Therapeutically targeting type I interferon directly to XCR1+ dendritic cells reveals the role of cDC1s in anti-drug antibodies.

Conventional type 1 dendritic cells (cDC1s) are superior in antigen cross-presentation and priming CD8+ T cell anti-tumor immunity and thus, are a target of high interest for cancer immunotherapy. Type I interferon (IFN) is a potent inducer of antigen cross-presentation, but, unfortunately, shows only modest results in the clinic given the short half-life and high toxicity of current type I IFN therapies, which limit IFN exposure in the tumor. CD8+ T cell immunity is dependent on IFN signaling in cDC1s and preclinical studies suggest targeting IFN directly to cDC1s may be sufficient to drive anti-tumor immunity. Here, we engineered an anti-XCR1 antibody (Ab) and IFN mutein (IFNmut) fusion protein (XCR1Ab-IFNmut) to determine whether systemic delivery could drive selective and sustained type I IFN signaling in cDC1s leading to anti-tumor activity and, in parallel, reduced systemic toxicity. We found that the XCR1Ab-IFNmut fusion specifically enhanced cDC1 activation in the tumor and spleen compared to an untargeted control IFN. However, multiple treatments with the XCR1Ab-IFNmut fusion resulted in robust anti-drug antibodies (ADA) and loss of drug exposure. Using other cDC1-targeting Ab-IFNmut fusions, we found that localizing IFN directly to cDC1s activates their ability to promote ADA responses, regardless of the cDC1 targeting antigen. The development of ADA remains a major hurdle in immunotherapy drug development and the cellular and molecular mechanisms governing the development of ADA responses in humans is not well understood. Our results reveal a role of cDC1s in ADA generation and highlight the potential ADA challenges with targeting immunostimulatory agents to this cellular compartment.

Brief report: STING expressed in tumor and non-tumor compartments has distinct roles in regulating anti-tumor immunity.

Type I interferon-mediated activation of immune cells can facilitate the generation of productive tumor antigen-specific T cell responses in solid tumors. The cGAS/STING DNA sensing pathway is a critical upstream mediator of type I interferon production and is an important regulator of anti-tumor immunity. Numerous STING pathway agonists are now being tested in clinical trials, but the effectiveness of this approach is not yet clear and a better understanding of the relative importance of this pathway in various tumor settings is needed. We have evaluated syngeneic tumor models with different baseline inflammatory states to determine the contributions of STING activity in both tumor and non-tumor cellular compartments to anti-tumor immune responses. We find that productive anti-tumor immune responses in the poorly immunogenic B16F10 model show a strong dependence on STING expression in non-tumor cells. In the immunogenic MC38 model, constitutive STING activation in tumor cells can partially bypass the requirement for STING-dependent activity from immune cells. Our findings reveal multiple, context-dependent roles for STING activity in the regulation of anti-tumor immunity and the response to immunotherapy. In preclinical models where STING is basally active, checkpoint inhibition is more likely to have a therapeutic effect and removal of STING signaling from either the tumor or the non-tumor compartment has a minimal effect. Removal of STING signaling in both, however, diminishes the efficacy derived from checkpoint therapy. Further work is needed to understand the heterogeneity of STING signaling in patients, both in tumor cells and the tumor microenvironment, and the best means of harnessing this pathway to generate anti-tumor immunity and improve therapeutic outcomes.

Neutrophil-specific deletion of Syk results in recruitment-independent stabilization of the barrier and a long-term improvement in cognitive function after traumatic injury to the developing brain.

While traumatic brain injury (TBI) is the leading cause of death and disability in children, we have yet to identify those pathogenic events that determine the extent of recovery. Neutrophils are best known as "first responders" to sites of infection and trauma where they become fully activated, killing pathogens via proteases that are released during degranulation. However, this activational state may generate substantial toxicity in the young brain after TBI that is partially due to developmentally regulated inadequate antioxidant reserves. Neutrophil degranulation is triggered via a downstream signaling pathway that is dependent on spleen tyrosine kinase (Syk). To test the hypothesis that the activational state of neutrophils is a determinant of early pathogenesis and long-term recovery, we compared young, brain-injured conditional knockouts of Syk (sykf/fMRP8-cre+) to congenic littermates (sykf/f). Based upon flow cytometry, there was an extended recruitment of distinct leukocyte subsets, including Ly6G+/Ly6C- and Ly6G+/Ly6Cint, over the first several weeks post-injury which was similar between genotypes. Subsequent assessment of the acutely injured brain revealed a reduction in blood-brain barrier disruption to both high and low molecular weight dextrans and reactive oxygen species in sykf/fMRP8-cre+ mice compared to congenic littermates, and this was associated with greater preservation of claudin 5 and neuronal integrity, as determined by Western blot analyses. At adulthood, motor learning was less affected in brain-injured sykf/fMRP8-cre+ mice as compared to sykf/f mice. Performance in the Morris Water Maze revealed a robust improvement in hippocampal-dependent acquisition and short and long-term spatial memory retention in sykf/fMRP8-cre+ mice. Subsequent analyses of swim path lengths during hidden platform training and probe trials showed greater thigmotaxis in brain-injured sykf/f mice than sham sykf/f mice and injured sykf/fMRP8-cre+ mice. Our results establish the first mechanistic link between the activation state of neutrophils and long-term functional recovery after traumatic injury to the developing brain. These results also highlight Syk kinase as a novel therapeutic target that could be further developed for the brain-injured child.

Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

HIF signaling in osteoblast-lineage cells promotes systemic breast cancer growth and metastasis in mice.

Bone metastasis involves dynamic interplay between tumor cells and the local stromal environment. In bones, local hypoxia and activation of the hypoxia-inducible factor (HIF)-1α in osteoblasts are essential to maintain skeletal homeostasis. However, the role of osteoblast-specific HIF signaling in cancer metastasis is unknown. Here, we show that osteoprogenitor cells (OPCs) are located in hypoxic niches in the bone marrow and that activation of HIF signaling in these cells increases bone mass and favors breast cancer metastasis to bone locally. Remarkably, HIF signaling in osteoblast-lineage cells also promotes breast cancer growth and dissemination remotely, in the lungs and in other tissues distant from bones. Mechanistically, we found that activation of HIF signaling in OPCs increases blood levels of the chemokine C-X-C motif ligand 12 (CXCL12), which leads to a systemic increase of breast cancer cell proliferation and dissemination through direct activation of the CXCR4 receptor. Hence, our data reveal a previously unrecognized role of the hypoxic osteogenic niche in promoting tumorigenesis beyond the local bone microenvironment. They also support the concept that the skeleton is an important regulator of the systemic tumor environment.

NLRP3 Inflammasome Mediates Dormant Neutrophil Recruitment following Sterile Lung Injury and Protects against Subsequent Bacterial Pneumonia in Mice.

Sterile lung injury is an important clinical problem that complicates the course of severely ill patients. Interruption of blood flow, namely ischemia-reperfusion (IR), initiates a sterile inflammatory response in the lung that is believed to be maladaptive. The rationale for this study was to elucidate the molecular basis for lung IR inflammation and whether it is maladaptive or beneficial. Using a mouse model of lung IR, we demonstrate that sequential blocking of inflammasomes [specifically, NOD-, LRR-, and pyrin domain-containing 3 (NLRP3)], inflammatory caspases, and interleukin (IL)-1ß, all resulted in an attenuated inflammatory response. IL-1ß production appeared to predominantly originate in conjunction with alveolar type 2 epithelial cells. Lung IR injury recruited unactivated or dormant neutrophils producing less reactive oxygen species thereby challenging the notion that recruited neutrophils are terminally activated. However, lung IR inflammation was able to limit or reduce the bacterial burden from subsequent experimentally induced pneumonia. Notably, inflammasome-deficient mice were unable to alter this bacterial burden following IR. Thus, we conclude that the NLRP3 inflammasome, through IL-1ß production, regulates lung IR inflammation, which includes recruitment of dormant neutrophils. The sterile IR inflammatory response appears to serve an important function in inducing resistance to subsequent bacterial pneumonia and may constitute a critical part of early host responses to infection in trauma.

Adaptive Immune Regulation of Mammary Postnatal Organogenesis.

Postnatal organogenesis occurs in an immune competent environment and is tightly controlled by interplay between positive and negative regulators. Innate immune cells have beneficial roles in postnatal tissue remodeling, but roles for the adaptive immune system are currently unexplored. Here we show that adaptive immune responses participate in the normal postnatal development of a non-lymphoid epithelial tissue. Since the mammary gland (MG) is the only organ developing predominantly after birth, we utilized it as a powerful system to study adaptive immune regulation of organogenesis. We found that antigen-mediated interactions between mammary antigen-presenting cells and interferon-γ (IFNγ)-producing CD4+ T helper 1 cells participate in MG postnatal organogenesis as negative regulators, locally orchestrating epithelial rearrangement. IFNγ then affects luminal lineage differentiation. This function of adaptive immune responses, regulating normal development, changes the paradigm for studying players of postnatal organogenesis and provides insights into immune surveillance and cancer transformation.

Delineating CSF-1-dependent regulation of myeloid cell diversity in tumors.

Myeloid cells contribute to increased malignancy and poor prognosis in breast cancer. We demonstrate that anti-CSF-1R therapy depletes a cell population sharing characteristics of tumor-associated macrophages (TAMs) and dendritic cells (DCs). Intravital imaging combined with cellular characterization has refined our understanding of anti-CSF-1R therapy on the tumor microenvironment.

Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.

Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.

Balancing the innate immune system in tumor development.

Cells of the innate immune system have a dual role in cancer development in both tumor initiation and progression. Innate immune cells can, on the one hand, aid malignant transformation and tumor outgrowth and, on the other hand, prevent tumor progression. The innate immune system has the ability to tune the inflammatory response and is a key player in cancer-related inflammation, which can precede the development of malignancy or be induced by oncogenic changes promoting a protumor inflammatory milieu. In this review, we discuss the emerging cellular and molecular mechanisms of the innate immune system and inflammation in tumor initiation and progression, and point to the outstanding questions that remain.

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations.

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R-dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti-CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1(+) neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.

Protective effects of matrix metalloproteinase-12 following corneal injury.

Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers α-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12(-/-) mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12(-/-) corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.

Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558.

Flavocytochrome b(558), the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b(558) is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91(phox), and a smaller subunit, p22(phox). We recently showed in murine macrophages that flavocytochrome b(558) localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91(phox) and p22(phox) reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b(558) expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91(phox) and p22(phox) but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91(phox) and p22(phox) from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b(558) and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated.

Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes.

Flavocytochrome b(558), the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91(phox) (NOX2) and a smaller subunit, p22(phox). Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91(phox) and p22(phox), we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22(phox) and gp91(phox) subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity.

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.