Tissue-resident FOLR2+ macrophages associate with CD8+ T cell infiltration in human breast cancer.

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.

Checkpoint inhibition enhances cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells of classic Hodgkin lymphoma.

Although checkpoint molecules like CTLA-4 and PD1 have been described several years ago, checkpoint inhibitors such as Nivolumab (an anti-PD-1 antibody) have only recently been used to treat classic Hodgkin lymphoma (cHL). Several studies have shown convincing therapeutic effects of Nivolumab in cHL. However, the mechanism of action of Nivolumab in cHL is not fully understood. The aim of this study was to monitor changes in cell motility and cell contacts after administration of Nivolumab to an in vitro model of cHL as well as to native hyperplastic lymphoid tissue and native human tissue from cHL. In both tissue and in vitro, CD4+, CD8+, CD30+ and CD20+ cell velocities were unchanged after Nivolumab incubation. In contrast, in primary cHL tissue, the duration of cell contacts between CD4+ T cells and HRS cells was significantly increased after 5 h Nivolumab treatment, and the number of contacts with HRS cells was also slightly increased for CD4+ T cells (not significant), suggesting that CD4+ T cells in particular contribute to the cytotoxicity observed as a result of Nivolumab therapy. There was no change in the duration of cell contacts in the hyperplastic lymphoid tissue after Nivolumab incubation. In conclusion, we show here for the first time by imaging of native lymphoma tissue an enhanced interaction of CD4+ T cells and HRS cells in cHL after Nivolumab administration.

Extracellular Release of Antigen by Dendritic Cell Regurgitation Promotes B Cell Activation through NF-κB/cRel.

Dendritic cells (DCs) are professional APCs, which sample Ags in the periphery and migrate to the lymph node where they activate T cells. DCs can also present native Ag to B cells through interactions observed both in vitro and in vivo. However, the mechanisms of Ag transfer and B cell activation by DCs remain incompletely understood. In this study, we report that murine DCs are an important cell transporter of Ag from the periphery to the lymph node B cell zone and also potent inducers of B cell activation both in vivo and in vitro. Importantly, we highlight a novel extracellular mechanism of B cell activation by DCs. In this study, we demonstrate that Ag released upon DC regurgitation is sufficient to efficiently induce early B cell activation, which is BCR driven and mechanistically dependent on the nuclear accumulation of the transcription factor NF-κB/cRel. Thus, our study provides new mechanistic insights into Ag delivery and B cell activation modalities by DCs and a promising approach for targeting NF-κB/cRel pathway to modulate the DC-elicited B cell responses.

Landscape of T Follicular Helper Cell Dynamics in Human Germinal Centers.

T follicular helper (Tfh) cells play a very important role in mounting a humoral response. Studies conducted in mouse models have revealed with good kinetic and spatial resolution the dynamics of these cells in germinal centers (GC) and their cross-talk with B cells upon an immune response. However, whether a similar migratory behavior is performed by human Tfh cells is unclear, as technology to track them in situ has been lacking. In this study, we combined traditional immunohistochemistry and real-time fluorescent imaging approaches on fresh human adenoid slices to provide static and dynamic information on Tfh cells. Our data indicate that GC light zones are composed of two distinct areas in terms of Tfh cell distribution and migration. In the outer GC light zones, Tfh cells migrate actively and with a high ability to form dynamic clusters showing intense and rapid reorganization. In these outer regions, Tfh cells demonstrate multiple interactions between each other. Conversely, in central regions of GC light zones, Tfh cells are much more static, forming long-lasting conjugates. These findings reveal for the first time, to our knowledge, the dynamic behavior whereby Tfh cells migrate in human GC and highlight the heterogeneity of GC for Tfh cell motility.

Is adaptive therapy natural?

Research suggests that progression-free survival can be prolonged by integrating evolutionary principles into clinical cancer treatment protocols. The goal is to prevent or slow the proliferation of resistant malignant cell populations. The logic behind this therapy relies on ecological and evolutionary processes. These same processes would be available to natural selection in decreasing the probability of an organism's death due to cancer. We propose that organisms' anticancer adaptions include not only ones for preventing cancer but also ones for directing and retarding the evolution of life-threatening cancer cells. We term this last strategy natural adaptive therapy (NAT). The body's NAT might include a lower than otherwise possible immune response. A restrained immune response might forego maximum short-term kill rates. Restraint would forestall immune-resistant cancer cells and produce long-term durable control of the cancer population. Here, we define, develop, and explore the possibility of NAT. The discovery of NAT mechanisms could identify new strategies in tumor prevention and treatments. Furthermore, we discuss the potential risks of immunotherapies that force the immune system to ramp up the short-term kill rates of malignant cancer cells in a manner that undermines the body's NAT and accelerates the evolution of immune resistance.

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.

Identification of a new subset of lymph node stromal cells involved in regulating plasma cell homeostasis.

Antibody-secreting plasma cells (PCs) arise rapidly during adaptive immunity to control infections. The early PCs are retained within the reactive lymphoid organ where their localization and homeostasis rely on extrinsic factors, presumably produced by local niche cells. While myeloid cells have been proposed to form those niches, the contribution by colocalizing stromal cells has remained unclear. Here, we characterized a subset of fibroblastic reticular cells (FRCs) that forms a dense meshwork throughout medullary cords of lymph nodes (LNs) where PCs reside. This medullary FRC type is shown to be anatomically, phenotypically, and functionally distinct from T zone FRCs, both in mice and humans. By using static and dynamic imaging approaches, we provide evidence that medullary FRCs are the main cell type in contact with PCs guiding them in their migration. Medullary FRCs also represent a major local source of the PC survival factors IL-6, BAFF, and CXCL12, besides also producing APRIL. In vitro, medullary FRCs alone or in combination with macrophages promote PC survival while other LN cell types do not have this property. Thus, we propose that this FRC subset, together with medullary macrophages, forms PC survival niches within the LN medulla, and thereby helps in promoting the rapid development of humoral immunity, which is critical in limiting early pathogen spread.

TCR-engineered T cells to treat tumors: Seeing but not touching?

Adoptive transfer of T cells gene-engineered with T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to accurately select TCRs that demonstrate antigen-selective responses that are restricted to tumor cells and, at the same time, include strategies that restore or enhance the entry, migration and local accumulation of T cells in tumor tissues. Here, we present the current standing of TCR-engineered T cell therapy, discuss and propose procedures to select TCRs as well as strategies to sensitize the tumor to T cell trafficking, and provide a rationale for combination therapies with TCR-engineered T cells.

Essential role of immobilized chemokine CXCL12 in the regulation of the humoral immune response.

Chemokines control the migration of a large array of cells by binding to specific receptors on cell surfaces. The biological function of chemokines also depends on interactions between nonreceptor binding domains and proteoglycans, which mediate chemokine immobilization on cellular or extracellular surfaces and formation of fixed gradients. Chemokine gradients regulate synchronous cell motility and integrin-dependent cell adhesion. Of the various chemokines, CXCL12 has a unique structure because its receptor-binding domain is distinct and does not overlap with the immobilization domains. Although CXCL12 is known to be essential for the germinal center (GC) response, the role of its immobilization in biological functions has never been addressed. In this work, we investigated the unexplored paradigm of CXCL12 immobilization during the germinal center reaction, a fundamental process where cellular traffic is crucial for the quality of humoral immune responses. We show that the structure of murine germinal centers and the localization of GC B cells are impaired when CXCL12 is unable to bind to cellular or extracellular surfaces. In such mice, B cells carry fewer somatic mutations in Ig genes and are impaired in affinity maturation. Therefore, immobilization of CXCL12 is necessary for proper trafficking of B cells during GC reaction and for optimal humoral immune responses.

IL4-induced gene 1 is secreted at the immune synapse and modulates TCR activation independently of its enzymatic activity.

Amino-acid catabolizing enzymes produced by mononuclear phagocytes play a central role in regulating the immune response. The mammalian phenylalanine-catabolizing enzyme IL4-induced gene 1 (IL4I1) inhibits effector T lymphocyte proliferation and facilitates regulatory T-cell development. IL4I1 expression by macrophages of various human tumors may affect patient prognosis as it facilitates tumor escape from the T-cell response in murine models. Its enzymatic activity appears to participate in its effects, but some actions of IL4I1 remain unclear. Here, we show that the presence of IL4I1 during T-cell activation decreases early signaling events downstream of TCR stimulation, resulting in global T-cell inhibition which is more pronounced when there is CD28 costimulation. Surprisingly, the enzymatic activity of IL4I1 is not involved. Focal secretion of IL4I1 into the immune synaptic cleft and its binding to CD3+ lymphocytes could be important in IL4I1 immunosuppressive mechanism of action.

Positive and negative influence of the matrix architecture on antitumor immune surveillance.

The migration of T cells and access to tumor antigens is of utmost importance for the induction of protective anti-tumor immunity. Once having entered a malignant site, T cells encounter a complex environment composed of non-tumor cells along with the extracellular matrix (ECM). It is now well accepted that a deregulated ECM favors tumor progression and metastasis. Recent progress in imaging technologies has also highlighted the impact of the matrix architecture found in solid tumor on immune cells and especially T cells. In this review, we argue that the ability of T cells to mount an antitumor response is dependent on the matrix structure, more precisely on the balance between pro-migratory reticular fiber networks and unfavorable migration zones composed of dense and aligned ECM structures. Thus, the matrix architecture, that has long been considered to merely provide the structural framework of connective tissues, can play a key role in facilitating or suppressing the antitumor immune surveillance. A new challenge in cancer therapy will be to develop approaches aimed at altering the architecture of the tumor stroma, rendering it more permissive to antitumor T cells.

Protocol for real-time monitoring of CD8+ T and myeloid cell behavior in human high-grade serous ovarian cancer slices.

Studying cell behavior in live human tumors is crucial to understand and improve response to immunotherapies. Here, we present a protocol to slice human ovarian tumors ex vivo, maintain their viability for 24 h, and monitor the behavior of CD8+ T and myeloid cells in real time. Furthermore, we detail procedures to semi-automatically analyze cell movements and aggregate and process behavior data. This protocol can potentially be applied for multiple tumor types and mouse cancer models. For complete details on the use and execution of this protocol, please refer to Laforets et al.1.

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.

CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.

The molecular mechanisms responsible for the sustained basal motility of T cells within lymph nodes (LNs) remain elusive. To study T cell motility in a LN environment, we have developed a new experimental system based on slices of LNs that allows the assessment of T cell trafficking after adoptive transfer or direct addition of T cells to the slice. Using this experimental system, we show that T cell motility is highly sensitive to pertussis toxin and strongly depends on CCR7 and its ligands. Our results also demonstrate that, despite its established role in myeloid cell locomotion, phosphoinositide 3-kinase (PI3K) activity does not contribute to the exploratory behavior of the T lymphocytes within LN slices. Likewise, although PI3K activation is detectable in chemokine-treated T cells, PI3K plays only a minor role in T cell polarization and migration in vitro. Collectively, our results suggest that the common amplification system that, in other cells, facilitates large phosphatidylinositol 3,4,5-trisphosphate increases at the plasma membrane is absent in T cells. We conclude that T cell motility within LNs is not an intrinsic property of T lymphocytes but is driven in a PI3K-independent manner by the lymphoid chemokine-rich environment.

CAR T-cell behavior and function revealed by real-time imaging.

Adoptive transfer of T-cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies. Nonetheless, the field of CAR T-cells is currently facing several major challenges. In particular, the CAR T-cell strategy has not yet produced favorable clinical responses when targeting solid tumors. In this context, it is of paramount importance to understand the determinants that limit the efficacy of T-cell-based immunotherapy. Characterization of CAR T-cells is usually based on flow cytometry and whole-transcriptome profiling. These approaches have been very valuable to determine intrinsic elements that condition T-cell ability to proliferate and expand. However, they do not take into account spatial and kinetic aspects of T-cell responses. In particular, in order to control tumor growth, CAR T-cells need to enter into the tumor, migrate within a complex tumor environment, and form productive conjugates with their targets. Advanced imaging techniques combined with innovative preclinical models represent promising tools to uncover the dynamics of CAR T-cells. In this review, we will discuss recent results on the biology of engineered T-cells that have been obtained with real-time imaging microscopy. Important notions have emerged from these imaging-based studies, such as the multi-killing potential of CAR T-cells. Finally, we will highlight how imaging techniques combined with other tools can solve remaining unresolved questions in the field of engineered T-cells.

Live Imaging of CAR T Cell Ca2+ Signals in Tumor Slices Using Confocal Microscopy.

The immune synapse is a key structure organizing T-cell activation against foreign entities, such as cancer cells expressing neoantigens. One crucial step in this activation cascade is the intracellular Ca2+ ([Ca2+]i) response that shapes T cells for proliferation, differentiation, and cytotoxicity. The development of calcium probes coupled to real-time fluorescence microscopy has allowed a close study of this phenomenon in vitro. Such systems have provided valuable insights on the consequences of Ca2+ responses on T cells, including cytotoxicity and cytoskeletal remodeling. However, in vitro models do not recapitulate the tissue architecture that T cells come in contact with in vivo. Thus, there is a growing necessity for better understanding the factors influencing Ca2+ response in T cells including in genetically modified T cells (e.g., CAR T cells). In this methodology chapter, we describe an experimental system to measure [Ca2+]i signals of CAR T cells loaded with the calcium probe Fluo-4 on fresh tumor slices. Combined with confocal fluorescent imaging, this model offers an approach to image early T-cell activation in a three-dimensional (3D) tissue environment.

Semi-supervised analysis of myeloid and T cell behavior in ex vivo ovarian tumor slices reveals changes in cell motility after treatments.

Studies of the high-grade serous ovarian cancer (HGSOC) tumor microenvironment, the most lethal gynecological cancer, aim to enhance the efficiency of established therapies. Cell motility is an important process of anti-tumor response. Using ex vivo human and mouse HGSOC tumor slices combined with time-lapse imaging, we assessed the motility of CD8+ T and myeloid cells. We developed a semi-supervised analysis of cell movements, identifying four cell behaviors: migrating, long migrating, static, and wobbling. Tumor slices were maintained 24h ex vivo, retaining viability and cell movements. Ex vivo treatments with lipopolysaccharide altered CD8+ T and myeloid cell behavior. In vivo chemotherapy reduced ex vivo cell movements in human and mouse tumors and differentially affected CD8+ T and myeloid cells in chemo-sensitive and chemo-resistant mouse models. Ex vivo tumor slices can extend in vivo mouse studies to human, providing a stepping stone to translate mouse cancer studies to clinical trials.

The RHOA Mutation G17V Does Not Lead to Increased Migration of Human Malignant T Cells but Is Associated with Matrix Remodelling.

Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (AITL), is characterized by constitutional symptoms, advanced-stage disease, and generalized lymphadenopathy. A genetic hallmark of this lymphoma is the frequent occurrence of the RHOA mutation G17V in neoplastic cells, which is observed in around 60% of patients. Because RHOA is involved in both T-cell receptor downstream signalling and cell migration, we hypothesized that the characteristic presentation of AITL could be the result of enhanced tumor cell migration. Therefore, this study aimed to elucidate the impact of the RHOA variant G17V on the migration of neoplastic T cells. We transfected the T-cell lymphoma cell lines HH and HuT78 to stably express the RHOA-G17V variant. RHOA-G17V-expressing T cells did not exhibit enhanced motility compared to empty-vector-transfected cells in microchannels, a 3D collagen gel, or primary human lymphatic tissue. Cells of the HH cell line expressing RHOA-G17V had an increased number of cells with cleaved collagen compared with the empty-vector-transfected cells. Therefore, we hypothesized that the early spread of AITL tumor cells may be related to remodelling of the extracellular matrix. Accordingly, we observed a significant negative correlation between the relative area of collagen in histological sections from 18 primary AITL and the allele frequency of the RHOA-G17V mutation. In conclusion, our results suggest that the characteristic presentation of AITL with early, widespread dissemination of lymphoma cells is not the result of an enhanced migration capacity due to the RHOA-G17V mutation; instead, this feature may rather be related to extracellular matrix remodelling.

ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma.

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.