Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies

Regulation of Endothelial Cell Adhesion Molecule Expression by Mast Cells, Macrophages, and Neutrophils

Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.Using bone marrow-derived mast cells from wild-type, Tnf(-/-), Ifng(-/-), Il6(-/-) mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases

Marginal zone B cells control the response of follicular helper T cells to a high-cholesterol diet

Splenic marginal zone B (MZB) cells, positioned at the interface between circulating blood and lymphoid tissue, detect and respond to blood-borne antigens. Here we show that MZB cells in mice activate a homeostatic program in response to a high-cholesterol diet (HCD) and regulate both the differentiation and accumulation of T follicular helper (TFH) cells. Feeding mice an HCD resulted in upregulated MZB cell surface expression of the immunoregulatory ligand PDL1 in an ATF3-dependent manner and increased the interaction between MZB cells and pre-TFH cells, leading to PDL1-mediated suppression of TFH cell motility, alteration of TFH cell differentiation, reduced TFH abundance and suppression of the proatherogenic TFH response. Our findings reveal a previously unsuspected role for MZB cells in controlling the TFH–germinal center response to a cholesterol-rich diet and uncover a PDL1-dependent mechanism through which MZB cells use their innate immune properties to limit an exaggerated adaptive immune response.This work was supported by BHF grant no. PG/15/76/31756, BHF grant no. PG/13/73/30466, ERC grant no. 2891164 and EC FP7 VIA grant no. HEALTH-F4- 2013-603131 to Z.M. and by SAF2013-45543-R from the Spanish Ministry of Economy and Competitiveness (MINECO) to J.L.d.l.P. M.N. was first supported by a Sara Borrell grant (CD09/00452) from the Instituto Nacional de Salud Carlos III (Spain) and then by a 2-year BHF Project Grant. M.N. has also received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. 608765. The Wellcome Trust supported the Cambridge Mouse Biochemistry Laboratory

Tracking the fate of adoptively transferred myeloid-derived suppressor cells in the primary breast tumor microenvironment.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that are expanded in cancer and act as potent suppressors of the anti-tumor immune response. MDSCs consist of two major subsets, namely monocytic (M-) MDSCs and granulocytic (G-) MDSCs that differ with respect to their phenotype, morphology and mechanisms of suppression. Here, we cultured bone marrow cells with IL-6 and GM-CSF in vitro to generate a population of bone marrow MDSCs (BM-MDSCs) similar to G-MDSCs from tumor-bearing mice in regards to phenotype, morphology and suppressive-function. Through fluorescent labeling of these BM-MDSCs and optical imaging, we could visualize the recruitment and localization of BM-MDSCs in breast tumor-bearing mice in vivo. Furthermore, we were able to demonstrate that BM-MDSCs home to primary and metastatic breast tumors, but have no significant effect on tumor growth or progression. Ex vivo flow cytometry characterization of BM-MDSCs after adoptive transfer demonstrated both organ-and tumor-specific effects on their phenotype and differentiation, demonstrating the importance of the local microenvironment on MDSC fate and function. In this study, we have developed a method to generate, visualize and detect BM-MDSCs in vivo and ex vivo through optical imaging and flow cytometry, in order to understand the organ-specific changes rendered to MDSCs in breast cancer

Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy

Tumorimmunolog

Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy

Tumorimmunolog

The Potential of <i>In Vivo</i> Imaging for Optimization of Molecular and Cellular Anti-cancer Immunotherapies

This review aims to emphasize the potential of in vivo imaging to optimize current and upcoming anti-cancer immunotherapies: spanning from preclinical to clinical applications. Immunotherapies are an emerging class of treatments for a variety of diseases. The agents include molecular and cellular therapeutics, which aim to treat the disease through re-education of the host immune system, often via complex mechanisms of action. In vivo imaging has the potential to contribute in several different ways: (1) as a drug development tool to improve our understanding of their complex mechanisms of action, (2) as a tool to predict efficacy, for example, to stratify patients into probable responders and likely non-responders, and (3) as a non-invasive treatment response biomarker to guide efficient immunotherapy use and to recognize early signs of potential loss of efficacy or resistance in patients. Areas where in vivo imaging is already successfully implemented in onco-immunology research will be discussed and domains where its use offers great potential will be highlighted. The focus of this article is on anti-cancer immunotherapy as it currently is the most advanced immunotherapy area. However, the described concepts can also be paralleled in other immune-mediated disorders and for conditions requiring immunotherapeutic intervention. Importantly, we introduce a new study group within the European Society of Molecular Imaging with the goal to facilitate and enhance immunotherapy development through the use of in vivo imaging

Noninvasive, longitudinal imaging-based analysis of body adipose tissue and water composition in a melanoma mouse model and in immune checkpoint inhibitor-treated metastatic melanoma patients.

Background As cancer cachexia (CC) is associated with cancer progression, early identification would be beneficial. The aim of this study was to establish a workflow for automated MRI-based segmentation of visceral (VAT) and subcutaneous adipose tissue (SCAT) and lean tissue water (LTW) in a B16 melanoma animal model, monitor diseases progression and transfer the protocol to human melanoma patients for therapy assessment. Methods For in vivo monitoring of CC B16 melanoma-bearing and healthy mice underwent longitudinal three-point DIXON MRI (days 3, 12, 17 after subcutaneous tumor inoculation). In a prospective clinical study, 18 metastatic melanoma patients underwent MRI before, 2 and 12 weeks after onset of checkpoint inhibitor therapy (CIT; n = 16). We employed an in-house MATLAB script for automated whole-body segmentation for detection of VAT, SCAT and LTW. Results B16 mice exhibited a CC phenotype and developed a reduced VAT volume compared to baseline (B16 - 249.8 mu l, - 25%; controls + 85.3 mu l, + 10%, p = 0.003) and to healthy controls. LTW was increased in controls compared to melanoma mice. Five melanoma patients responded to CIT, 7 progressed, and 6 displayed a mixed response. Responding patients exhibited a very limited variability in VAT and SCAT in contrast to others. Interestingly, the LTW was decreased in CIT responding patients (- 3.02% +/- 2.67%; p = 0.0034) but increased in patients with progressive disease (+ 1.97% +/- 2.19%) and mixed response (+ 4.59% +/- 3.71%). Conclusion MRI-based segmentation of fat and water contents adds essential additional information for monitoring the development of CC in mice and metastatic melanoma patients during CIT or other treatment approaches

Imaging the Rewired Metabolism in Lung Cancer in Relation to Immune Therapy

Metabolic reprogramming is recognized as one of the hallmarks of cancer. Alterations in the micro-environmental metabolic characteristics are recognized as important tools for cancer cells to interact with the resident and infiltrating T-cells within this tumor microenvironment. Cancer-induced metabolic changes in the micro-environment also affect treatment outcomes. In particular, immune therapy efficacy might be blunted because of somatic mutation-driven metabolic determinants of lung cancer such as acidity and oxygenation status. Based on these observations, new onco-immunological treatment strategies increasingly include drugs that interfere with metabolic pathways that consequently affect the composition of the lung cancer tumor microenvironment (TME). Positron emission tomography (PET) imaging has developed a wide array of tracers targeting metabolic pathways, originally intended to improve cancer detection and staging. Paralleling the developments in understanding metabolic reprogramming in cancer cells, as well as its effects on stromal, immune, and endothelial cells, a wave of studies with additional imaging tracers has been published. These tracers are yet underexploited in the perspective of immune therapy. In this review, we provide an overview of currently available PET tracers for clinical studies and discuss their potential roles in the development of effective immune therapeutic strategies, with a focus on lung cancer. We report on ongoing efforts that include PET/CT to understand the outcomes of interactions between cancer cells and T-cells in the lung cancer microenvironment, and we identify areas of research which are yet unchartered. Thereby, we aim to provide a starting point for molecular imaging driven studies to understand and exploit metabolic features of lung cancer to optimize immune therapy

Lactate production precedes inflammatory cell recruitment in arthritic ankles : an imaging study

Purpose: Inflammation is involved in many disease processes. However, accurate imaging tools permitting diagnosis and characterization of inflammation are still missing. As inflamed tissues exhibit a high rate of glycolysis, pyruvate metabolism may offer a unique approach to follow the inflammatory response and disease progression. Therefore, the aim of the study was to follow metabolic changes and recruitment of inflammatory cells after onset of inflammation in arthritic ankles using hyperpolarized 1-13C-pyruvate magnetic resonance spectroscopy (MRS) and 19F magnetic resonance imaging (MRI), respectively. Procedure: Experimental rheumatoid arthritis (RA) was induced by intraperitoneal injection of glucose-6-phosphate-isomerase-specific antibodies (GPI) containing serum. To monitor pyruvate metabolism, the transformation of hyperpolarized 1-13C-pyruvate into hyperpolarized 1-13C-lactate was followed using MRS. To track phagocytic immune cell homing, we intravenously injected a perfluorocarbon emulsion 48 h before imaging. The animals were scanned at days 1, 3, or 6 after GPI-serum injection to examine the different stages of arthritic inflammation. Finally, to confirm the pyruvate metabolic activity and the link to inflammatory cell recruitment, we conducted hematoxylin-eosin histopathology and monocarboxylase transporter (MCT-1) immune histochemistry (IHC) of inflamed ankles. Results: Hyperpolarized 1-13C-pyruvate MRS revealed a high rate of lactate production immediately at day 1 after GPI-serum transfer, which remained elevated during the progression of the disease, while 19F-MRI exhibited a gradual recruitment of phagocytic immune cells in arthritic ankles, which correlated well with the course of ankle swelling. Histopathology and IHC revealed that MCT-1 was expressed in regions with inflammatory cell recruitment, confirming the metabolic shift identified in arthritic ankles. Conclusions: Our study demonstrated the presence of a very early metabolic shift in arthritic joints independent of phagocytic immune cell recruitment. Thus, hyperpolarized 1-13C-pyruvate represents a promising tracer to monitor acute arthritic joint inflammation, even with minor ankle swelling. Furthermore, translated to the clinics, these methods add a detailed characterization of disease status and could substantially support patient stratification and therapy monitoring