Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

NK cells and solid tumors: therapeutic potential and persisting obstacles.

Natural killer (NK) cells, which are innate lymphocytes endowed with potent cytotoxic activity, have recently attracted attention as potential anticancer therapeutics. While NK cells mediate encouraging responses in patients with leukemia, the therapeutic effects of NK cell infusion in patients with solid tumors are limited. Preclinical and clinical data suggest that the efficacy of NK cell infusion against solid malignancies is hampered by several factors including inadequate tumor infiltration and persistence/activation in the tumor microenvironment (TME). A number of metabolic features of the TME including hypoxia as well as elevated levels of adenosine, reactive oxygen species, and prostaglandins negatively affect NK cell activity. Moreover, cancer-associated fibroblasts, tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells actively suppress NK cell-dependent anticancer immunity. Here, we review the metabolic and cellular barriers that inhibit NK cells in solid neoplasms as we discuss potential strategies to circumvent such obstacles towards superior therapeutic activity.

Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality.

Successful translation of chimeric antigen receptor (CAR) T cell therapy for the treatment of solid tumors has proved to be troublesome, mainly due to the complex tumor microenvironment promoting T cell dysfunction and antigen heterogeneity. Mesothelin (MSLN) has emerged as an attractive target for CAR T cell therapy of several solid malignancies, including ovarian cancer. To improve clinical response rates with MSLN-CAR T cells, a better understanding of the mechanisms impacting CAR T cell functionality in vitro is crucial. Here, we demonstrated superior cytolytic capacity of CD28-costimulated MSLN-CAR T cells (M28z) relative to 4-1BB-costimulated MSLN-CAR T cells (MBBz). Furthermore, CD28-costimulated MSLN CAR T cells displayed enhanced cytolytic capacity against tumor spheroids with heterogeneous MSLN expression compared to MBBz CAR T cells. In this study, we identified CAR-mediated trogocytosis as a potential impeding factor for successful MSLN-CAR T cell therapy due to fratricide killing and contributing to tumor antigen heterogeneity. Moreover, we link antigen-dependent upregulation of LAG-3 with reduced CAR T cell functionality. Taken together, our study highlights the therapeutic potential and bottlenecks of MSLN-CAR T cells, providing a rationale for combinatorial treatment strategies.

MHC Class I-Dependent Shaping of the NK Cell Ly49 Receptor Repertoire Takes Place Early during Maturation in the Bone Marrow.

MHC class I (MHC I) expression in the host influences NK cells in a process termed education. The result of this education is reflected in the responsiveness of NK cells at the level of individual cells as well as in the repertoire of inhibitory MHC I-specific receptors at the NK cell system level. The presence of MHC I molecules in the host environment gives rise to a skewed receptor repertoire in spleen NK cells where subsets expressing few (one or two) inhibitory receptors are expanded whereas subsets with many (three or more) receptors are contracted. It is not known whether this MHC I-dependent skewing is imposed during development or after maturation of NK cells. In this study, we tested the hypothesis that the NK cell receptor repertoire is shaped already early during NK cell development in the bone marrow. We used mice with a repertoire imposed by a single MHC I allele, as well as a C57BL/6 mutant strain with exaggerated repertoire skewing, to investigate Ly49 receptor repertoires at different stages of NK cell differentiation. Our results show that NK cell inhibitory receptor repertoire skewing can indeed be observed in the bone marrow, even during the earliest developmental steps where Ly49 receptors are expressed. This may partly be accounted for by selective proliferation of certain NK cell subsets, but other mechanisms must also be involved. We propose a model for how repertoire skewing is established during a developmental phase in the bone marrow, based on sequential receptor expression as well as selective proliferation.

Targeting of Nrf2 improves antitumoral responses by human NK cells, TIL and CAR T cells during oxidative stress.

BACKGROUND: Adoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair NK cell and T cell function. Auranofin, a gold (I)-containing phosphine compound, is a strong activator of the transcription factor Nrf2. Nrf2 controls a wide range of downstream targets important for the cells to obtain increased resistance to ROS. In this study, we present a strategy using auranofin to render human cytotoxic lymphocytes resistant toward oxidative stress. METHODS: Melanoma patient-derived tumor infiltrating lymphocytes (TIL) and healthy donor-derived NK cells and CD19-directed CAR T cells were pretreated with a low dose of auranofin. Their resistance toward oxidative stress was assessed by measuring antitumoral responses (killing-assay, degranulation/CD107a, cytokine production) and intracellular ROS levels (flow cytometry) in conditions of oxidative stress. To confirm that the effects were Nrf2 dependent, the transcription level of Nrf2-driven target genes was analyzed by qPCR. RESULTS: Pretreatment of human TIL and NK cells ex vivo with a low-dose auranofin significantly lowered their accumulation of intracellular ROS and preserved their antitumoral activity despite high H2O2 levels or monocyte-derived ROS. Furthermore, auranofin pretreatment of CD19 CAR-T cells or TIL increased their elimination of CD19 +tumor cells or autologous tumor spheroids, respectively, especially during ROS exposure. Analysis of Nrf2-driven target genes revealed that the increased resistance against ROS was Nrf2 dependent. CONCLUSION: These novel findings suggest that Nrf2 activation in human cytotoxic lymphocytes could be used to enhance the efficacy of adoptive cell therapy.

Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads.

For the past decade, adoptive cell therapy including tumor-infiltrating lymphocytes, genetically modified cytotoxic lymphocytes expressing a chimeric antigen receptor, or a novel T-cell receptor has revolutionized the treatment of many cancers. Progress within exome sequencing and neoantigen prediction technologies provides opportunities for further development of personalized immunotherapies. In this study, we present a novel strategy to deliver in silico predicted neoantigens to autologous dendritic cells (DCs) using paramagnetic beads (EpiTCer beads). DCs pulsed with EpiTCer beads are superior in enriching for healthy donor and patient blood-derived tumor-specific CD8+ T cells compared to DC loaded with whole-tumor lysate or 9mer neoantigen peptides. A dose-dependent effect was observed, with higher EpiTCer bead per DC being favorable. We concluded that CD8+ T cells enriched by DC loaded with EpiTCer beads are tumor specific with limited tumor cross-reactivity and low recognition of autologous non-activated monocytes or CD8+ T cells. Furthermore, tumor specificity and recognition were improved and preserved after additional expansion using our Good Manufacturing Process (GMP)-compatible rapid expansion protocol. Phenotypic analysis of patient-derived EpiTCer DC expanded CD8+ T cells revealed efficient maturation, with high frequencies of central memory and effector memory T cells, similar to those observed in autologous expanded tumor-infiltrating lymphocytes. These results indicate that DC pulsed with EpiTCer beads enrich for a T-cell population with high capacity of tumor recognition and elimination, which are features needed for a T-cell product to be used for personalized adoptive cell therapy.

Interleukin-33 is a Novel Immunosuppressor that Protects Cancer Cells from TIL Killing by a Macrophage-Mediated Shedding Mechanism.

Recognition of specific antigens expressed in cancer cells is the initial process of cytolytic T cell-mediated cancer killing. However, this process can be affected by other non-cancerous cellular components in the tumor microenvironment. Here, it is shown that interleukin-33 (IL-33)-activated macrophages protect melanoma cells from tumor-infiltrating lymphocyte-mediated killing. Mechanistically, IL-33 markedly upregulates metalloprotease 9 (MMP-9) expression in macrophages, which acts as a sheddase to trim NKG2D, an activating receptor expressed on the surface of natural killer (NK) cells, CD8+ T cells, subsets of CD4+ T cells, iNKT cells, and γδ T cells. Further, MMP-9 also cleaves the MHC class I molecule, cell surface antigen-presenting complex molecules, expressed in melanoma cells. Consequently, IL-33-induced macrophage MMP-9 robustly mitigates the tumor killing-effect by T cells. Genetic and pharmacological loss-of-function of MMP-9 sheddase restore T cell-mediated cancer killing. Together, these data provide compelling in vitro and in vivo evidence showing novel mechanisms underlying the IL-33-macrophage-MMP-9 axis-mediated immune tolerance against cancer cells. Targeting each of these signaling components, including IL-33 and MMP-9 provides a new therapeutic paradigm for improving anticancer efficacy by immune therapy.

Regulatory T Cells Inhibit T Cell Activity by Downregulating CD137 Ligand via CD137 Trogocytosis.

CD137 is a costimulatory molecule expressed on activated T cells. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC), which use the CD137-CD137L system to enhance immune responses. It was, therefore, surprising to discover CD137 expression on regulatory T cells (Treg). The function of CD137 in Treg are controversial. While some studies report that CD137 signalling converts Treg to effector T cells (Teff), other studies find that CD137-expressing Treg display a stronger inhibitory activity than CD137- Treg. Here, we describe that CD137 on Treg binds to CD137L on APC, upon which one of the two molecules is transferred via trogocytosis to the other cell, where CD137-CD137L forms a complex that is internalized and deprives APC of the immune-stimulatory CD137L. Truncated forms of CD137 that lack the cytoplasmic domain of CD137 are also able to downregulate CD137L, demonstrating that CD137 signalling is not required. Comparable data have been obtained with human and murine cells, indicating that this mechanism is evolutionarily conserved. These data describe trogocytosis of CD137 and CD137L as a new mechanism employed by Treg to control immune responses by downregulating the immunostimulatory CD137L on APC.

Counteracting CAR T cell dysfunction.

In spite of high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, the efficacy of this approach is limited by generation of dysfunctional CAR T cells in vivo, conceivably induced by immunosuppressive tumor microenvironment (TME) and excessive antigen exposure. Exhaustion and senescence are two critical dysfunctional states that impose a pivotal hurdle for successful CAR T cell therapies. Recently, modified CAR T cells with an "exhaustion-resistant" phenotype have shown superior antitumor functions and prolonged lifespan. In addition, several studies have indicated the feasibility of senescence delay in CAR T cells. Here, we review the latest reports regarding blockade of CAR T cell exhaustion and senescence with a particular focus on the exhaustion-inducing pathways. Subsequently, we describe what potential these latest insights offer for boosting the potency of adoptive cell transfer (ACT) therapies involving CAR T cells. Furthermore, we discuss how induction of costimulation, cytokine exposure, and TME modulation can impact on CAR T cell efficacy and persistence, while potential safety issues associated with reinvigorated CAR T cells will also be addressed.

Complete and long-lasting clinical responses in immune checkpoint inhibitor-resistant, metastasized melanoma treated with adoptive T cell transfer combined with DC vaccination.

Development of T cell-directed immune checkpoint inhibitors (ICI) has revolutionized metastatic melanoma (MM) therapy, but <50% of treated patients experience durable responses. This phase I trial (NCT01946373) investigates the safety/feasibility of tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) combined with dendritic cell (DC) vaccination in MM patients progressing on ICI. An initial cohort (5 patients) received TIL therapy alone to evaluate safety and allow for optimization of TIL expansion protocols. A second cohort (first-in-man, 5 patients) received TIL combined with autologous tumor lysate-loaded DC vaccination. All patients received cyclophosphamide/fludarabine preconditioning prior to, and intravenous (i.v.) IL-2 after, TIL transfer. The DC vaccine was given as five intradermal injections after TIL and IL-2 administration. [18F]-FDG PET/CT radiology was performed to evaluate clinical response, according to RECIST 1.1 (on the CT part). Immunological monitoring was performed by flow cytometry and T-cell receptor (TCR) sequencing. In the safety/optimization cohort, all patients had a mixed response or stable disease, but none durable. In the combination cohort, two patients experienced complete responses (CR) that are still ongoing (>36 and >18 months, respectively). In addition, two patients had partial responses (PR), one still ongoing (>42 months) with only a small bone-lesion remaining, and one of short duration (<4 months). One patient died early during treatment and did not receive DC. Long-lasting persistency of the injected TILs was demonstrated in blood. In summary, we report clinical responses by TIL therapy combined with DC vaccination in 4 out of 4 treated MM patients who previously failed ICI.

Visualization of human T lymphocyte-mediated eradication of cancer cells in vivo.

Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.

Genetically modified immune cells targeting tumor antigens.

Immunotherapy approaches consisting of genetically modified immune cells have become a promising platform for cancer treatment. Such 'living' therapies targeting tumor antigens have shown success in many cancer patients in the form of durable responses in a growing number of clinical studies. Besides, a large number of ongoing studies have been designed to introduce reliable methods for identification of tumor antigens. In addition, technical and biotechnological developments are being applied to the generation and expansion of genetically modified immune cells. In this review, we summarize and discuss the latest progress and current challenges in the tumor antigen landscape and in the generation of genetically modified immune cells in view of their clinical efficacy, either as monotherapy or combinational therapy.

The Outcome of Ex Vivo TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic In Vitro Growth Capacity between T-Cell Clones.

PURPOSE: During our efforts to develop tumor-infiltrating lymphocyte (TIL) therapy to counter the devastating recurrence rate in patients with primary resectable pancreatic ductal adenocarcinoma (PDA), we found that PDA TILs can readily be expanded in vitro and that the majority of resulting TIL cultures show reactivity against the autologous tumor. However, the fraction of tumor-reactive T cells is low. We investigated to which extent this was related to the in vitro expansion. EXPERIMENTAL DESIGN: We compared the clonal composition of TIL preparations before and after in vitro expansion using T-cell receptor (TCR) deep sequencing. Our findings for PDA were benchmarked to experiments with melanoma TILs. RESULTS: We found that the TIL TCR repertoire changes dramatically during in vitro expansion, leading to loss of tumor- dominant T-cell clones and overgrowth by newly emerging T-cell clones that are barely detectable in the tumor. These changes are primarily driven by differences in the intrinsic in vitro expansion capacity of T-cell clones. Single-cell experiments showed an association between poor proliferative capacity and expression of markers related to antigen experience and dysfunction. Furthermore, we found that spatial heterogeneity of the TIL repertoire resulted in TCR repertoires that are greatly divergent between TIL cultures derived from distant tumor samples of the same patient. CONCLUSIONS: Culture-induced changes in clonal composition are likely to affect tumor reactivity of TIL preparations. TCR deep sequencing provides important insights into the factors that govern the outcome of in vitro TIL expansion and thereby a path toward optimization of the production of TIL preparations with high therapeutic efficacy.See related commentary by Lozano-Rabella and Gros, p. 4177.

Expansion of Tumor-Infiltrating Lymphocytes from Melanoma Tumors.

Autologous, in vitro-expanded tumor-infiltrating lymphocytes (TIL) have been successfully used for treatment of melanoma patients. Expansion of TIL from tumors is usually performed in two steps. The details of the procedure differ between different laboratories, but the general concept remains the same. In the first step, small fragments of a tumor are placed in culture medium containing one or more T cell stimulating growth factors. Here the most common protocol using interleukin (IL)-2 is described. The length of the first step is flexible to allow generation of enough cell to start the second step of the procedure. The second step is a so-called rapid expansion protocol (REP) where harvested TIL from the first step are induced to massive proliferation via triggering of their T cell receptor (TCR) complex in presence of an excess of feeder cells and, again, T cell stimulating growth factors. This second expansion step is usually around 2 weeks in length. Here will be described a REP that uses soluble anti-CD3 antibodies for TCR triggering, irradiated peripheral blood mononuclear cells (PBMC) as feeder cells, and IL-2 as the T cell growth factor. Furthermore, the described protocol utilizes gas-permeable cell culture flasks that yield large number of cells similar to conventional bioreactors but using standard laboratory equipment.

Assessment of Antitumor T-Cell Responses by Flow Cytometry After Coculture of Tumor Cells with Autologous Tumor-Infiltrating Lymphocytes.

As a consequence of the ever-increasing use of immunotherapy for the treatment of cancer, interest in the direct interaction between T cells and tumors has surged tremendously. In vitro coculture of tumor cells with autologous tumor-infiltrating lymphocytes (TIL) is a highly physiological and clinically relevant model to study functional T-cell responses that result from the array of activatory and inhibitory signals that naturally occur on the patient's own tumor cells. This chapter describes a detailed protocol to set up a tumor-TIL coculture and assess ensuing functional T-cell responses, in order to establish the strength of tumor recognition by T cells and identify key determinants that govern this.

Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor-Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display.

Tumor-infiltrating lymphocytes (TIL) are considered enriched for T cells recognizing shared tumor antigens or mutation-derived neoepitopes. We performed exome sequencing and HLA-A*02:01 epitope prediction from tumor cell lines from two HLA-A2-positive melanoma patients whose TIL displayed strong tumor reactivity. The potential neoepitopes were screened for recognition using autologous TIL by immunological assays and presentation on tumor major histocompatibility complex class I (MHC-I) molecules by Poisson detection mass spectrometry (MS). TIL from the patients recognized 5/181 and 3/49 of the predicted neoepitopes, respectively. MS screening detected 3/181 neoepitopes on tumor MHC-I from the first patient but only one was also among those recognized by TIL. Consequently, TIL enriched for neoepitope specificity failed to recognize tumor cells, despite being activated by peptides. For the second patient, only after IFN-γ treatment of the tumor cells was one of 49 predicted neoepitopes detected by MS, and this coincided with recognition by TIL sorted for the same specificity. Importantly, specific T cells could be expanded from patient and donor peripheral blood mononuclear cells (PBMC) for all neoepitopes recognized by TIL and/or detected on tumor MHC-I. In summary, stimulating the appropriate inflammatory environment within tumors may promote neoepitope MHC presentation while expanding T cells in blood may circumvent lack of specific TIL. The discordance in detection between physical and functional methods revealed here can be rationalized and used to improve neoantigen-targeted T cell immunotherapy.

Retuning of Mouse NK Cells after Interference with MHC Class I Sensing Adjusts Self-Tolerance but Preserves Anticancer Response.

Natural killer (NK) cells are most efficient if their targets do not express self MHC class I, because NK cells carry inhibitory receptors that interfere with activating their cytotoxic pathway. Clinicians have taken advantage of this by adoptively transferring haploidentical NK cells into patients to mediate an effective graft-versus-leukemia response. With a similar rationale, antibody blockade of MHC class I-specific inhibitory NK cell receptors is currently being tested in clinical trials. Both approaches are challenged by the emerging concept that NK cells may constantly adapt or "tune" their responsiveness according to the amount of self MHC class I that they sense on surrounding cells. Hence, these therapeutic attempts would initially result in increased killing of tumor cells, but a parallel adaptation process might ultimately lead to impaired antitumor efficacy. We have investigated this question in two mouse models: inhibitory receptor blockade in vivo and adoptive transfer to MHC class I-disparate hosts. We show that changed self-perception via inhibitory receptors in mature NK cells reprograms the reactivity such that tolerance to healthy cells is always preserved. However, reactivity against cancer cells lacking critical MHC class I molecules (missing self-reactivity) still remains or may even be increased. This dissociation between activity against healthy cells and tumor cells may provide an answer as to why NK cells mediate graft-versus-leukemia effects without causing graft-versus-host disease and may also be utilized to improve immunotherapy.

Dynamic Regulation of NK Cell Responsiveness.

Natural killer (NK) cells deliver cytotoxic granules and immunomodulatory cytokines in response to transformed and virally infected cells. NK cell functions are under the control of a large number of germline-encoded receptors that recognize various ligands on target cells, but NK cells also respond to cytokines in the surrounding environment. The interaction between NK cell receptors and their ligands delivers either inhibitory or activating signals. The cytokine milieu further shapes NK cell responses, either directly or by influencing the way inhibitory or activating signals are perceived by NK cells. In this review, we discuss how NK cell function is controlled by inhibitory receptors and MHC-I molecules, how activating receptors contribute to NK cell education, and finally, how cytokines secreted by the surrounding cells affect NK cell responsiveness. Inputs at these three levels involve different cell types and are seamlessly integrated to form a functional NK cell population.

A genetic defect in mice that impairs missing self recognition despite evidence for normal maturation and MHC class I-dependent education of NK cells.

In studies of a CD1d1-deficient mouse strain, we unexpectedly observed a severely impaired capacity for NK cell-mediated rejection of MHC class I-deficient (spleen or tumor) cells. Studies of another CD1-defective strain, as well as intercrosses with C57BL/6 mice, indicated that the impaired missing self rejection (IMSR) NK cell defect was a recessive trait, independent from the targeted CD1 locus. Studies with mixed bone marrow chimeras indicated that the defect is intrinsic to NK cells. The IMSR mice had normal proportions of NK cells, displaying a typical cell surface phenotype, as evaluated using a panel of Abs to developmental markers and known receptors. The impaired missing self recognition could not be overcome through cytokine stimulation. There was also an impaired capacity with respect to NKG2D-dependent cytotoxicity, whereas the mice exhibited normal Ly49D/DAP12-dependent responses in vivo and in vitro. The NK cell system of IMSR mice showed two hallmarks of MHC-dependent education: skewing of the Ly49 receptor repertoire and differential in vitro responsiveness between NK cells with and without inhibitory receptors for self-MHC ("licensing"). We conclude that these mice have a recessive trait that perturbs the missing self reaction, as well as NKG2D-dependent responses, whereas other aspects of the NK system, such as development, capacity to sense MHC molecules during education, and Ly49D/DAP12-dependent responses, are largely intact.