The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling ...agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca²âº-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2â»/â» mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2â»/â» mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2â»/â» mice displayed a higher degree of susceptibility than IL-12-unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2â»/â» mice. The severity of listeriosis we observed in TRPM2â»/â» mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.
Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites ...of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase- and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved.
Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and ...resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the alpha-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.
Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and ...promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects.
Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma.
In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy.
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•Apoptotic lymphoma cells promote tumor growth, angiogenesis, and TAM accumulation•Unbiased “in situ transcriptomics” analysis shows TAMs promote pro-tumor pathways•Apoptotic tumor cells express and process matrix remodeling proteins•The oncogenic potential of apoptotic tumor cells extends beyond lymphoma
Apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. Ford et al. demonstrate apoptotic lymphoma cells can promote tumor growth, angiogenesis, TAM accumulation, and TAM activation to potentiate cancer progression. These results have important implications for apoptosis-inducing anti-cancer therapies.
Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the "professional phagocyte" of the body, are responsible for efficient, ...non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity in vivo. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways.
Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of ...apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.
Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) ...are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.
Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance ...is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14-/-macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14-/-macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.
In this phase 1/2 study, brentuximab vedotin (BV) and nivolumab (Nivo) administered in combination were evaluated as initial salvage therapy in patients with relapsed or refractory (R/R) classical ...Hodgkin lymphoma (HL). Patients received up to 4 cycles of combination treatment, with BV administered on day 1 and Nivo on day 8 of the first cycle. For cycles 2 to 4, BV and Nivo were both administered on day 1. After study treatment, responses were evaluated by investigators per the 2014 Lugano classification, and patients could proceed to autologous stem cell transplantation (ASCT). Sixty-two patients were enrolled; the complete response rate among all treated patients (n = 61) was 61%, with an objective response rate of 82%. Before ASCT, adverse events (AEs) occurred in 98% of patients, mostly grades 1 and 2. Infusion-related reactions (IRRs) occurred in 44% of patients overall, with 41% of patients experiencing an IRR during at least 1 infusion of BV. Five patients (8%) were treated with systemic steroids for immune-related AEs. A reduction of peripheral T-cell subsets including regulatory T cells was observed after the first dose of BV, and reduced serum levels of thymus- and activation-regulated chemokine concurrent with an increase in proinflammatory cytokines and chemokines were seen after the first BV plus Nivo infusions. The combination of BV plus Nivo was an active and well-tolerated first salvage regimen, potentially providing patients with R/R HL an alternative to traditional chemotherapy. This trial was registered at www.clinicaltrials.gov as #NCT02572167.
•BV and Nivo were well-tolerated in patients with R/R HL, with less than 10% of patients treated with systemic steroids for immune-related AEs.•The complete response rate was 61% (82% objective response rate), and patients were able to undergo stem cell transplant without adverse impact.
Introduction: In recently-reported interim results from a phase 1/2, open-label, multicenter study (NCT02572167), we showed that 61% of relapsed/refractory Hodgkin Lymphoma (HL) patients achieved ...complete responses (CRs) after treatment with Brentuximab Vedotin (BV) in combination with Nivolumab (Nivo) (Herrera et al., 2018). To gain insight into tumor microenvironment driven disease characteristics associated with CRs after combined BV+Nivo therapy, we performed RNA sequencing (RNA-Seq) transcriptome analysis of formalin-fixed paraffin-embedded tumor biopsies obtained from 37 study participants (23 CR, 14 PR/SD/PD) prior to the start of treatment.
Results: Within a set of 132 candidate genes comprised of BV+Nivo therapeutic targets (CD30, PD-1, PD-L1), the markers for immune cells, inflammatory response factors, and potential resistance mechanism factors within the tumor microenvironment, the gene most strongly associated with CRs was the BV target CD30 (TNFRSF8) (p=4x10-4, FDR=5%). Baseline tumor expression of CD30 above 16 counts per million mapped reads (CPM) was strongly associated with achieving CR after BV+Nivo (65% sensitivity and 93% specificity). CRs were associated with several other factors, including higher baseline tumor expression of macrophage markers CD14 and CD163 and lower expression of the Nivo target PD-1 (PDCD1) and multidrug resistance-associated protein 2 (MRP2/ABCC2) in the tumor microenvironment. Both PD-1 and MRP2 were significantly complementary to CD30 for discriminating CRs from the other patients on the basis of baseline gene expression profiling in tumor microenvironment (p<0.01, FDR<20%), and a simple baseline gene expression ratio of CD30/PD-1 achieved high accuracy (area under the receiver operating characteristic curve = 0.93). Interestingly, baseline expression of PD-L1 (CD274) was not significantly associated with development of CRs after BV+Nivo, either by itself (p=0.13) or in combination with CD30 (p=0.22), which may be expected given that consistently high PD-L1 expression has been reported for HL. To gain further insight into the role of the tumor microenvironment (TME) in determining HL responsiveness to BV+Nivo, we performed mathematical deconvolution of the baseline HL biopsy transcriptomes. This analysis suggested that both macrophages and T cells were major leukocyte constituents of the HL biopsy TMEs, with higher baseline macrophage abundance and lower baseline T cell abundance being associated with higher CR rates after BV+Nivo treatment.
Conclusions: The CR rate for combination therapy of BV+Nivo in relapsed/refractory HL is nearly double the rate reported for this patient population for either agent individually, suggesting the possibility of complementary mechanisms between BV-targeted and Nivo-targeted pathways. By performing RNA-Seq analysis of patient baseline tumor biopsies from the BV+Nivo trial (NCT02572167), we were able to identify specific tumor characteristics that are associated with favorable responses to treatment. In particular, the gene expression ratio of CD30/PD-1 was an accurate discriminator for patients that would achieve CRs. This result suggests that BV+Nivo efficacy may be observed in tumors in which the potential for BV-driven immunogenic cell death (ICD) - as indicated by CD30 expression - exceeds a threshold set by the immunosuppressive potential of the tumor (as indicated by PD-1 expression). This hypothesis is supported by the deconvolution analysis results that implicated a favorable role for macrophages (which may become activated in response to BV-induced ICD) in the tumor and a detrimental role for immunosuppressive tumor T cells. Immunohistochemical analyses of biopsies from the study are underway to quantify target abundance and to further characterize the TMEs. These data will be integrated with the transcriptomics to validate and refine the model for BV+Nivo efficacy in relapsed/refractory HL.
Zak:Seattle Genetics: Employment, Equity Ownership. Ogden:Seattle Genetics: Employment, Equity Ownership. Herrera:Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Research Funding; Immune Design: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Merck, Inc.: Consultancy, Research Funding; AstraZeneca: Research Funding; Genentech: Consultancy, Research Funding; KiTE Pharma: Consultancy, Research Funding; Gilead Sciences: Research Funding. Sacchi:Bristol-Myers Squibb: Employment, Equity Ownership. Onsum:Seattle Genetics: Employment, Equity Ownership.
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