improves CD8+ T-cell Priming and Cancer Vaccine Efficacy.

ß-Adrenergic receptor (ß-AR) signaling exerts protumoral effects by acting directly on tumor cells and angiogenesis. In addition, ß-AR expression on immune cells affects their ability to mount antitumor immune responses. However, how ß-AR signaling impinges antitumor immune responses is still unclear. Using a mouse model of vaccine-based immunotherapy, we showed that propranolol, a nonselective ß-blocker, strongly improved the efficacy of an antitumor STxBE7 vaccine by enhancing the frequency of CD8+ T lymphocytes infiltrating the tumor (TIL). However, propranolol had no effect on the reactivity of CD8+ TILs, a result further strengthened by ex vivo experiments showing that these cells were insensitive to adrenaline- or noradrenaline-induced AR signaling. In contrast, naïve CD8+ T-cell activation was strongly inhibited by ß-AR signaling, and the beneficial effect of propranolol mainly occurred during CD8+ T-cell priming in the tumor-draining lymph node. We also demonstrated that the differential sensitivity of naïve CD8+ T cells and CD8+ TILs to ß-AR signaling was linked to a strong downregulation of ß2-AR expression related to their activation status, since in vitro-activated CD8+ T cells behaved similarly to CD8+ TILs. These results revealed that ß-AR signaling suppresses the initial priming phase of antitumor CD8+ T-cell responses, providing a rationale to use clinically available ß-blockers in patients to improve cancer immunotherapies.

TGFß blocks IFNα/ß release and tumor rejection in spontaneous mammary tumors.

Type I interferons (IFN) are being rediscovered as potent anti-tumoral agents. Activation of the STimulator of INterferon Genes (STING) by DMXAA (5,6-dimethylxanthenone-4-acetic acid) can induce strong production of IFNα/ß and rejection of transplanted primary tumors. In the present study, we address whether targeting STING with DMXAA also leads to the regression of spontaneous MMTV-PyMT mammary tumors. We show that these tumors are refractory to DMXAA-induced regression. This is due to a blockade in the phosphorylation of IRF3 and the ensuing IFNα/ß production. Mechanistically, we identify TGFß, which is abundant in spontaneous tumors, as a key molecule limiting this IFN-induced tumor regression by DMXAA. Finally, blocking TGFß restores the production of IFNα by activated MHCII+ tumor-associated macrophages, and enables tumor regression induced by STING activation. On the basis of these findings, we propose that type I IFN-dependent cancer therapies could be greatly improved by combinations including the blockade of TGFß.

The Remarkable Plasticity of Macrophages: A Chance to Fight Cancer.

It is well established that tumor-associated macrophages (TAM) found in most advanced tumors have a pro-tumoral role. In this context, TAM limit the activity of tumor-infiltrating lymphocytes (TIL), and a number of mechanisms have been described including a trapping in the stroma, impeding TIL to reach malignant cells. Based on these results, a number of therapeutic approaches have been designed to deplete TAM. However, during tumor regression induced by immunotherapeutic treatments, recent studies revealed that TAM can switch from pro-tumoral to anti-tumoral and actively cooperate with TIL. Here, we will review the two faces of TAM in their interaction with TIL. We will summarize how they can inhibit T cell activities in growing tumors, and how they may also, together with T cells, successfully contribute to tumor eradication after an appropriate stimulation. Finally, we will discuss current promising therapies combining TAM reprogramming with T cell-based immunotherapy.

Macrophages of distinct origins contribute to tumor development in the lung.

Tissue-resident macrophages can self-maintain without contribution of adult hematopoiesis. Herein we show that tissue-resident interstitial macrophages (Res-TAMs) in mouse lungs contribute to the pool of tumor-associated macrophages (TAMs) together with CCR2-dependent recruited macrophages (MoD-TAMs). Res-TAMs largely correlated with tumor cell growth in vivo, while MoD-TAMs accumulation was associated with enhanced tumor spreading. Both cell subsets were depleted after chemotherapy, but MoD-TAMs rapidly recovered and performed phagocytosis-mediated tumor clearance. Interestingly, anti-VEGF treatment combined with chemotherapy inhibited both Res and Mod-TAM reconstitution without affecting monocyte infiltration and improved its efficacy. Our results reveal that the developmental origin of TAMs dictates their relative distribution, function, and response to cancer therapies in lung tumors.

Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment.

In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment.

The STING agonist DMXAA triggers a cooperation between T lymphocytes and myeloid cells that leads to tumor regression.

Regressing tumors are usually associated with a large immune infiltrate, but the molecular and cellular interactions that govern a successful anti-tumor immunity remain elusive. Here, we have triggered type I Interferon (IFN) signaling in a breast tumor model (MMTV-PyMT) using 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a ligand of the STimulator of Interferon Genes, STING. The 2 main events rapidly triggered by DMXAA in transplanted PyMT tumors are 1) the disruption of the tumor vasculature, followed by hypoxia and cell death; 2) the release of chemokines. Both events converged to trigger the recruitment of 2 waves of immune cells: a swift, massive recruitment of neutrophils, followed by a delayed rise in monocytes and CD8 T cells in the tumor mass. Depletion experiments in vivo revealed that myeloid cell subsets and T cells need to cooperate to achieve full-blown recruitment and activation at the tumor site and to induce effective secondary cell death leading to tumor regression (Illustration 1). Altogether, our study highlights that the tumor regression induced by the STING agonist DMXAA results from a cascade of events, with an initial vessel destruction followed by several infiltration waves of immune cells which have to cooperate to amplify and sustain the initial effect. We thus provide the first global and detailed kinetic analysis of the anti-tumoral effect of DMXAA and of its different articulated steps.

Nanoprobe Synthesized by Magnetotactic Bacteria, Detecting Fluorescence Variations under Dissociation of Rhodamine B from Magnetosomes following Temperature, pH Changes, or the Application of Radiation.

We report a method of fabrication of fluorescent magnetosomes, designated as MCR400, in which 400 µM of rhodamine B are introduced in the growth medium of AMB-1 magnetotactic bacteria and fluorescent magnetosomes are then extracted from these bacteria. These fluorescent magnetosomes behave differently from most fluorescent nanoprobes, which often lead to fluorescence losses over time due to photobleaching. Indeed, when MCR400 are heated to 30-90 °C, brought to an acidic pH, or exposed to radiations, we observed that their fluorescence intensity increased. We attributed this behavior to the dissociation of rhodamine B from the magnetosomes. Interestingly, enhanced fluorescence was also observed in vitro when MCR400 were mixed with either primary macrophages or tumor cells (TC1-GFP or RG2-Cells) or in vivo when MCR400 were introduced in rat glioblastoma. We showed that MCR400 internalize in tumor and immune cells (macrophages) leading to enhanced fluorescence, suggesting that fluorescent magnetosomes could be used during cancer treatments such as magnetic hyperthermia to image cells of interest such as immune or tumor cells.

Vaccine-induced tumor regression requires a dynamic cooperation between T cells and myeloid cells at the tumor site.

Most cancer immunotherapies under present investigation are based on the belief that cytotoxic T cells are the most important anti-tumoral immune cells, whereas intra-tumoral macrophages would rather play a pro-tumoral role. We have challenged this antagonistic point of view and searched for collaborative contributions by tumor-infiltrating T cells and macrophages, reminiscent of those observed in anti-infectious responses. We demonstrate that, in a model of therapeutic vaccination, cooperation between myeloid cells and T cells is indeed required for tumor rejection. Vaccination elicited an early rise of CD11b+ myeloid cells that preceded and conditioned the intra-tumoral accumulation of CD8+ T cells. Conversely, CD8+ T cells and IFNγ production activated myeloid cells were required for tumor regression. A 4-fold reduction of CD8+ T cell infiltrate in CXCR3KO mice did not prevent tumor regression, whereas a reduction of tumor-infiltrating myeloid cells significantly interfered with vaccine efficiency. We show that macrophages from regressing tumors can kill tumor cells in two ways: phagocytosis and TNFα release. Altogether, our data suggest new strategies to improve the efficiency of cancer immunotherapies, by promoting intra-tumoral cooperation between macrophages and T cells.

Revisiting the role of T cells in tumor regression.

This note challenges the current idea that a key role of T cells in tumor regression is to directly kill tumor cells. It favors the view that TIL are keys but act indirectly by helping other immune cells to damage the tumor and its stroma.

T cells contribute to tumor progression by favoring pro-tumoral properties of intra-tumoral myeloid cells in a mouse model for spontaneous melanoma.

Tumors affect myelopoeisis and induce the expansion of myeloid cells with immunosuppressive activity. In the MT/ret model of spontaneous metastatic melanoma, myeloid cells are the most abundant tumor infiltrating hematopoietic population and their proportion is highest in the most aggressive cutaneous metastasis. Our data suggest that the tumor microenvironment favors polarization of myeloid cells into type 2 cells characterized by F4/80 expression, a weak capacity to secrete IL-12 and a high production of arginase. Myeloid cells from tumor and spleen of MT/ret mice inhibit T cell proliferation and IFNγ secretion. Interestingly, T cells play a role in type 2 polarization of myeloid cells. Indeed, intra-tumoral myeloid cells from MT/ret mice lacking T cells are not only less suppressive towards T cells than corresponding cells from wild-type MT/ret mice, but they also inhibit more efficiently melanoma cell proliferation. Thus, our data support the existence of a vicious circle, in which T cells may favor cancer development by establishing an environment that is likely to skew myeloid cell immunity toward a tumor promoting response that, in turn, suppresses immune effector cell functions.

Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors.

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.

Progressive upregulation of PD-1 in primary and metastatic melanomas associated with blunted TCR signaling in infiltrating T lymphocytes.

Programmed death-1 (PD-1) is involved in T-cell tolerance to self-antigens. For some cancers, it has been suggested that the expression of a ligand of PD-1, namely PD-L1, could contribute to tumor escape from immune destruction. Nevertheless, the relationship between PD-1 expression on tumor-infiltrating T lymphocytes (TILs), disease stage, and TIL responsiveness is still poorly documented. In this study, we show that freshly isolated CD4(+) and CD8(+) TILs express substantial levels of PD-1 in primary melanomas. The expression of PD-1 was further increased at later stages in distant cutaneous metastases, especially on CD8(+) TILs. The expression of PD-1 ligands was frequent only in metastases, on both tumor cells and tumor-derived myeloid cells. TILs isolated from these cutaneous tumors are poorly reactive ex vivo, with blunted calcium response and IFN-γ production after TCR stimulation. Surprisingly, in distinct parts of a primary melanoma, either invasive or regressing, we show that TILs similarly express PD-1 and remain dysfunctional. The expressions of PD-1 and PD-L1 in metastatic melanoma lesions could be considered as witnesses of an unsuccessful anti-tumoral immune response, but the direct involvement of PD-1 in the severity of the disease, and the importance of TILs in tumor regression, remain to be established.